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WO1999032622A2 - Semaphorin zsmf-3 - Google Patents

Semaphorin zsmf-3 Download PDF

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Publication number
WO1999032622A2
WO1999032622A2 PCT/US1998/026793 US9826793W WO9932622A2 WO 1999032622 A2 WO1999032622 A2 WO 1999032622A2 US 9826793 W US9826793 W US 9826793W WO 9932622 A2 WO9932622 A2 WO 9932622A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
amino acid
zsmf
semaphorin
Prior art date
Application number
PCT/US1998/026793
Other languages
French (fr)
Other versions
WO1999032622A3 (en
Inventor
James L. Holloway
Original Assignee
Zymogenetics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zymogenetics, Inc. filed Critical Zymogenetics, Inc.
Priority to AU20002/99A priority Critical patent/AU2000299A/en
Publication of WO1999032622A2 publication Critical patent/WO1999032622A2/en
Publication of WO1999032622A3 publication Critical patent/WO1999032622A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Neuronal cell outgrowths grow away from the cell body to form synaptic connections.
  • Long, thin processes which carry information away from the cell body are called axons, and short, thicker processes which carry information to and from the cell body are called dendrites.
  • Axons and dendrites are collectively referred to as neurites .
  • Neurites are extended by means of growth cones, the growing tip of the neurite, which is highly motile and is ultimately responsible for increasing and extending the neuronal network in the body.
  • the growth cones are able to navigate their way to their targets using environmental cues or signals, which encourage or discourage the growth cone from extending the neurite in a particular direction.
  • Such cues and signals include older neurons and orienting glial fibers, chemicals such as nerve growth factor released by astrocytes and other attracting or repelling substances released by target cells.
  • the membrane of the growth cone bears molecules such as N-CAM (nerve cell adhesion molecule) which are attracted or repelled by environmental cues and thus influence the direction and degree of neurite growth.
  • N-CAM nerve cell adhesion molecule
  • the growth cone also engulfs molecules from the environment which are transported to the cell body and influence growth. A number of proteins from vertebrates and invertebrates have been identified as influencing the guidance of neurite growth, either through repulsion or chemoattraction.
  • netrins EPH-related receptor tyrosine kinases and their ligands, vitronectin, thrombospondin, human neuronal attachment factor- 1 (NAF-1) , connectin, adhesion molecules such as CAM (cell adhesion molecule) and the semaphorins/collapsins (Neugebauer et al . , Neuron 6 . :345-58, 1991; 0 ' Shea et al . , Neuron 1:231-7, 1991; Osterhout et al . , Devel . Biol . 150 : 256-65 , 1992; Goodman, Cell 78 .
  • Semaphorins/collapsins are a family of related transmembrane and secreted molecules.
  • Invertebrate, vertebrate and viral semaphorins are known (Kolodkin et al., Cell 25_: 1389-99 , 1993; Luo et al . , Cell 75:217-27, 1993; Ensser and Fleckenstein, J. Gen. Virol. 76 :1063-7, 1995; Luo et al , Neuron 14.: 1131-40, 1995; Adams et al . , Mech. Devel. 5_7:33-45, 1996; Hall et al . , Proc. Natl. Acad. Sci. USA 93:11780-8, 1996; Roche et al .
  • the semaphorins comprise a variable region of. 30- 60 amino acids that includes a signal sequence, followed by a conserved region of about 500 amino acid residues called the semaphorin (Sema) domain. This extracellular domain contains between 14-16 conserved cysteine residues, an N- linked glycosylation site and numerous blocks of conserved amino acids.
  • Sema semaphorin
  • Membrane-bound and soluble semaphorin proteins have been described, see for example, Kolodkin et al . , ibid. ; Adams et al . , ibid, and Goodman et al . , US Patent No : 5 , 639 , 856.
  • Group I includes semaphorins having a transmembrane domain followed by a cytoplasmic domain.
  • Group II and III have no transmembrane domain, but have a region with Ig homology
  • Group IV has a region of Ig homology C-terminal of the semaphorin domain followed by a transmembrane and cytoplasmic domain.
  • Group V has a series of thrombospondin repeats C-terminal to the semaphorin domain followed by a transmembrane and cytoplasmic domain.
  • G-Sema I, T-Sema I and D- Sema I have a region of 80 amino acid residues following the semaphorin domain, which is followed by a transmembrane domain and an 80-110 amino acid cytoplasmic domain.
  • Murine Sema IVa has a transmembrane domain followed by a 216 amino acid cytoplasmic domain.
  • Murine sema F and G are members of Group V and have an approximately 400 amino acid thrombospondin repeat domain between the semaphorin and transmembrane domains.
  • Group II secreted proteins such as D-sema II
  • Group III secreted proteins such as H-Sema III
  • the viral semaphorin- like gene, ahv-sema is truncated immediately following the Ig-domain.
  • the viral semaphorin, sema IV has a truncated, 441 amino acid residue, semaphorin domain and no Ig region . See Kolodkin et al .
  • semaphorins share the greatest degree of homology within the semaphorin domain, between, 25-93%, with a greater degree of divergence in all other regions and domains suggesting distinct roles for various sub-groups within the semaphorin family.
  • the viral semaphorins are the most diverse, sharing only 25% identity with vertebrate semaphorins . Between vertebrate and invertebrate semaphorins, the percent identity varies between 30-40%.
  • the invention also provides an isolated polypeptide having the amino acid sequence of SEQ ID NO : 2.
  • the invention further provides an isolated polypeptide comprising the amino acid sequence of amino acid residue 1 to amino acid residue 19 of SEQ ID NO : 2.
  • the invention provides an isolated polypeptide consisting of a semaphorin domain, a transmembrane domain and a cytoplasmic domain, wherein said semaphorin domain comprises the amino acid sequence of SEQ ID NO: 2.
  • a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion comprising a polypeptide wherein said polypeptide comprises a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; wherein said polypeptide is a semaphorin; and said second portion comprising another polypeptide.
  • the second polypeptide is a transmembrane domain of a semaphorin protein.
  • the second polypeptide further comprises a cytoplasmic domain of a semaphorin protein.
  • the invention also provides a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1 to 19 of SEQ ID N0:2, wherein said secretory signal sequence is operably linked to an additional polypeptide.
  • an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; b) degenerate polynucleotide sequences of a) ; and c) a polynucleotide sequence complementary to a) or b) ; wherein said polynucleotide encodes a polypeptide which is a semaphorin.
  • the polypeptide comprises residue ' s 20 through 321 of SEQ ID NO : 2.
  • the protein comprises residues 1 through 321 of SEQ ID NO : 2.
  • the invention also provides a method of making a semaphorin protein comprising: (a) culturing a host cell containing an expression vector as described herein; and
  • the expression vector further comprises a secretory signal sequence operably linked to said DNA segment, the cell secretes the protein into a culture medium, and the protein is recovered from the medium.
  • the expression vector further comprises an affinity tag operably linked to said
  • the invention provides a pharmaceutical composition comprising a polypeptide as described herein, in combination with a pharmaceutically acceptable vehicle.
  • Figure 2 shows an alignment of human ZSMF-3 (SEQ ID NO:l) with murine Sema IVa (SEQ ID NO: 37) .
  • Allelic variant Any of two or more alternative forms of a gene occupying the same chromosomal locus.
  • Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations.
  • Gene mutations can be silent (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Also included are the same protein from the same species which differs from a reference amino acid sequence due to allelic variation. Allelic variation refers to naturally occurring differences among individuals in genes encoding a given protein.
  • Amino-terminal and carboxyl -terminal are used herein to denote positions within polypeptides and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.
  • Complement/anti-complement pair Denotes non- identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti -complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 -9 M.
  • Complements of polynucleotide molecules Denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • Degenerate As applied to a nucleotide sequence such as a probe or primer, denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) . Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • Expression vector A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • Isolated When applied to a polynucleotide molecule, the term "isolated” indicates that the molecule is removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316 :774-78, 1985) .
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • Ortholog or species homolog A polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation .
  • Polynucleotide denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
  • Promoter Denotes a portion of a gene containing
  • Receptor A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell.
  • Metabolic events that are linked to receptor- ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • Secretory signal sequence A DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • Soluble receptor/soluble ligand A receptor/ligand polypeptide that is not bound to a cell membrane. Many cell-surface receptors/ligands have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. They may also be produced by cleavage or by recombinant methods .
  • Soluble receptors are most commonly ligand-binding receptor polypeptides and soluble ligands are most commonly receptor-binding polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors/ligands can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate. Receptor/ligand polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
  • Splice variant As used herein, denote alternative forms of RNA transcribed from a gene.
  • Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules,, and may result in several mRNAs transcribed from the same gene.
  • Splice variants may encode polypeptides having altered amino acid sequence.
  • the term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene .
  • Molecular weights and lengths of polymers determined by imprecise analytical methods e.g., gel electrophoresis
  • the present invention is based in part upon- the discovery of a novel human member of the semaphorin family, designated "ZSMF-3".
  • ZSMF-3 novel human member of the semaphorin family
  • the human ZSMF-3 nucleotide sequence is represented in SEQ ID NO : 1 and the deduced amino acid sequence in SEQ ID NO : 2.
  • a murine homologue is represented in SEQ ID NO: 3 and the deduced amino acid sequence in SEQ ID NO:4.
  • Murine and human ZSMF-3 share 90% nucleotide identity and 95% amino acid identity.
  • novel human ZSMF-3 semaphorin proteins and polypeptides encoded by polynucleotides of the present invention were initially identified by querying an EST (Expressed Sequence Tag) database for sequences homologous to conserved motifs within the semaphorin family.
  • Sequence analysis of the deduced amino acid sequence as represented in SEQ ID NO: 2 indicate the presence of a 19 amino acid residue signal sequence (amino acid residues 1-19 of SEQ ID NO: 2) followed by at least 302 amino acid residues of a semaphorin domain (amino acid residues 20-321 of SEQ ID
  • amino acid residues 107, 117, 135, 144, 165, 258 and 283 of SEQ ID NO: 2 amino acid residues 107, 117, 135, 144, 165, 258 and 283 of SEQ ID NO: 2 .
  • Sema IVa The sequence of human and murine ZSMF-3, as represented in SEQ ID NOs : 2 and 4, are homologous to murine Sema IVa identified by Zhou et al . , (Mol. Cell. Neurosci. 3_ : 26-41, 1997. Sema IVa has been described as having a 16 amino acid residue signal sequence, a 648 amino acid residue extracellular region containing a 500 amino acid residue semaphorin domain. Within the semaphorin domain are 13 conserved cysteine residues and 7 N-linked glycosylation sites. The extracellular region is followed by a 24 amino acid transmembrane domain and a 216 amino acid residue cytoplasmic domain having one protein kinase C site.
  • human ZSMF-3 protein encoded by the nucleotide sequence described in SEQ ID NO : 1 represents an alternative splicing product or "splice variant" of a corresponding full length human ZSMF- 3 protein.
  • Splice variants of a full length DNA sequence include DNA sequences that result from mature RNA molecules created by known eukaryotic RNA splicing processes wherein the intron sequence is removed and exon sequence is joined.
  • a well defined splice site which begins at nucleotide 963 of SEQ ID N0:1, a poly A region (nucleotide 1033-1038 of SEQ ID NO:l) followed by a poly A tail (beginning at nucleotide 1054 of SEQ ID NO:l) confirm that this sequence indeed represents a viable splice variant.
  • Northern blot analysis indicates a predominant transcript in stomach tissue of 2 kb which agrees with the splice variant size. Two faint transcripts of 4 kb and 3 kb were detected in spinal cord and adrenal gland which would indicate ⁇ the existence of a full length ZSMF-3 protein.
  • Sema IVa represents a new class of mammalian semaphorins which have structural homology to the insect Sema I proteins.
  • murine Sema IVa shares 25-36% amino acid identity with murine Sema III within the semaphorin domain
  • Sem IVa has a transmembrane domain and a cytoplasmic domain, although much larger, as are seen in the insect Sema I proteins.
  • Previously described transmembrane containing vertebrate semaphorins also have regions of Ig homology or transponder repeats which are not present in murine Sema IVa.
  • human ZSMF-3 is a human hom ' olog of murine Sema IVa and a member of this new semaphorin family. A final determination can only be made once it is determined what sequence lies C terminal to the ZSMF-3 semaphorin domain. Over a similar area in the semaphorin domain, murine ZSMF-3 (SEQ ID NO: ) and human ZSMF-3 (SEQ ID NO:2) share 100% and 95% identity respectively, with murine SEMA IVa described by Zhou et al . , ibid.
  • Figure 1 shows an alignment of human and mouse ZSMF-3 with murine semaphorins Sem F and Sem G.
  • Figure 2 shows an alignment of human ZSMF-3 with murine SEMA IVa.
  • the truncated semaphorin domain of ZSMF-3 may suggest that it also shares an association with the viral semaphorin Sema IV, which has a truncated semaphorin domain of about 400 amino acids.
  • An alternatively spliced 3.5 kb human semaphorin, SEMA3F,- was described by Xiang et al . (Genomics 32:39-48, 1996). The splice resulted in the addition of 96 bases to the message and retained the correct reading frame.
  • Such features set human ZSMF-3 apart from previously described semaphorins and may be characteristic qualities which further define a new sub-group within the semaphorin family.
  • the DNA sequence shown in SEQ ID NO:l or portions thereof can be used as probes or primers to obtain the corresponding full length ZSMF-3 DNA from cells or libraries (including cDNA and genomic libraries) from human tissues.
  • Methods for isolating polynucleotide sequences are known in the art.
  • One example for obtaining the ZSMF-3 sequence uses mRNA obtained from a tissue or cell types that are likely to express the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. Suitable tissue sources include human spinal cord, adrenal gland, stomach and brain.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • the cD ⁇ A can then be isolated by a variety of methods, such as by probing with the sequence disclosed in SEQ ID ⁇ O:l or some portion thereof.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences disclosed herein and templates derived from appropriate tissues as discussed above. Additional oligonucleotides may be generated from the PCR derived sequence and SEQ ID N0:1, as needed, to obtain a complete full length cDNA encoding a ZSMF-3 polypeptide of about 890 amino acid residues.
  • full length ZSMF-3 polypeptides would have about 700-750 amino acid residues.
  • Such a full length ZSMF-3 molecule would include a semaphorin domain of approximately 500 amino acid residues, a transmembrane domain of approximately 24 amino acid residues and a cytoplasmic domain of approximately 216 amino acid residues.
  • Other methods such as 3' RACE using oligo sequences from SEQ ID NO:l and templates derived from spinal cord, adrenal gland, brain or stomach tissue could also be used.
  • Northern blot analysis of various human tissues was performed using a 200 bp human DNA probe (SEQ ID, NO: 6) .
  • a 2 kb transcript was detected corresponding to ZSMF-3 in stomach tissue.
  • the transcript size agrees with the predicted size of the ZSMF-3 protein as disclosed in SEQ ID NO: 2.
  • Two faint transcripts of 4 kb and 3 kb were detected in spinal cord and adrenal gland tissue.
  • a degenerate polynucleotide sequence that encompasses all polynucleotides that encode the ZSMF-3 polypeptides of SEQ ID NOs: 2 or 4 , amino acid residues 1- 321, are disclosed in SEQ ID NO: 5 (human sequence) and SEQ ID NO:17 (mouse sequence).
  • ZSMF-3 polypeptide- encoding polynucleotides ranging from nucleotide 1-963 of SEQ ID N0:5 and nucleotides 1-963 of SEQ ID N0:17 are contemplated by the present invention.
  • fragments as described above with respect to SEQ ID NO : 1 which are formed from analogous regions of SEQ ID NO : 5 , wherein nucleotides 1-963 of SEQ ID NO:l correspond to nucleotides 57-963 of SEQ ID NO: 5.
  • the nucleotide base codes in SEQ ID NOs : 5 and 17 are summarized in Table 1.
  • degenerate codons used in SEQ ID NOs : 5 and 17, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR)
  • the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) .
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • amino acid threonine amino acid threonine
  • Thr may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.
  • the degenerate codon sequences disclosed in SEQ ID NOs : 5 and 17 serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or to sequences complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe .
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • RNA DNA and RNA are well known in the art. It is generally preferred to isolate RNA from stomach tissue, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry 18 : 52-94 , 1979) .
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 6_9: 1408-12, 1972) .
  • Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods.
  • Polynucleotides encoding ZSMF-3 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • SEQ ID NO : 1 represents a single allele of human ZSMF-3
  • sequence disclosed in SEQ ID NO : 3 represents a single allele of murine ZSMF-3
  • allelic variation and alternative splicing are expected to occur.
  • Allelic variants and splice variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • Allelic variants of the DNA sequence shown in SEQ ID NOs : 1 or 3, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs : 2 or 4.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZSMF-3 polypeptides are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • the present invention further provides counterpart ligands and polynucleotides from other species ("species orthologs"). These species would include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are ZSMF-3 polypeptides from other mammalian species, including porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Species orthologs of human ZSMF-3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the ligand. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a ligand- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on- the disclosed sequence.
  • a cDNA can also be cloned by PCR, using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the ligand. Similar techniques can also be applied to the isolation of genomic clones.
  • the present invention also provides isolated polypeptides and proteins that are substantially homologous to the polypeptides and proteins of SEQ ID NO: 2 and their species orthologs.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated protein or polypeptide is substantially free of other proteins or polypeptides, particularly other proteins or polypeptides of animal origin. It is preferred to provide the proteins or polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • substantially homologous is used herein to denote proteins or polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO: 2 or its species orthologs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or its species orthologs. Particularly preferred are polypeptides which are 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 2 or its species orthologs. Within one embodiment, any difference between the amino acid sequence of the polypeptide and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution. Percent sequence identity is determined by conventional methods.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, Glu-Glu affinity tags, protein A (Nilsson et al . , EMBO J .
  • an affinity tag such as a poly-histidine tract, Glu-Glu affinity tags, protein A (Nilsson et al . , EMBO J .
  • Aromatic phenylalanine tryptophan tyrosine
  • non-standard amino acids such as 4-hydroxyproline, 6 -N-methyl lysine, 2-aminoisobutyric acid, isovaline and ⁇ -me.thyl serine
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for ZSMF-3 amino acid residues.
  • "Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain (s) different from that of the standard amino acids.
  • Unnatural amino acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3- dimethylproline .
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non- naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis- 4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine , allo-threonine, methylthreonine, hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine, homo-glutamine, pipecolic acid, tert-leucine, norvaline, 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine .
  • an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs .
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am. Chem. Soc . 113 :2722, 1991; Ellman et al .
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) .
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33 : 7470- 6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 1:395-3, 1993) .
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 : 1081-5, 1989; Bass et al . , Proc. Natl. Acad. Sci. USA . 88 . : 4498-502, 1991) .
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244 : 1081-5, 1989; Bass et al . , Proc. Natl. Acad. Sci. USA . 88 . : 4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., collapase assay) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity; in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al . , Science 255:306-12, 1992; Smith et al . , J. Mol. Biol. 2Z4:899-904, 1992; Wlodaver et al . , FEBS Lett . 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
  • Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active ligands or portions thereof e.g., receptor-binding fragments
  • These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the semaphorin polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al .
  • a DNA sequence encoding a ZSMF-3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art . Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the ZSMF-3 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
  • the secretory signal sequence is joined to the ZSMF-3 DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-19 of SEQ ID NOs : 2 or 4 is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14 . : 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 1:603, 1981; Graham and Van der Eb, Virology 52 . :456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid. ) , and liposome-mediated transfection (Hawley-Nelson et al . , Focus 15 .
  • CHO-K1 ATCC No. CCL 61
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants . " A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select ' for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222; Bang et al . , U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463.
  • Agroba cterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. (Bangalore) 11:47-58, 1987.
  • Insect cells can be infected with recombinant baculovirus vectors, which are commonly derived from Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV) .
  • DNA encoding the polypeptide of interest is inserted into the viral genome in place of the polyhedrin gene coding sequence by homologous recombination in cells infected with intact, wild-type AcMNPV and transfected with a transfer vector comprising the cloned gene operably linked to polyhedrin gene promoter, terminator, and flanking sequences.
  • the resulting recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and
  • Fungal cells including yeast cells, and particularly cells of the genus Sa ccharomyces, can also be used within the present invention, such as for producing fragments or polypeptide fusions.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in yeast is the POTl vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Transformation systems for other yeasts including Hansenula polymorpha , Schizosaccharomyces pombe, Kl uyveromyces lactis , Kl uyveromyces fragilis , Us tilago maydis , Pichia pa storis, Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol .
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a ZSMF-3 polypeptide in bacteria such as E . col i , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • the invention provides soluble ZSMF-3 ligand polypeptides.
  • Soluble ZSMF-3 polypeptides can be prepared by expressing a DNA encoding amino acid residues 20-32 . 1 of a human ZSMF-3 polypeptide (SEQ ID NO: 2) .
  • SEQ ID NO: 2 a human ZSMF-3 polypeptide
  • these soluble, extracellular receptor-binding domain polypeptides be prepared in a form substantially free of transmembrane and intracellular polypeptide segments.
  • the C- terminus of the ligand polypeptide may be at residue 32.1 of SEQ ID NO: 2 or the corresponding region of an allelic variant or at the C-terminal end of a semaphorin domain.
  • Expressed recombinant ZSMF-3 polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast- Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl , butyl, or octyl groups, such as Phenyl -Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • the polypeptides of the present invention can be isolated by exploitation of their physical properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3_:l-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • ZSMF-3 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZSMF-3 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Polypeptides containing the receptor-binding region of the ligand can be used for purification of receptor.
  • the ligand polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting media will generally be configured in the form of a column, and fluids containing receptors are passed through the column one or more times to allow receptor to bind to the ligand polypeptide.
  • the 'receptor is then eluted using changes in salt concentration, chaotropic agents (MnCl2), or pH to disrupt ligand-receptor binding.
  • Soluble ZSMF-3 or ZSMF-3 fusion proteins are used, for example, to identify the ZSMF-3 receptor.
  • cells expressing the receptor are identified by fluorescence immunocytometry or immunohistochemistry. The soluble receptor is useful in studying the distribution of receptor on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • ZSMF-3 fusion proteins consist essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion comprising a polypeptide wherein the polypeptide comprises a sequence of amino acid residues selected from the group consisting of: a) the sequence of amino acids as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; b) a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO: 2 from Phe, residue 20, through Arg, residue 321; and c) allelic variants of a) or b) ; wherein said polypeptide is a semaphorin.
  • the second portion comprises another polypeptide.
  • the second portion polypeptide may be another polypeptide such as a semaphorin or a polypeptide fragment of a semaphorin protein, such as a transmembrane domain or cytoplasmic domain.
  • ZSMF-3 proteins, agonists and antagonists may be used to modulate neurite growth and development and demarcate nervous system structures.
  • ZSMF-3 and antagonists would be useful as a treatment of peripheral neuropathies by increasing spinal cord and sensory neurite outgrowth.
  • Such an antagonist could be part of a therapeutic treatment for the regeneration of neurite outgrowths following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries.
  • Application may also be made in treating neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease.
  • Application may also be made in mediating development and innervation pattern of stomach tissue . Semaphorins have also been associated with mediation of other non-neuronal biological functions.
  • Semaphorin E which is homologous to viral cytokine inhibiting proteins contains conserved regions of amino acid residues that have been found in the viral semaphorins .
  • Semaphorin E was found to be upregulated in rheumatoid synovial fibroblastoid cells which suggests that it may have a role as a regulator of inflammatory processes and an involvement in the development of rheumatoid arthritis (Mangasser- Stephan et al . , Biochem. Bio hys . Res. Comm. 234 : 153-6 , 1997) .
  • Semaphorin G (Sema G) is expressed on lymphocytes and may play a role in the immune system (Furuyama et al .
  • Semaphorin CD100 promotes B-cell growth and aggregation and may be involved in lymphocyte activation (Hall et al . , Proc. Natl. Acad. Sci. USA 93 : 11780-5, 1996) .
  • Semaphorins such as Semaphorin III
  • G-Sema I and collapsin are hypothesized to act in vivo as repulsive or inhibitory molecules that prevent neighboring ventral motorneurons from innervating extra thoracic muscle.
  • G-Sema I and collapsin may also act as an attractive agent to promote innervation (Kolodkin et al . , Cell 15:1389-99, 1993) .
  • the truncated human ZSMF-3 may play a similar role in the stomach.
  • the activity of semaphorin polypeptides, agonists, antagonists and antibodies of the present invention can be measured, and compounds screened to identify agonists and antagonists, using a variety of assays, in particular assays that measure axon guidance and growth.
  • assays that indicate changes in neuron growth patterns, see for example, Hastings, WIPO Patent Application No: 97/29189 and Walter et al . , Development 101 :685-96 , 1987.
  • Assays to measure the effects of semaphorins on neuron growth are well known in the art. For example, the C assay (see for example, Raper and Kapfhammer, Neuron 4 . : 21-9, 1990 and Luo et al .
  • Conditioned media from cells expressing a semaphorin, semaphorin agonist or semaphorin antagonist, or aggregates of such cells can be placed in a gel matrix near suitable neural cells, such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been cocultured with nerve growth factor.
  • DRG dorsal root ganglia
  • neuronal cell suspensions grown in the presence of molecules of the present invention see for example, O'Shea et al . , Neuron 1:231-7, 1991 and DeFreitas et al . , Neuron 15:333-43, 1995.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art . Such systems include Scatchard analysis for determination of binding affinity (see, Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 251:545-8, 1991; Cunningham et al . , Science 245:821-5, 1991) .
  • Proteins of the present invention may also be assayed using viral delivery systems.
  • Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see
  • the adenovirus system can also be used for protein production in vi tro .
  • the cells can produce proteins for extended periods of time.
  • BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest.
  • the cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see A. Gamier et al., Cvtotechnol . 15:145-55, 1994).
  • ZSMF-3 polypeptides can also be used to prepare antibodies that specifically bind to ZSMF-3 epitopes, peptides or polypeptides. Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning-: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R.
  • polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a ZSMF-3 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZSMF-3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 anc ⁇ Fa ⁇ proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies (scFv) , single complementarity- determining region peptides ("minimal recognition units") and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Humanized monoclonal antibodies directed against ZSMF-3 polypeptides could be used as a protein therapeutic, in particular for use as an immunotherapy.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZSMF-3 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSMF-3 protein or peptide) .
  • Antibodies are defined to be specifically binding if they bind to a ZSMF-3 polypeptide with a binding affinity (K a ) of IO 6 M “1 or greater, preferably 10 M ⁇ or greater, more preferably IO 8 M “1 or greater, and most preferably 10 M "1 or greater.
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis) .
  • Genes encoding polypeptides having potential ZSMF-3 binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al .
  • Random peptide display libraries can be screened using the ZSMF-3 sequences disclosed herein to identify proteins which bind to ZSMF-3.
  • These "binding proteins" which interact with ZSMF-3 polypeptides may be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • a variety of assays known to those skilled in the art can be utilized to detect antibodies and binding proteins which specifically bind to ZSMF-3 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) , Cold Spring Harbor Laboratory Press, 1988.
  • assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay.
  • ELISA enzyme-linked immunosorbent assay
  • dot blot or Western blot assay inhibition or competition assay
  • sandwich assay antibodies can be screened for binding to wild-type versus mutant ZSMF-3 protein or peptide.
  • Antibodies and binding proteins to ZSMF-3 may be used for immunohistochemical tagging of cells that express human ZSMF-3, for example, to use in diagnostic assays; for isolating ZSMF-3 by affinity purification; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZSMF-3 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti -complement pairs as intermediates.
  • Antibodies and binding proteins herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • the invention also provides isolated and purified ZSMF-3 polynucleotide probes.
  • polynucleotide probes can be RNA or DNA.
  • DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire ZSMF-3 gene or cDNA.
  • the synthetic oligonucleotides of the present invention have at least 80% identity to a representative ZSMF-3 DNA sequence (SEQ ID NOs : 1 or 3) or its complements.
  • Preferred regions from which to construct probes include the 5' and/or 3' coding sequences, receptor binding regions, semaphorin, extracellular, transmembrane and/or cytoplasmic domains and signal sequences, and the like.
  • Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991.
  • the molecules can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • a detectable signal such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZSMF-3 gene or mRNA transcript in a sample.
  • ZSMF-3 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in si tu hybridization (FISH) or immunohistochemistry .
  • Polynucleotide probes can be used to identify genes encoding ZSMF-3 -like proteins.
  • ZSMF-3 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the semaphorin family.
  • Such probes can also be used to screen libraries for related sequences encoding novel semaphorin receptors.
  • Such screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence.
  • Such methods and conditions are well known in the art, see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989.
  • Such low stringency conditions could include hybridization temperatures less than 42°C, formamide concentrations of less than 50% and moderate to low concentrations of salt.
  • Libraries may be made of genomic DNA or cDNA. Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridization and in si tu hybridization.
  • ZSMF-3 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems.
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • ZSMF-3 RNA can be detected with a nonradioactive hybridization method (see, for example,
  • Nonradioactive Probes Humana Press, Inc., 1993.
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative non-radioactive moieties include biotin, fluorescein, and digoxigenin.
  • PCR polymerase chain reaction
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; White (ed.), PCR Protocols: Current Methods and Applications, Humana Press, Inc., 1993; Cotter (ed.), Molecular Diagnosis of Cancer, Humana Press, Inc., 1996; Hanausek and Walaszek (eds.), Tumor Marker Protocols, Humana Press, Inc., 1998; Lo (ed.), Clinical Applications of PCR, Humana Press, Inc., 1998 and Meltzer (ed.), PCR in Bioanalysis, Humana Press, Inc., 1998).
  • PCR primers can be designed to amplify a sequence encoding a particular ZSMF-3 region, such as the semaphorin domain.
  • PCR reverse transcriptase-PCR
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZSMF-3 primers (see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28, CRC Press, Inc. 1997) .
  • ZSMF-3 primers see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28, CRC Press, Inc. 1997) .
  • ZSMF-3 primers see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28, CRC Press, Inc. 1997) .
  • PCR is
  • RNA is isolated from a biological sample using, for example, the guanidinium- thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or ZSMF-3 anti- sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • ZSMF-3 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled ZSMF-3 probe, and examined by autoradiography .
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers.
  • the reporter dye is separated from the quencher dye and a sequence-specific signal is generated and increases as amplification increases.
  • the fluorescence intensity can be continuously monitored and quantified during the PCR reaction.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • ZSMF-3 probes and primers can also be used to detect and to localize ZSMF-3 gene expression in tissue samples.
  • Methods for such in si tu hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), Jn Si tu Hybridization Protocols, Humana Press, Inc., 1994; Wu et al . (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridization (RISH) , " in Methods in Gene Biotechnology, pages 259-278, CRC Press, Inc., 1997; and Wu et al .
  • Such polynucleotide probes could be used to hybridize to counterpart sequences on individual chromosomes.
  • Chromosomal identification and/or mapping of the ZSMF-3 gene could provide useful information about gene function and disease association.
  • Many mapping techniques are available to one skilled in the art, for example, mapping somatic cell hybrids, and fluorescence in si tu hybridization (FISH) .
  • FISH fluorescence in si tu hybridization
  • a preferred method is radiation hybrid mapping. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 25_0: 245-50 , 1990).
  • STSs non-polymorphic- and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • the precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross- referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.
  • Chromosomal localization can also be done using STSs.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome .
  • An STS can be defined by a pair of oligonucleotide primers that can be used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences.
  • STSs are based solely on DNA sequence they can be completely described within a database, for example, Database of Sequence Tagged Sites (dbSTS) , GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), they can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences .
  • ZSMF-3 was mapped to the 5q23.1-q23.2 region on the integrated LDB chromosome 5 map as is described in more detail below.
  • the present invention also contemplates use of such chromosomal localization for diagnostic applications.
  • the ZSMF-3 gene a probe comprising ZSMF-3 DNA or RNA or a subsequence thereof, can be used to determine if the ZSMF-3 gene is present on human chromosome 5 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ZSMF-3 gene locus include, but are not limited, to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (.STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108 :255- 65, 1995) .
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (.STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108 :255- 65, 1995) .
  • RFLP restriction fragment length polymorphism
  • .STR short tandem repeat
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient;- (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l * , or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108 :255-65,
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase . Hybridized regions of the RNA are protected from digestion.
  • PCR assays a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • ZSMF-3 polypeptides and polynucleotides may be used within diagnostic systems.
  • Antibodies or other agents that specifically bind to ZSMF-3 may be used to detect the presence of circulating ligand or receptor polypeptides.
  • detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • Immunohistochemically labeled ZSMF-3 antibodies can be used to detect ZSMF-3 receptor and/or ligands in tissue samples and identify ZSMF-3 receptors.
  • ZSMF-3 levels can also be monitored by such methods as RT-PCR, where ZSMF-3 mRNA can be detected and quantified.
  • the information derived from such detection methods would provide insight into the significance of ZSMF-3 polypeptides in various diseases and biological processes, and as a would serve as diagnostic tools for diseases for which altered levels of ZSMF-3 are significant .
  • the ZSMF-3 polynucleotides and/or polypeptides disclosed herein can be useful as therapeutics, wherein ZSMF-3 agonists and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids.
  • ZSMF-3 antagonists provided by the invention, bind to ZSMF-3 polypeptides or, alternatively, to a receptor to which ZSMF-3 polypeptides bind, thereby inhibiting or eliminating the function of ZSMF-3.
  • ZSMF-3 antagonists would include antibodies; oligonucleotides which bind either to the ZSMF-3 polypeptide or to its ligand; natural or synthetic analogs of ZSMF-3 ligands which retain the ability to bind the receptor but do- not result in either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZSMF-3 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZSMF-3 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZSMF-3 receptor or ligand would be beneficial.
  • the invention also provides nucleic acid-based therapeutic treatment. If a mammal lacks or has a mutated ZSMF-3 gene, the ZSMF-3 gene can be introduced into the cells of the mammal. Using such methods, cells altered to express the nerve growth factor neurotrophin-3 (NT-3) were grafted to a rat model for spinal injury and stimulated axon regrowth at the lesion site and the rats thus treated recovered some ability to walk (Grill et al . , J. Neuroscience 11:5560-72, 1997). In one embodiment, a gene encoding a ZSMF-3 polypeptide is introduced in vivo in a viral vector.
  • NT-3 nerve growth factor neurotrophin-3
  • Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus , Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is ' not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al . , Molec. Cell. Neurosci.
  • adenovirus vector such as the vector described by Stratford-Perricaudet et al . (J. Clin. Invest. 91:626-30, 1992), and a defective adeno- associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , J. Virol. £:3822-8,
  • the gene can be introduced in a retroviral vector, e . g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al . , Cell 33 :153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al . , J . Virol .
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci. USA £4:7413-7, 1987; and Mackey et al., Proc. Natl. Acad. Sci.
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents • one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al . , J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988).
  • 7 ⁇ nother aspect of the present invention involves antisense polynucleotide compositions that are complementary to a segment of the polynucleotide set forth in SEQ ID NO:l.
  • Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZSMF-3 polypeptides and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides are used to inhibit expression of ZSMF-3 polypeptide-encoding genes in cell culture or in a subject.
  • mice engineered to express the ZSMF-3 gene, and mice that exhibit a complete absence of ZSMF-3 gene function, referred to as "knockout mice” (Snouwaert et al., Science 257 : 1083 , 1992), may also be generated (Lowell et al., Nature 366 : 740-2, 1993). These mice may be employed to study the ZSMF-3 gene and the protein encoded thereby in an in vivo system.
  • Novel ZSMF-3 encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for sequences homologous to conserved motifs within the semaphorin family. To identify the corresponding cDNA, a clone from which an identified EST was derived that was considered likely to contain the entire murine ZSMF-3 sequence was used for sequencing. Using a QIAwell 8 plasmid kit
  • Oligonucleotides ZC14201 (SEQ ID NO: 9) and ZC14202 (SEQ ID NO: 10) were used to complete the sequence from the clone. Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System
  • a clone from which the identified human EST was derived and considered likely to contain the entire human ZSMF-3 sequence was used for sequencing.
  • a QIAwell 8 plasmid kit as above an overnight culture was prepared.
  • the template was sequenced as above using oligos ZC694 (SEQ ID N0:7), ZC976 (SEQ ID NO: 8) as sequencing primers.
  • This clone contained only about 600 bp of 3' sequence, so 5' RACE (rapid amplification of cDNA ends) was done to identify the remaining 5' portion.
  • 5' RACE was carried out at 94°C, for 1.5 minutes, followed by 35 cycles at 94°C for 15 seconds, 56°C for 30 seconds and 72°C for 1 minute, followed by a 10 minute extension at 72°C. A band of approximately 700 bp fragment was resolved by gel electrophoresis.
  • the 5' RACE fragment was ligated into the TA vector (Invitrogen Inc., San Diego, CA) according to manufacturer's instructions. The sequence of the 5 ' fragment was confirmed by sequence analysis as described above.
  • ZSMF-3/pCZF199 expression vectors the following DNA fragments were transformed into S. cerevisiae : Sna BI digested pCZR199 as an acceptor vector, the 3' ZSMF-3 Xho I-Not I restriction fragment, the 5' Eco RI restriction fragment, and two, double stranded linker segment.
  • the expression vector, pCZR199 has yeast replication elements, (CEN, ARS) , the selectable marker, URA3 , E.
  • coli replication elements e.g., AMP R and ori
  • Sna BI blunt-ended cloning site
  • the vectors were used to create ZSMF-3 polypeptides having either end of the expressed protein Glu-Glu tagged.
  • the double stranded linker segments were prepared using PCR.
  • the linkers served to join the vector to the insert fragments at both the 5' and 3' ends. Two sets of linkers were prepared. One set of linkers joined the insert to a vector placing the Glu-Glu tag on the 5' end of the insert sequence.
  • the 5' linker of this set was made from oligos ZC14,397 (SEQ ID NO:27), ZC14,396 (SEQ ID NO:28), ZC16,463 (SEQ ID NO:29) and ZC16,442 (SEQ ID NO:30).
  • the 3' linker was made from oligos ZC14,455 (SEQ ID N0:31), ZC14,394 (SEQ ID NO:32), ZC16,461 (SEQ ID NO:33) and ZC16,444 (SEQ ID NO:26) .
  • the second set of linkers was used to join the ZSMF-3 insert into a vector placing a 3' Glu-Glu tag.
  • the 5' linker was made from oligos ZC14,454 (SEQ ID N0:21), ZC14,392 (SEQ ID NO:22), ZC16,443 (SEQ ID N0:19) and ZC16,462 (SEQ ID NO:20) .
  • the 3' linker was made from oligos ZC14,395 (SEQ ID NO:24), ZC14,393 (SEQ ID NO:23), ZC16,441 (SEQ ID NO:25) and ZC16,444 (SEQ ID NO:26).
  • the oligos were joined using standard PCR reaction conditions and heated to 94°C for 1.5 minutes followed by 10 cycles at 94°C for 30 seconds, 50°C for 1 minute and 72°C for 1 minute, then a 10 minute extension at 72°C.
  • the DNA fragments were added to 100 ⁇ l competent yeast (Genetic strain SF83890, Roffman et al . , EMBO J . £:2057-65, 1989) and electroporated .
  • the yeast cells were immediately diluted in 600 ⁇ l 1.5 M sorbitol and plated on Ura D plates and incubated at 30°C for 48 hours. .Ura + colonies were selected from both the N-terminally-tagged and C-terminally-tagged ZSMF-3 proteins and the DNA from the resulting yeast colonies was extracted and transformed into E. coli . Individual clones harboring the correct expression construct were identified by PCR screening.
  • CA human stomach poly A+ RNA
  • the probe was amplified in a polymerase chain reaction as follows: 1 cycle at 94°C for 1 minute 30 seconds; 35 cycles of 94°C for 15 seconds and 64°C for 30 seconds, followed by 1 cycle at 72°C for 10 minutes.
  • the resulting DNA fragment was electrophoresed on a 2% low melt agarose gel (SEA PLAQUE GTG low melt agarose, FMC Corp., Rockland, ME), the fragment was purified using the QIAquickTM method (Qiagen, Chatsworth, CA) , and the sequence was confirmed by sequence analysis.
  • the probe was radioactively labeled using the random priming MULTIPRIME DNA labeling system (Amersham, Arlington Heights, IL) , according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene, La Jolla, CA) .
  • ExpressHybTM
  • RNA Master Dot Blot (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C using 2 x IO 6 cpm/ml of labeled probe. The blots were then washed at 25°C in 2X SSC, 0.05% SDS for 1 hour, then at 50°C in 0. IX SSC, 0.1% SDS for 1 hour. A transcript of approximately 2 kb was seen in stomach. A Human RNA Master Dot Blot (Clontech) that contained RNAs from various tissues that were normalized to 8 housekeeping genes was also probed and hybridized as described above. Expression was seen only in stomach.
  • Mouse Multiple Choice ⁇ Tissue Northern Blots (OriGene Technologies, Inc., Rockville, MD) were probed to determine the tissue distribution of mouse ZSMF-3 expression.
  • the 200 bp stomach derived probe (SEQ ID NO: 6) described above was used as a probe. Hybridization took place overnight at 65°C using 1 x 10 ⁇ cpm/ml of labeled probe.
  • the blots were then washed at 25°C in 2X SSC, 0.1% SDS for 1 hour, then at 50°C in 0. IX SSC, 0.1% SDS for 1 hour.
  • a transcript of approximately 1.5 kb was seen in all tissues, stomach, small intestine, muscle, lung, skin, brain, heart, kidney, spleen, thymus and liver.
  • mice RNA Master BlotTM (Clontech) was probed as described above. Expression was detected only in epididymus .
  • ZSMF-3 was mapped to chromosome 5 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) .
  • the GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
  • a publicly available WWW server http : //www-genome .wi .mit . edu/cgi-bin/contig/ rhmapper.pl
  • WICGR Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome
  • Each of the 95 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR reaction buffer (Clontech), 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin- Elmer, Foster City, CA) , 1 ⁇ l sense primer, 15,398 (SEQ ID N0:15), 1 ⁇ l antisense primer, ZC 15,397 (SEQ ID N0:16), 2 ⁇ l flediLoad (Research Genetics, Inc.), 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech) , 25 ng of DNA from an individual hybrid clone or control and ddH 2 0 for a total volume of 20 ⁇ l .
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 62°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .
  • the results showed that ZSMF-3 maps 384.88 cR_3000 from the top of the human chromosome 5 linkage group on the WICGR radiation hybrid map.
  • the proximal and distal framework markers were D5S492 and WI-3394, respectively.
  • the oligos added two unique restriction sites, Fsel and Ascl.
  • the reaction proceeded a 95°C for 5 minutes, followed by 25 cycles at 95°C for 1 minute, 58°C for 1 minute and 72°C for 1.5 minutes, with a 7 minute extension at 72°C.
  • the PCR product was visualized by gel electrophoresis, excised and purified.
  • the zsmf3 cDNA was digested with the restriction enzymes Fsel and Ascl, phenol/chloroform extracted, ethanol precipitated, and rehydrated in 20 ⁇ l TE (10 mM Tris HCl/l mM EDTA pH 8.0).
  • the zsmf3 fragment was then ligated into the Fsel -Ascl sites of the transgenic vector pTG12-8 and transformed into DH10B competent cells by electroporation. Clones containing zsmf3 were identified by mini prep followed by digestion with Fsel-Ascl. Identity of the clone was confirmed by sequence analysis.
  • a 966 base pair ZSMF-3 fragment (nucleotide 1-966 of SEQ ID NO:l) was released from the TG12-8 vector using Fsel and Ascl restriction enzymes.
  • the cDNA was isolated by electrophoresis on a 1% low melt SeaPlaque GTG gel. A band of the appropriate size was excised from the gel and the DNA extracted.
  • the zsmf3 cDNA was cloned into the Fsel-Ascl sites of pAdTrack CMV vector (He, et al . , ibid. ) in which the native polylinker was replaced with Fsel, EcoRV, Ascl sites. Ligation was performed using the Fast-Link DNA ligation and screening kit by Epicentre Technologies (Madison, WI) . Five micrograms of the pAdTrack CMV zsmf3 plasmid was digested with Pmel to linearize the plasmid. One microgram of the linearized plasmid was co-transformed with 200 ng of supercoiled pAdEasy (He et al .
  • adenoviral DNA Five micrograms of recombinant adenoviral DNA was digested with Pad enzyme (NEBiolabs, Beverly, MA) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20- 30U of Pad. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitate with ethanol. The DNA pellet was resuspended in 10 ⁇ l distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc . Canada) set up the day before which were 60-70% confluent were transfected with the Pad digested DNA. The Pad-digested DNA was diluted up to a total volume of 50 ⁇ l with sterile HBS (150 mM NaCl, 20 mM HEPES) . In a separate tube, 20 ⁇ l DOTAP
  • the DNA/lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours . After 4 hours the media containing the DNA/lipid mixture was aspirated off and replaced with 5 mis complete MEM containing 5% fetal bovine serum. The transfected cells were monitored for Green Fluorescent Protein (GFP) expression and formation of viral plaques.
  • GFP Green Fluorescent Protein
  • the crude lysate was amplified to obtain a working "stock" of rAdV lysate.
  • Ten, 10 cm plates of nearly confluent (80-90%) 293A cells were set up 20 hours previously, 200 ⁇ l of crude rAdV lysate added to each 10 cm plate and monitored for 48 to 72 hours looking for CPE
  • NP-40 detergent was added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells. Bottles were placed on a rotating platform for 10 minutes agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatant was transferred to 250 ml polycarbonate centrifuge bottles and 0.5 volumes of 20% PEG8000/2.5M NaCl solution added. The supernatant containing bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes and supernatant discarded.
  • the white precipitate (precipitated virus/PEG) was collected using a sterile cell scraper and the precipitate from 2 bottles was resuspended in 2.5 mis PBS.
  • the virus solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G in the microfuge for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2 ml microcentrifuge tubes was transferred into a 15 ml polypropylene snapcap tube and adjusted to a density of 1.34g/ml with Cesium Chloride (CsCl) .
  • the solution was transferred polycarbonate thick-walled centrifuge tubes 3.2 ml (Beckman Instruments, Palo Alto, CA) and spun at 80,000 rpm (348,000 X G) for 3-4 hours at 25°C in a Beckman Optima TLX micro ultracentrifuge with a TLA-100.4 rotor.
  • the virus formed a white band. Using wide bore pipette tips virus band was collected.
  • the virus was loaded and allow it to run into the column.
  • Five milliliters of PBS was added to the column and fractions of 8-10 drop collected.
  • the optical densities of 1:50 dilutions of each fraction was determined at 260 nm on a spectrophotometer . Those fractions having virus were pooled and the optical density of a 1:50 dilution determined.
  • Recombinant virus infectivity was measured according to the method of Quantum Biotechnologies, Inc.
  • the zsmf3 adenovirus had a titer of 8.0 X 10 11 pfu/ml .

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Abstract

Semaphorin polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed. The polypeptides include a soluble, truncated semaphorin that is highly expressed in stomach tissue. The polypeptides may be used within methods for detecting receptors that mediate neurite outgrowth, and may also have immunological value.

Description

DESCRIPTION SEMAPHORIN ZSMF-3
BACKGROUND OF THE INVENTION
Neuronal cell outgrowths, known as processes, grow away from the cell body to form synaptic connections. Long, thin processes which carry information away from the cell body are called axons, and short, thicker processes which carry information to and from the cell body are called dendrites. Axons and dendrites are collectively referred to as neurites . Neurites are extended by means of growth cones, the growing tip of the neurite, which is highly motile and is ultimately responsible for increasing and extending the neuronal network in the body. The growth cones are able to navigate their way to their targets using environmental cues or signals, which encourage or discourage the growth cone from extending the neurite in a particular direction. Such cues and signals include older neurons and orienting glial fibers, chemicals such as nerve growth factor released by astrocytes and other attracting or repelling substances released by target cells. The membrane of the growth cone bears molecules such as N-CAM (nerve cell adhesion molecule) which are attracted or repelled by environmental cues and thus influence the direction and degree of neurite growth. The growth cone also engulfs molecules from the environment which are transported to the cell body and influence growth. A number of proteins from vertebrates and invertebrates have been identified as influencing the guidance of neurite growth, either through repulsion or chemoattraction. Among those molecules are netrins, EPH-related receptor tyrosine kinases and their ligands, vitronectin, thrombospondin, human neuronal attachment factor- 1 (NAF-1) , connectin, adhesion molecules such as CAM (cell adhesion molecule) and the semaphorins/collapsins (Neugebauer et al . , Neuron 6.:345-58, 1991; 0 ' Shea et al . , Neuron 1:231-7, 1991; Osterhout et al . , Devel . Biol . 150 : 256-65 , 1992; Goodman, Cell 78.:353-6, 1993; DeFreitas et al . , Neuron 15 :333-43 , 1995; Dodd and Schuchardy Cell J3JL: 471-4, 1995; Keynes and Cook, Cell 81:161-9, 1995; Mϋller et al . , Cur. Qpin. Genet, and Devel . 6.:469-74, 1996, Goodman, Annu . Rev. Neurosci . 19:341-77, 1996; WIPO Patent Application No: 97/29189 and Goodman et al . , US Patent No. 5,639,856). Semaphorins/collapsins are a family of related transmembrane and secreted molecules. Invertebrate, vertebrate and viral semaphorins are known (Kolodkin et al., Cell 25_: 1389-99 , 1993; Luo et al . , Cell 75:217-27, 1993; Ensser and Fleckenstein, J. Gen. Virol. 76 :1063-7, 1995; Luo et al , Neuron 14.: 1131-40, 1995; Adams et al . , Mech. Devel. 5_7:33-45, 1996; Hall et al . , Proc. Natl. Acad. Sci. USA 93:11780-8, 1996; Roche et al . , Oncoqene 12:1289- 97, 1996; Skeido et al . , Proc. Natl. Acad. Sci. USA 93:4120-5, 1996; Xiang et al . , Genomics 32:39-48, 1996; Eckhardt et al . , Mol. Cell Neurosci. 1:409-19, 1997 . and Zhou et al., Mol. Cell. Neurosci. 9:26-41, 1997). Invertebrate semaphorins include but are not limited to G- sema I (grasshopper semaphorin) , D-sema I and D-sema II (Drosophila semaphorin) and T-sema I (Tribolium semaphorin) . Vertebrate semaphorins include but are not limited to Sema A - Se a G, the collapsins 1-5, murine .Sema IVa and IVb, and human sema III, human semaphorin A(V), human semaphorin IV, human semaphorin I and CD 100. Viral semaphorins include Sema IV from both vaccinia and variola viruses and a semaphorin-like gene (ahv-sema) has been reported from alcelaphine herpesvirus type 1.
The semaphorins comprise a variable region of. 30- 60 amino acids that includes a signal sequence, followed by a conserved region of about 500 amino acid residues called the semaphorin (Sema) domain. This extracellular domain contains between 14-16 conserved cysteine residues, an N- linked glycosylation site and numerous blocks of conserved amino acids. Membrane-bound and soluble semaphorin proteins have been described, see for example, Kolodkin et al . , ibid. ; Adams et al . , ibid, and Goodman et al . , US Patent No : 5 , 639 , 856. Classification of five subgroups within the semaphorin family has made based on the sequence C terminal of the semaphorin domain. Both soluble (lacking a transmembrane domain) and membrane-bound (having a transmembrane domain and localized to the membrane) semaphorins have been described. Group I includes semaphorins having a transmembrane domain followed by a cytoplasmic domain. Group II and III have no transmembrane domain, but have a region with Ig homology, Group IV has a region of Ig homology C-terminal of the semaphorin domain followed by a transmembrane and cytoplasmic domain. Group V has a series of thrombospondin repeats C-terminal to the semaphorin domain followed by a transmembrane and cytoplasmic domain.
Most insect semaphorins are membrane bound proteins and belong to Group I. G-Sema I, T-Sema I and D- Sema I have a region of 80 amino acid residues following the semaphorin domain, which is followed by a transmembrane domain and an 80-110 amino acid cytoplasmic domain. Murine Sema IVa has a transmembrane domain followed by a 216 amino acid cytoplasmic domain. Murine sema F and G are members of Group V and have an approximately 400 amino acid thrombospondin repeat domain between the semaphorin and transmembrane domains. Group II secreted proteins, such as D-sema II, have no membrane linkage but instead have a block of less than 20 amino acids following the semaphorin domain which is followed by an Ig domain and a 70 amino acid C-terminal region. Group III secreted proteins, such as H-Sema III, are similar to Group II except the C- terminal region is longer, in excess of 100 amino acids. The viral semaphorin- like gene, ahv-sema, is truncated immediately following the Ig-domain. The viral semaphorin, sema IV, has a truncated, 441 amino acid residue, semaphorin domain and no Ig region . See Kolodkin et al . , ibid. ; Adams et al . ibid. ; Zhou et al . ibid, and Ensser and Fleckenstein, ibid. Overall semaphorins share the greatest degree of homology within the semaphorin domain, between, 25-93%, with a greater degree of divergence in all other regions and domains suggesting distinct roles for various sub-groups within the semaphorin family. The viral semaphorins are the most diverse, sharing only 25% identity with vertebrate semaphorins . Between vertebrate and invertebrate semaphorins, the percent identity varies between 30-40%.
Isolating and characterizing neurite growth cues would be of great value, for example, in modulating neurite growth and development; treatment of peripheral neuropathies; for use as therapeutics for the regeneration of neurons following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries; diagnosing neurological diseases and in treating neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. The present invention addresses these needs and others by providing novel semaphorins and related compositions and methods.
SUMMARY OF THE INVENTION
The present invention provides a novel semaphorin polypeptide and related compositions and methods.
Within one aspect is provided an isolated polypeptide comprising a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO:2 from Phe, residue 20, through Arg, residue .321, wherein said polypeptide is a semaphorin. Within one embodiment any difference between said amino acid sequence of said isolated polypeptide and said corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution. Within another embodiment the polypeptide comprises amino acid residues 20 through 32.1 of SEQ ID NO: 2. Within another embodiment the polypeptide further comprises a heterologous affinity tag or binding domain. Within a related embodiment the affinity tag is a Glu-Glu tag.
The invention also provides an isolated polypeptide having the amino acid sequence of SEQ ID NO : 2. The invention further provides an isolated polypeptide comprising the amino acid sequence of amino acid residue 1 to amino acid residue 19 of SEQ ID NO : 2.
Additionally, the invention provides an isolated polypeptide consisting of a semaphorin domain, a transmembrane domain and a cytoplasmic domain, wherein said semaphorin domain comprises the amino acid sequence of SEQ ID NO: 2.
Within another aspect is provided a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion comprising a polypeptide wherein said polypeptide comprises a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; wherein said polypeptide is a semaphorin; and said second portion comprising another polypeptide. Within one embodiment the second polypeptide is a transmembrane domain of a semaphorin protein. Within a related embodiment the second polypeptide further comprises a cytoplasmic domain of a semaphorin protein.
The invention also provides a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1 to 19 of SEQ ID N0:2, wherein said secretory signal sequence is operably linked to an additional polypeptide.
Within another aspect of the invention is provided an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; b) degenerate polynucleotide sequences of a) ; and c) a polynucleotide sequence complementary to a) or b) ; wherein said polynucleotide encodes a polypeptide which is a semaphorin. Within one embodiment the polypeptide comprises residue's 20 through 321 of SEQ ID NO : 2. Within another embodiment the protein comprises residues 1 through 321 of SEQ ID NO : 2.
Within another aspect is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described herein; and a transcription terminator. Within one embodiment the expression vector further comprises secretory signal sequence operably linked to said DNA segment . Within yet another embodiment the expression vector further comprises an affinity tag operably linked to said DNA segment. The invention also provided a cultured cell containing an expression vector as described herein, wherein said cell expresses said DNA segment. Within one embodiment the expression vector further comprises a secretory signal sequence operably linked to said DNA segment and the cell secretes said polypeptide. Within yet another embodiment the expression vector further comprises an affinity tag operably linked to said DNA segment.
The invention also provides a method of making a semaphorin protein comprising: (a) culturing a host cell containing an expression vector as described herein; and
(b) recovering said protein encoded by said DNA segment.
Within another embodiment the expression vector further comprises a secretory signal sequence operably linked to said DNA segment, the cell secretes the protein into a culture medium, and the protein is recovered from the medium. Within a related embodiment the expression vector further comprises an affinity tag operably linked to said
DNA segment .
Within a further aspect, the invention provides an antibody that specifically binds to a polypeptide as described herein. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody. Within one embodiment the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described herein. Within another embodiment is provided a binding protein that specifically binds to a polypeptide as described herein.
Within another aspect the invention provides a pharmaceutical composition comprising a polypeptide as described herein, in combination with a pharmaceutically acceptable vehicle. These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawing.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows an alignment of human (SEQ ID NO: 2) and mouse (SEQ ID NO:4) ZSMF-3 with mouse semaphorin F (SEQ ID NO: 14) and mouse semaphorin G (SEQ ID NO: 13) . The starting methionine, signal sequence and beginning of the mature peptide are noted as are the conserved cysteine residues .
Figure 2 shows an alignment of human ZSMF-3 (SEQ ID NO:l) with murine Sema IVa (SEQ ID NO: 37) .
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter:
Allelic variant : Any of two or more alternative forms of a gene occupying the same chromosomal locus.
Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations.
Gene mutations can be silent (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence. The term "allelic variant" is also used herein to denote a protein encoded by an allelic variant of a gene. Also included are the same protein from the same species which differs from a reference amino acid sequence due to allelic variation. Allelic variation refers to naturally occurring differences among individuals in genes encoding a given protein.
Amino-terminal and carboxyl -terminal : are used herein to denote positions within polypeptides and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.
Complement/anti-complement pair: Denotes non- identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti -complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10-9 M.
Complements of polynucleotide molecules: Denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
Degenerate : As applied to a nucleotide sequence such as a probe or primer, denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) . Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
Expression vector: A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
Isolated: When applied to a polynucleotide molecule, the term "isolated" indicates that the molecule is removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316 :774-78, 1985) . Operably linked: As applied to nucleotide segments, the term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
Ortholog or species homolog: A polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation .
Polynucleotide : denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Promoter : Denotes a portion of a gene containing
DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. Receptor : A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell. Metabolic events that are linked to receptor- ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
Secretory signal sequence : A DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. Soluble receptor/soluble ligand: A receptor/ligand polypeptide that is not bound to a cell membrane. Many cell-surface receptors/ligands have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. They may also be produced by cleavage or by recombinant methods . Soluble receptors are most commonly ligand-binding receptor polypeptides and soluble ligands are most commonly receptor-binding polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors/ligands can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate. Receptor/ligand polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively. Splice variant: As used herein, denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules,, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene . Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to +10%.
The present invention is based in part upon- the discovery of a novel human member of the semaphorin family, designated "ZSMF-3". The human ZSMF-3 nucleotide sequence is represented in SEQ ID NO : 1 and the deduced amino acid sequence in SEQ ID NO : 2. A murine homologue is represented in SEQ ID NO: 3 and the deduced amino acid sequence in SEQ ID NO:4. Murine and human ZSMF-3 share 90% nucleotide identity and 95% amino acid identity.
The novel human ZSMF-3 semaphorin proteins and polypeptides encoded by polynucleotides of the present invention were initially identified by querying an EST (Expressed Sequence Tag) database for sequences homologous to conserved motifs within the semaphorin family. Sequence analysis of the deduced amino acid sequence as represented in SEQ ID NO: 2 indicate the presence of a 19 amino acid residue signal sequence (amino acid residues 1-19 of SEQ ID NO: 2) followed by at least 302 amino acid residues of a semaphorin domain (amino acid residues 20-321 of SEQ ID
NO: 2), a stop codon followed by a poly A region. Within the semaphorin domain are 7 conserved cysteine residues
(amino acid residues 107, 117, 135, 144, 165, 258 and 283 of SEQ ID NO: 2) . Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding.
The sequence of human and murine ZSMF-3, as represented in SEQ ID NOs : 2 and 4, are homologous to murine Sema IVa identified by Zhou et al . , (Mol. Cell. Neurosci. 3_ : 26-41, 1997. Sema IVa has been described as having a 16 amino acid residue signal sequence, a 648 amino acid residue extracellular region containing a 500 amino acid residue semaphorin domain. Within the semaphorin domain are 13 conserved cysteine residues and 7 N-linked glycosylation sites. The extracellular region is followed by a 24 amino acid transmembrane domain and a 216 amino acid residue cytoplasmic domain having one protein kinase C site.
This information suggests that the human ZSMF-3 protein encoded by the nucleotide sequence described in SEQ ID NO : 1 represents an alternative splicing product or "splice variant" of a corresponding full length human ZSMF- 3 protein. Splice variants of a full length DNA sequence include DNA sequences that result from mature RNA molecules created by known eukaryotic RNA splicing processes wherein the intron sequence is removed and exon sequence is joined. A well defined splice site which begins at nucleotide 963 of SEQ ID N0:1, a poly A region (nucleotide 1033-1038 of SEQ ID NO:l) followed by a poly A tail (beginning at nucleotide 1054 of SEQ ID NO:l) confirm that this sequence indeed represents a viable splice variant. Northern blot analysis indicates a predominant transcript in stomach tissue of 2 kb which agrees with the splice variant size. Two faint transcripts of 4 kb and 3 kb were detected in spinal cord and adrenal gland which would indicate ■ the existence of a full length ZSMF-3 protein.
Zhou et al . (ibid. ) suggest that their Sema IVa, represents a new class of mammalian semaphorins which have structural homology to the insect Sema I proteins. Although murine Sema IVa shares 25-36% amino acid identity with murine Sema III within the semaphorin domain, Sem IVa has a transmembrane domain and a cytoplasmic domain, although much larger, as are seen in the insect Sema I proteins. Previously described transmembrane containing vertebrate semaphorins also have regions of Ig homology or transponder repeats which are not present in murine Sema IVa. It is possible that human ZSMF-3 is a human hom'olog of murine Sema IVa and a member of this new semaphorin family. A final determination can only be made once it is determined what sequence lies C terminal to the ZSMF-3 semaphorin domain. Over a similar area in the semaphorin domain, murine ZSMF-3 (SEQ ID NO: ) and human ZSMF-3 (SEQ ID NO:2) share 100% and 95% identity respectively, with murine SEMA IVa described by Zhou et al . , ibid. Over a similar area, human ZSMF-3 shares 31% nucleotide identity with Vaccinia virus semaphorin Sema IV, 36% identity with Drosophila semaphorin II (D Sema II) , 39% identity with Tribolium confusum semaphorin I (T Sema I) , Human semaphorin CD100, and chicken collapsin, 42% identity with Drosophila semaphorin I (D Sema I) and grasshopper semaphorin I (G Sema I) , 44% identity with murine semaphorin Sem G, 45% identity with murine semaphorin SemF, and 61% identity with murine semaphorin VIb. Figure 1 shows an alignment of human and mouse ZSMF-3 with murine semaphorins Sem F and Sem G. Figure 2 shows an alignment of human ZSMF-3 with murine SEMA IVa. The truncated semaphorin domain of ZSMF-3 may suggest that it also shares an association with the viral semaphorin Sema IV, which has a truncated semaphorin domain of about 400 amino acids. An alternatively spliced 3.5 kb human semaphorin, SEMA3F,- was described by Xiang et al . (Genomics 32:39-48, 1996). The splice resulted in the addition of 96 bases to the message and retained the correct reading frame. Such features set human ZSMF-3 apart from previously described semaphorins and may be characteristic qualities which further define a new sub-group within the semaphorin family. The DNA sequence shown in SEQ ID NO:l or portions thereof can be used as probes or primers to obtain the corresponding full length ZSMF-3 DNA from cells or libraries (including cDNA and genomic libraries) from human tissues. Methods for isolating polynucleotide sequences are known in the art. One example for obtaining the ZSMF-3 sequence uses mRNA obtained from a tissue or cell types that are likely to express the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. Suitable tissue sources include human spinal cord, adrenal gland, stomach and brain. A library is then prepared from mRNA of a positive tissue or cell line. The cDΝA can then be isolated by a variety of methods, such as by probing with the sequence disclosed in SEQ ID ΝO:l or some portion thereof. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences disclosed herein and templates derived from appropriate tissues as discussed above. Additional oligonucleotides may be generated from the PCR derived sequence and SEQ ID N0:1, as needed, to obtain a complete full length cDNA encoding a ZSMF-3 polypeptide of about 890 amino acid residues. More preferably such full length ZSMF-3 polypeptides would have about 700-750 amino acid residues. Such a full length ZSMF-3 molecule would include a semaphorin domain of approximately 500 amino acid residues, a transmembrane domain of approximately 24 amino acid residues and a cytoplasmic domain of approximately 216 amino acid residues. Other methods such as 3' RACE using oligo sequences from SEQ ID NO:l and templates derived from spinal cord, adrenal gland, brain or stomach tissue could also be used.
Northern blot analysis of various human tissues was performed using a 200 bp human DNA probe (SEQ ID, NO: 6) . A 2 kb transcript was detected corresponding to ZSMF-3 in stomach tissue. The transcript size agrees with the predicted size of the ZSMF-3 protein as disclosed in SEQ ID NO: 2. Two faint transcripts of 4 kb and 3 kb were detected in spinal cord and adrenal gland tissue.
Chromosomal localization results showed .that ZSMF-3 maps 384.88 cR_3000 from the top of the human chromosome 5 linkage group on the WICGR radiation hybrid map. The proximal and distal framework markers were D5S492 and WI-3394, respectively. The use of surrounding markers positions ZSMF-3 in the 5q23.1-q23.2 region on the integrated LDB chromosome 5 map. Among the genes mapping to this region are a neurogenic differentiation factor, NEUROD3, (Tamimi et al . , Genomics 40.:355-7, 1997); type IV calmodulin-dependent protein kinase, CAMK4 , (Sikela et al . , Genomics 4.:21-7, 1989); genes associated with Charcot- Marie-Tooth disease, a demyelinating peripheral motor and sensory neuropathy LeGuern et al . , Hum. Molec. Genet. 5.: 1685-8, 1996) and ileal lipid-binding protein (Oelkers and Dawson, Biochim. Biophys . Acta 1257:199-202, 1995) . A degenerate polynucleotide sequence that encompasses all polynucleotides that encode the ZSMF-3 polypeptides of SEQ ID NOs: 2 or 4 , amino acid residues 1- 321, are disclosed in SEQ ID NO: 5 (human sequence) and SEQ ID NO:17 (mouse sequence). Thus, ZSMF-3 polypeptide- encoding polynucleotides ranging from nucleotide 1-963 of SEQ ID N0:5 and nucleotides 1-963 of SEQ ID N0:17 are contemplated by the present invention. Also contemplated by the present invention are fragments as described above with respect to SEQ ID NO : 1 , which are formed from analogous regions of SEQ ID NO : 5 , wherein nucleotides 1-963 of SEQ ID NO:l correspond to nucleotides 57-963 of SEQ ID NO: 5. The nucleotide base codes in SEQ ID NOs : 5 and 17 are summarized in Table 1.
T/ABLE 1
Nucleotide
Base Code Nucleotide Base Code Complement
A A T T
C C G G
G G C C
T T A A
R A|G Y C|T
Y C|T R A|G
M A|C K G|T
K G|T M A|C
S C|G S C|G
W A|T W A|T
H A|C|T D A|G|T
B C|G|T V A|C|G
V A|C|G B C|G|T
D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
The degenerate codons used in SEQ ID NOs : 5 and 17, encompassing all possible codons for a given amino acid, are set forth in Table 2.
TABLE 2
Three One
Letter Letter Synonymous Codons Degenerate
Code Code Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT SN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gin Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
He I ATA ATC ATT ATH
Leu L CTA CTC CTG cπ TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F πc TTT TTY
Tyr Y TAC TAT TAY
Trp TGG TGG
Ter . TAA TAG TGA TRR
Asn |Asp B RAY
Glu|Gln Z SAR
Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) , and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) . A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Such variant sequences can be tested for functionality as disclosed herein. One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al . , Nuc . Acids Res. .13:1893-912, 1980; Haas, et al . Curr. Biol. 6.: 315-24, 1996; Wain-Hobson, et al . , Gene lJ3_:355-64, 1981; Grosjean and Fiers, Gene .18.: 199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) . For example, the amino acid threonine
(Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ ID NOs : 5 and 17 serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
Within preferred embodiments of the invention, isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or to sequences complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe . Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from stomach tissue, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry 18 : 52-94 , 1979) . Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 6_9: 1408-12, 1972) . Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Polynucleotides encoding ZSMF-3 polypeptides are then identified and isolated by, for example, hybridization or PCR. Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO : 1 represents a single allele of human ZSMF-3 and the sequence disclosed in SEQ ID NO : 3 represents a single allele of murine ZSMF-3, and that allelic variation and alternative splicing are expected to occur. Allelic variants and splice variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art. Allelic variants of the DNA sequence shown in SEQ ID NOs : 1 or 3, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs : 2 or 4. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZSMF-3 polypeptides are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
The present invention further provides counterpart ligands and polynucleotides from other species ("species orthologs"). These species would include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are ZSMF-3 polypeptides from other mammalian species, including porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Species orthologs of human ZSMF-3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the ligand. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A ligand- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on- the disclosed sequence. A cDNA can also be cloned by PCR, using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the ligand. Similar techniques can also be applied to the isolation of genomic clones.
The present invention also provides isolated polypeptides and proteins that are substantially homologous to the polypeptides and proteins of SEQ ID NO: 2 and their species orthologs. By "isolated" is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein or polypeptide is substantially free of other proteins or polypeptides, particularly other proteins or polypeptides of animal origin. It is preferred to provide the proteins or polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. The term "substantially homologous" is used herein to denote proteins or polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO: 2 or its species orthologs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or its species orthologs. Particularly preferred are polypeptides which are 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 2 or its species orthologs. Within one embodiment, any difference between the amino acid sequence of the polypeptide and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 8.9: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 3 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as:
Total number of identical matches x 100
[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]
Table ! 3
A R N D C Q E G H I L K M F P S T W Y V
A 4
R -1 5
N -2 0 6
D -2 -2 1 6
C 0 -3 -3 -3 9
Q -1 1 0 0 -3 5
E -1 0 0 2 -4 2 5
G 0 -2 0 -1 -3 -2 -2 6
H -2 0 1 -1 -3 0 0 -2 8
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4
L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4
K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5
M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7
S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11
Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7
V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1
Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, Glu-Glu affinity tags, protein A (Nilsson et al . , EMBO J . 1:1075, 1985; Nilsson et al . , Methods Enzymol . 198 : 3 , 1991), glutathione S transferase (Smith and Johnson, Gene 6.7:31, 1988), maltose binding protein (Kellerman and Ferenci, Meth. Enzymol. ,90.:459-63, 1982; Guan et al . , Gene 6_7:21-30, 1987), thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, an immunoglobulin heavy chain constant region or other antigenic epitope or binding domain. See, in general Ford et al . , Protein Expression and Purification 2.: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ) .
Table 4
Conservative amino acid substitutions
Basic : arginine lysine histidine
Acidic : glutamic acid aspartic acid
Polar: glutamine asparagine
Hydrophobic leucine isoleucine valine
Aromatic : phenylalanine tryptophan tyrosine
Small glycine alanine serine threonine methionine
In addition to the 20 standard amino acids, non- standard amino acids (such as 4-hydroxyproline, 6 -N-methyl lysine, 2-aminoisobutyric acid, isovaline and α-me.thyl serine) may be substituted for amino acid residues of ZSMF- 3. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for ZSMF-3 amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain (s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3- dimethylproline .
The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non- naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis- 4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine , allo-threonine, methylthreonine, hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine, homo-glutamine, pipecolic acid, tert-leucine, norvaline, 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine . Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs . Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am. Chem. Soc . 113 :2722, 1991; Ellman et al . , Methods Enzymol . 202 :301, 1991; Chung et al . , Science 259:806-9, 1993; and Chung et al . , Proc. Natl. Acad. Sci. USA .90.: 10145-9, 1993) . In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al . , J. Biol. Chem. 2J1 : 19991-8, 1996) . Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) . The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33 : 7470- 6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 1:395-3, 1993) .
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 : 1081-5, 1989; Bass et al . , Proc. Natl. Acad. Sci. USA .88.: 4498-502, 1991) . In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., collapase assay) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity; in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al . , Science 255:306-12, 1992; Smith et al . , J. Mol. Biol. 2Z4:899-904, 1992; Wlodaver et al . , FEBS Lett . 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer
(Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl.
Acad. Sci. USA 8j5: 2152-6, 1989) . Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al . , Biochem. 3_0:10832-7, 1991; Ladner et al . , U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene £6:145, 1986; Ner et al. , DNA 2:127, 1988) .
Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active ligands or portions thereof (e.g., receptor-binding fragments) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
Using the methods discussed above, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially homologous to amino acid residues 20-321 of SEQ ID NO: 2 or allelic variants thereof and retain the properties of the wild-type protein. Such polypeptides may include additional amino acids from the semaphorin, transmembrane and/or cytoplasmic domains; affinity tags; and the like. Such polypeptides may also include additional polypeptide segments as generally disclosed above.
The semaphorin polypeptides of the present invention, including full-length polypeptides, fragments (e.g., receptor-binding fragments, growth cone directing fragments, immune response provoking fragments) , and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a ZSMF-3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art . Many such elements are described in the literature and are available through commercial suppliers.
To direct a ZSMF-3 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the ZSMF-3 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo . The secretory signal sequence is joined to the ZSMF-3 DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest
(see, e.g., Welch et al . , U.S. Patent No. 5,037,743;
Holland et al . , U.S. Patent No. 5,143,830). Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-19 of SEQ ID NOs : 2 or 4 is operably linked to another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.
Cultured mammalian cells are preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14.: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 1:603, 1981; Graham and Van der Eb, Virology 52.:456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid. ) , and liposome-mediated transfection (Hawley-Nelson et al . , Focus 15.: 73, 1993; Ciccarone et al . , Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al . , U.S. Patent No. 4,784,950; Palmiter et al . , U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650) , COS-
7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570
(ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 3_6:59-72, 1977) and Chinese hamster ovary (e.g., CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants" . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants . " A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select ' for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate . Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology. Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222; Bang et al . , U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463. The use of Agroba cterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. (Bangalore) 11:47-58, 1987. Insect cells can be infected with recombinant baculovirus vectors, which are commonly derived from Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV) . DNA encoding the polypeptide of interest is inserted into the viral genome in place of the polyhedrin gene coding sequence by homologous recombination in cells infected with intact, wild-type AcMNPV and transfected with a transfer vector comprising the cloned gene operably linked to polyhedrin gene promoter, terminator, and flanking sequences. The resulting recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and
Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
Fungal cells, including yeast cells, and particularly cells of the genus Sa ccharomyces, can also be used within the present invention, such as for producing fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) . A preferred vector system for use in yeast is the POTl vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe, Kl uyveromyces lactis , Kl uyveromyces fragilis , Us tilago maydis , Pichia pa storis, Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol . 132:3459-65, 1986; and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al . , Yeast 14:11-23,
1998, and in WIPO Publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
Prokaryotic host cells, including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) . When expressing a ZSMF-3 polypeptide in bacteria such as E . col i , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
The invention provides soluble ZSMF-3 ligand polypeptides. Soluble ZSMF-3 polypeptides can be prepared by expressing a DNA encoding amino acid residues 20-32.1 of a human ZSMF-3 polypeptide (SEQ ID NO: 2) . In one embodiment, it is preferred that these soluble, extracellular receptor-binding domain polypeptides be prepared in a form substantially free of transmembrane and intracellular polypeptide segments. For example, the C- terminus of the ligand polypeptide may be at residue 32.1 of SEQ ID NO: 2 or the corresponding region of an allelic variant or at the C-terminal end of a semaphorin domain. To direct the export from the host cell, the ligand DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide. To facilitate purification of the secreted receptor domain, an N- or C- terminal extension, such as a poly-histidine tag, a Glu-Glu affinity tag, substance P, FLAG™ peptide (Hopp et al . , Biotechnology 6.: 1204-10, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the ligand polypeptide. Expressed recombinant ZSMF-3 polypeptides (or chimeric or fused ZSMF-3 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast- Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred. Exemplary chromatographic media include those media derivatized with phenyl , butyl, or octyl groups, such as Phenyl -Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be isolated by exploitation of their physical properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3_:l-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Alternatively, a fusion of the polypeptide of interest and an affinity tag (e.g., polyhistidine, maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.
It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
ZSMF-3 polypeptides or fragments thereof may also be prepared through chemical synthesis. ZSMF-3 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
Polypeptides containing the receptor-binding region of the ligand can be used for purification of receptor. The ligand polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting media will generally be configured in the form of a column, and fluids containing receptors are passed through the column one or more times to allow receptor to bind to the ligand polypeptide. The 'receptor is then eluted using changes in salt concentration, chaotropic agents (MnCl2), or pH to disrupt ligand-receptor binding. Soluble ZSMF-3 or ZSMF-3 fusion proteins are used, for example, to identify the ZSMF-3 receptor. Using labeled soluble ZSMF-3, cells expressing the receptor are identified by fluorescence immunocytometry or immunohistochemistry. The soluble receptor is useful in studying the distribution of receptor on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
ZSMF-3 fusion proteins consist essentially of a first portion and a second portion joined by a peptide bond. The first portion comprising a polypeptide wherein the polypeptide comprises a sequence of amino acid residues selected from the group consisting of: a) the sequence of amino acids as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; b) a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO: 2 from Phe, residue 20, through Arg, residue 321; and c) allelic variants of a) or b) ; wherein said polypeptide is a semaphorin. The second portion comprises another polypeptide. The second portion polypeptide may be another polypeptide such as a semaphorin or a polypeptide fragment of a semaphorin protein, such as a transmembrane domain or cytoplasmic domain.
ZSMF-3 proteins, agonists and antagonists may be used to modulate neurite growth and development and demarcate nervous system structures. As such, ZSMF-3 and antagonists would be useful as a treatment of peripheral neuropathies by increasing spinal cord and sensory neurite outgrowth. Such an antagonist could be part of a therapeutic treatment for the regeneration of neurite outgrowths following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries. Application may also be made in treating neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Application may also be made in mediating development and innervation pattern of stomach tissue . Semaphorins have also been associated with mediation of other non-neuronal biological functions. ZSMF-3 polypeptides may find use in mediating immune response, in particular ZSMF-3 polypeptides and ZSMF-3 agonists would be usef l as immunosuppressants . It has been suggested that viral proteins play a role in modulating immune response against viral infection by either binding to a cytokine or preventing activation of a cytokine. It is speculated that viral semaphorin, Sema' IV, plays the role of a natural immunosuppressor to reduce the immune response (Kolodkin et al . , ibid.). ZSMF-3 shares structural homology with sema IV and may have similar biological activity. Other non-viral semaphorins have also been associated with the immune system. Human semaphorin E, which is homologous to viral cytokine inhibiting proteins contains conserved regions of amino acid residues that have been found in the viral semaphorins . Semaphorin E was found to be upregulated in rheumatoid synovial fibroblastoid cells which suggests that it may have a role as a regulator of inflammatory processes and an involvement in the development of rheumatoid arthritis (Mangasser- Stephan et al . , Biochem. Bio hys . Res. Comm. 234 : 153-6 , 1997) . Semaphorin G (Sema G) is expressed on lymphocytes and may play a role in the immune system (Furuyama et al . , J. Biol. Chem. 271:33376-81, 1996) . The figure shows the homology ZSMF-3 shares with sema G. Semaphorin CD100 promotes B-cell growth and aggregation and may be involved in lymphocyte activation (Hall et al . , Proc. Natl. Acad. Sci. USA 93 : 11780-5, 1996) .
Semaphorins, such as Semaphorin III, have a role in the development of bones and heart by acting as a restraining signal during organ development (Behar et al . , Nature 383 :525-8, 1996). G-Sema I and collapsin are hypothesized to act in vivo as repulsive or inhibitory molecules that prevent neighboring ventral motorneurons from innervating extra thoracic muscle. In other situations, G-Sema I and collapsin may also act as an attractive agent to promote innervation (Kolodkin et al . , Cell 15:1389-99, 1993) . The truncated human ZSMF-3 may play a similar role in the stomach.
The activity of semaphorin polypeptides, agonists, antagonists and antibodies of the present invention can be measured, and compounds screened to identify agonists and antagonists, using a variety of assays, in particular assays that measure axon guidance and growth. Of particular interest are assays that indicate changes in neuron growth patterns, see for example, Hastings, WIPO Patent Application No: 97/29189 and Walter et al . , Development 101 :685-96 , 1987. Assays to measure the effects of semaphorins on neuron growth are well known in the art. For example, the C assay (see for example, Raper and Kapfhammer, Neuron 4.: 21-9, 1990 and Luo et al . , Cell 15_:217-27, 1993), can be used to determine collapsing activity semaphorins on growing neurons. Other methods which assess semaphorin induced inhibition of neurite extension or divert such extension are also known, see Goodman, Annu. Rev. Neurosci. 19:341-77, 1996. Conditioned media from cells expressing a semaphorin, semaphorin agonist or semaphorin antagonist, or aggregates of such cells, can be placed in a gel matrix near suitable neural cells, such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been cocultured with nerve growth factor. Compared to control cells, semaphorin- induced changes in neuron growth can be measured (see for example, Messersmith et al . , Neuron 14:949-59, 1995; Puschel et al . , Neuron 14.: 941-8, 1995). Likewise neurite outgrowth can be measured using neuronal cell suspensions grown in the presence of molecules of the present invention see for example, O'Shea et al . , Neuron 1:231-7, 1991 and DeFreitas et al . , Neuron 15:333-43, 1995.
Also available are assay systems that use a ligand-binding receptor (or an antibody, one member of a complement/anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument
(BIAcore™, Pharmacia Biosensor, Piscataway, NJ) . Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145 :229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 23_4: 554-63 , 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti- complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. Ligand-binding receptor polypeptides can also be used within other assay systems known in the art . Such systems include Scatchard analysis for determination of binding affinity (see, Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 251:545-8, 1991; Cunningham et al . , Science 245:821-5, 1991) .
In vi tro and in vivo response to soluble ZSMF-3 can also be measured using cultured cells or by administering molecules of the claimed invention to the appropriate animal model . Proteins of the present invention may also be assayed using viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus
(AAV) . Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see
Becker et al . , Meth. Cell Biol. 41:161-89, 1994; and Douglas and Curiel, Science & Medicine 4_:44-53, 1997). A preferred method is that of He et al . , Proc. Natl. Acad. Sci . USA 9J5:2509-14, 1998 and is described in more detail below.
The adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see A. Gamier et al., Cvtotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non- secreted proteins may also be effectively obtained. ZSMF-3 polypeptides can also be used to prepare antibodies that specifically bind to ZSMF-3 epitopes, peptides or polypeptides. Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning-: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R. , Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982) . As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
The immunogenicity of a ZSMF-3 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZSMF-3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier
(such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization. As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 anc^ Fa^ proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies (scFv) , single complementarity- determining region peptides ("minimal recognition units") and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody) .■ In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Humanized monoclonal antibodies directed against ZSMF-3 polypeptides could be used as a protein therapeutic, in particular for use as an immunotherapy. Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZSMF-3 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSMF-3 protein or peptide) .
Antibodies are defined to be specifically binding if they bind to a ZSMF-3 polypeptide with a binding affinity (Ka) of IO6 M"1 or greater, preferably 10 M~ or greater, more preferably IO8 M"1 or greater, and most preferably 10 M"1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis) .
Genes encoding polypeptides having potential ZSMF-3 binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli . Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US Patent NO : 5 , 223 , 409 ; Ladner et al . , US Patent NO: 4 , 946 , 778 ; Ladner et al . , US Patent NO: 5, 403, 484 and Ladner et al . , US Patent NO : 5 , 571, 698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) . Random peptide display libraries can be screened using the ZSMF-3 sequences disclosed herein to identify proteins which bind to ZSMF-3. These "binding proteins" which interact with ZSMF-3 polypeptides may be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. A variety of assays known to those skilled in the art can be utilized to detect antibodies and binding proteins which specifically bind to ZSMF-3 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) , Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ZSMF-3 protein or peptide. Antibodies and binding proteins to ZSMF-3 may be used for immunohistochemical tagging of cells that express human ZSMF-3, for example, to use in diagnostic assays; for isolating ZSMF-3 by affinity purification; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZSMF-3 in vi tro and in vivo . Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti -complement pairs as intermediates. Antibodies and binding proteins herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
The invention also provides isolated and purified ZSMF-3 polynucleotide probes. Such polynucleotide probes can be RNA or DNA. DNA can be either cDNA or genomic DNA. Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire ZSMF-3 gene or cDNA. The synthetic oligonucleotides of the present invention have at least 80% identity to a representative ZSMF-3 DNA sequence (SEQ ID NOs : 1 or 3) or its complements. Preferred regions from which to construct probes include the 5' and/or 3' coding sequences, receptor binding regions, semaphorin, extracellular, transmembrane and/or cytoplasmic domains and signal sequences, and the like. Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991. For use as probes, the molecules can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZSMF-3 gene or mRNA transcript in a sample. ZSMF-3 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in si tu hybridization (FISH) or immunohistochemistry . Polynucleotide probes can be used to identify genes encoding ZSMF-3 -like proteins. For example, ZSMF-3 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the semaphorin family. Such probes can also be used to screen libraries for related sequences encoding novel semaphorin receptors. Such screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence. Such methods and conditions are well known in the art, see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989. Such low stringency conditions could include hybridization temperatures less than 42°C, formamide concentrations of less than 50% and moderate to low concentrations of salt. Libraries may be made of genomic DNA or cDNA. Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridization and in si tu hybridization. Mixtures of different ZSMF-3 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems. Nucleic acid probes can be detectably labeled with radioisotopes such as 32P or 35S. Alternatively, ZSMF-3 RNA can be detected with a nonradioactive hybridization method (see, for example,
Isaac (ed.), Protocols for Nucleic Acid Analysis by
Nonradioactive Probes. Humana Press, Inc., 1993). Typically, nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative non-radioactive moieties include biotin, fluorescein, and digoxigenin.
Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods. Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; White (ed.), PCR Protocols: Current Methods and Applications, Humana Press, Inc., 1993; Cotter (ed.), Molecular Diagnosis of Cancer, Humana Press, Inc., 1996; Hanausek and Walaszek (eds.), Tumor Marker Protocols, Humana Press, Inc., 1998; Lo (ed.), Clinical Applications of PCR, Humana Press, Inc., 1998 and Meltzer (ed.), PCR in Bioanalysis, Humana Press, Inc., 1998). PCR primers can be designed to amplify a sequence encoding a particular ZSMF-3 region, such as the semaphorin domain.
One variation of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR) . In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZSMF-3 primers (see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, pages 15-28, CRC Press, Inc. 1997) . PCR is then performed and the products are analyzed using standard techniques .
As an illustration, RNA is isolated from a biological sample using, for example, the guanidinium- thiocyanate cell lysis procedure described above. Alternatively, a solid-phase technique can be used to isolate mRNA from a cell lysate. A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or ZSMF-3 anti- sense oligomers. Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences. ZSMF-3 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled ZSMF-3 probe, and examined by autoradiography . Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
Another approach is real time quantitative PCR (Perkin-Elmer Cetus, Norwalk, CT) . A fluorogenic probe, consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers. Using the 5' endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated and increases as amplification increases. The fluorescence intensity can be continuously monitored and quantified during the PCR reaction.
Another approach for detection of ZSMF-3 expression is cycling probe technology (CPT) , in which a single-stranded DNA target binds with an excess of DNA-RNA- DNA chimeric probe to form a complex, the RNA portion is cleaved with RNase H, and the presence of cleaved chimeric probe is detected (see, for example, Beggs et al . , J. Clin. Microbiol . 3_4:2985, 1996 and Bekkaoui et al . , Biotechniques 0.:240, 1996) . Alternative methods for detection of ZSMF-3 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA) , cooperative amplification of templates by cross-hybridization (CATCH) , and the ligase chain reaction (LCR) (see, for example, Marshall et al . , U.S. Patent No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 6_0:161, 1996; Ehricht et al . , Eur. J. Biochem. 243 :358, 1997; and Chadwick et al . , J. Virol. Methods ϋ:59, 1998). Other standard methods are known to those of skill in the art.
ZSMF-3 probes and primers can also be used to detect and to localize ZSMF-3 gene expression in tissue samples. Methods for such in si tu hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), Jn Si tu Hybridization Protocols, Humana Press, Inc., 1994; Wu et al . (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridization (RISH) , " in Methods in Gene Biotechnology, pages 259-278, CRC Press, Inc., 1997; and Wu et al . (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Si tu Hybridization (RISH) , " in Methods in Gene Biotechnology, pages 279-289, CRC Press, Inc., 1997). Various additional diagnostic approaches are well-known to those of skill in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics. Humana Press, Inc., 1996; and Elles, Molecular Diagnosis of Genetic Diseases, Humana Press, Inc., 1996) .
In addition, such polynucleotide probes could be used to hybridize to counterpart sequences on individual chromosomes. Chromosomal identification and/or mapping of the ZSMF-3 gene could provide useful information about gene function and disease association. Many mapping techniques are available to one skilled in the art, for example, mapping somatic cell hybrids, and fluorescence in si tu hybridization (FISH) . A preferred method is radiation hybrid mapping. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 25_0: 245-50 , 1990). Partial or full knowledge of a gene's sequence allows the designing of PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel
(Research Genetics, Inc., Huntsville, AL) , are available.
These panels enable rapid, PCR based, chromosomal localizations and ordering of genes, sequence-tagged sites
(STSs) , and other non-polymorphic- and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross- referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.
Chromosomal localization can also be done using STSs. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome . An STS can be defined by a pair of oligonucleotide primers that can be used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within a database, for example, Database of Sequence Tagged Sites (dbSTS) , GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), they can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences . ZSMF-3 was mapped to the 5q23.1-q23.2 region on the integrated LDB chromosome 5 map as is described in more detail below.
The present invention also contemplates use of such chromosomal localization for diagnostic applications. Briefly, the ZSMF-3 gene, a probe comprising ZSMF-3 DNA or RNA or a subsequence thereof, can be used to determine if the ZSMF-3 gene is present on human chromosome 5 or if a mutation has occurred. Detectable chromosomal aberrations at the ZSMF-3 gene locus include, but are not limited, to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (.STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108 :255- 65, 1995) .
In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient;- (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l*, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108 :255-65,
1995). Ribonuclease protection assays (see, e.g., Ausubel et al . , ibid. , ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase . Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) . ZSMF-3 polypeptides and polynucleotides may be used within diagnostic systems. Antibodies or other agents that specifically bind to ZSMF-3 may be used to detect the presence of circulating ligand or receptor polypeptides. Such detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. Immunohistochemically labeled ZSMF-3 antibodies can be used to detect ZSMF-3 receptor and/or ligands in tissue samples and identify ZSMF-3 receptors. ZSMF-3 levels can also be monitored by such methods as RT-PCR, where ZSMF-3 mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of ZSMF-3 polypeptides in various diseases and biological processes, and as a would serve as diagnostic tools for diseases for which altered levels of ZSMF-3 are significant . The ZSMF-3 polynucleotides and/or polypeptides disclosed herein can be useful as therapeutics, wherein ZSMF-3 agonists and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids. ZSMF-3 antagonists provided by the invention, bind to ZSMF-3 polypeptides or, alternatively, to a receptor to which ZSMF-3 polypeptides bind, thereby inhibiting or eliminating the function of ZSMF-3. Such ZSMF-3 antagonists would include antibodies; oligonucleotides which bind either to the ZSMF-3 polypeptide or to its ligand; natural or synthetic analogs of ZSMF-3 ligands which retain the ability to bind the receptor but do- not result in either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZSMF-3 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZSMF-3 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZSMF-3 receptor or ligand would be beneficial.
The invention also provides nucleic acid-based therapeutic treatment. If a mammal lacks or has a mutated ZSMF-3 gene, the ZSMF-3 gene can be introduced into the cells of the mammal. Using such methods, cells altered to express the nerve growth factor neurotrophin-3 (NT-3) were grafted to a rat model for spinal injury and stimulated axon regrowth at the lesion site and the rats thus treated recovered some ability to walk (Grill et al . , J. Neuroscience 11:5560-72, 1997). In one embodiment, a gene encoding a ZSMF-3 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus , Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is' not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al . , Molec. Cell. Neurosci. 1:320-30, 1991), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . (J. Clin. Invest. 91:626-30, 1992), and a defective adeno- associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , J. Virol. £3:3822-8,
1989) .
In another embodiment, the gene can be introduced in a retroviral vector, e . g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al . , Cell 33 :153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al . , J . Virol .
62:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; Dougherty et al . , WIPO Publication WO 95/07358; and Kuo et al., Blood 82:845-52, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci. USA £4:7413-7, 1987; and Mackey et al., Proc. Natl. Acad. Sci. USA £5:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents • one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
It is possible to remove the cells from the body and introduce the vector as a naked DNA plasmid and then re- implant the transformed cells into the body. Naked DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al . , J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988).
7Λnother aspect of the present invention involves antisense polynucleotide compositions that are complementary to a segment of the polynucleotide set forth in SEQ ID NO:l. Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZSMF-3 polypeptides and to inhibit translation of such mRNA. Such antisense oligonucleotides are used to inhibit expression of ZSMF-3 polypeptide-encoding genes in cell culture or in a subject.
Transgenic mice, engineered to express the ZSMF-3 gene, and mice that exhibit a complete absence of ZSMF-3 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257 : 1083 , 1992), may also be generated (Lowell et al., Nature 366 : 740-2, 1993). These mice may be employed to study the ZSMF-3 gene and the protein encoded thereby in an in vivo system.
The invention is further illustrated by the following non-limiting examples. EXAMPLES
Example 1 Identification of ZSMF-3
Novel ZSMF-3 encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for sequences homologous to conserved motifs within the semaphorin family. To identify the corresponding cDNA, a clone from which an identified EST was derived that was considered likely to contain the entire murine ZSMF-3 sequence was used for sequencing. Using a QIAwell 8 plasmid kit
(Qiagen, Inc., Chatsworth, CA) according to manufacturer's instructions, a 5 ml overnight culture in LB + 50 μg/ml ampicillin was prepared. The template was sequenced on an Applied Biosystems™ model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Ct . ) using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) according to the manufacturer's instructions. Oligonucleotides ZC694 (SEQ ID NO: 7) and ZC976 (SEQ ID NO: 8) to the T7 and SP6 promoters on the vector were used as sequencing primers. Oligonucleotides ZC14201 (SEQ ID NO: 9) and ZC14202 (SEQ ID NO: 10) were used to complete the sequence from the clone. Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System
(National Labnet Co., Woodbridge, NY). SequencherTM 3.0 sequence analysis software (Gene Codes Corporation, Ann
Arbor, MI) was used for data analysis. A 1047 bp sequence was identified which encoded a semaphorin protein having a truncated semaphorin domain, but no stop codon . was identified.
To identify the human ZSMF-3 cDNA, a clone from which the identified human EST was derived and considered likely to contain the entire human ZSMF-3 sequence was used for sequencing. Using a QIAwell 8 plasmid kit as above an overnight culture was prepared. The template was sequenced as above using oligos ZC694 (SEQ ID N0:7), ZC976 (SEQ ID NO: 8) as sequencing primers. This clone contained only about 600 bp of 3' sequence, so 5' RACE (rapid amplification of cDNA ends) was done to identify the remaining 5' portion. Using a fetal brain Marathon™ .cDNA library as a template and oligos ZC15,523 (SEQ ID NO: 18) to a conserved region of the mouse untranslated region and ZC14,095 (SEQ ID NO: 11) to an internal sequence of the 3' human fragment, 5' RACE was carried out at 94°C, for 1.5 minutes, followed by 35 cycles at 94°C for 15 seconds, 56°C for 30 seconds and 72°C for 1 minute, followed by a 10 minute extension at 72°C. A band of approximately 700 bp fragment was resolved by gel electrophoresis. The 5' RACE fragment was ligated into the TA vector (Invitrogen Inc., San Diego, CA) according to manufacturer's instructions. The sequence of the 5 ' fragment was confirmed by sequence analysis as described above.
Homologous recombination in yeast was used to create expression plasmids containing the polynucleotide encoding ZSMF-3 for expression in mammalian and yeast cells. To construct the ZSMF-3/pCZF199 expression vectors the following DNA fragments were transformed into S. cerevisiae : Sna BI digested pCZR199 as an acceptor vector, the 3' ZSMF-3 Xho I-Not I restriction fragment, the 5' Eco RI restriction fragment, and two, double stranded linker segment. The expression vector, pCZR199, has yeast replication elements, (CEN, ARS) , the selectable marker, URA3 , E. coli replication elements (e.g., AMPR and ori ) , a blunt-ended cloning site, Sna BI, and adds either a N- terminal or C-terminal Glu-Glu tag. The vectors were used to create ZSMF-3 polypeptides having either end of the expressed protein Glu-Glu tagged. The double stranded linker segments were prepared using PCR. The linkers served to join the vector to the insert fragments at both the 5' and 3' ends. Two sets of linkers were prepared. One set of linkers joined the insert to a vector placing the Glu-Glu tag on the 5' end of the insert sequence. The 5' linker of this set was made from oligos ZC14,397 (SEQ ID NO:27), ZC14,396 (SEQ ID NO:28), ZC16,463 (SEQ ID NO:29) and ZC16,442 (SEQ ID NO:30). The 3' linker was made from oligos ZC14,455 (SEQ ID N0:31), ZC14,394 (SEQ ID NO:32), ZC16,461 (SEQ ID NO:33) and ZC16,444 (SEQ ID NO:26) . The second set of linkers was used to join the ZSMF-3 insert into a vector placing a 3' Glu-Glu tag. The 5' linker was made from oligos ZC14,454 (SEQ ID N0:21), ZC14,392 (SEQ ID NO:22), ZC16,443 (SEQ ID N0:19) and ZC16,462 (SEQ ID NO:20) . The 3' linker was made from oligos ZC14,395 (SEQ ID NO:24), ZC14,393 (SEQ ID NO:23), ZC16,441 (SEQ ID NO:25) and ZC16,444 (SEQ ID NO:26). The oligos were joined using standard PCR reaction conditions and heated to 94°C for 1.5 minutes followed by 10 cycles at 94°C for 30 seconds, 50°C for 1 minute and 72°C for 1 minute, then a 10 minute extension at 72°C.
The DNA fragments were added to 100 μl competent yeast (Genetic strain SF83890, Roffman et al . , EMBO J . £:2057-65, 1989) and electroporated . The yeast cells were immediately diluted in 600 μl 1.5 M sorbitol and plated on Ura D plates and incubated at 30°C for 48 hours. .Ura+ colonies were selected from both the N-terminally-tagged and C-terminally-tagged ZSMF-3 proteins and the DNA from the resulting yeast colonies was extracted and transformed into E. coli . Individual clones harboring the correct expression construct were identified by PCR screening. DNA sequencing as described above using oligos ZC447 .(SEQ ID NO:34), ZC16296 (SEQ ID NO:35) and ZC16,292 (SEQ ID NO: 36) as sequencing primers confirmed that the desired sequences had been enjoined with one another.
Large scale plasmid DNA is isolated from one or more correct clones from both the N- and C-terminally tagged ZSMF-3 sequences, the expression cassette liberated from the vector and transformed into yeast or E. coli for large scale protein production. Example 2 Tissue Distribution
Human Multiple Tissue Northern Blots (MTN I, MTN II, and MTN III; Clontech) were probed to determine the tissue distribution of human ZSMF-3 expression. An approximately 200 bp PCR derived probe (SEQ ID NO: 6) was amplified from a human stomach derived Marathon™-ready cDNA library. Oligonucleotide primers ZC14,095 (SEQ ID NO: 11) and ZC14,096 (SEQ ID NO: 12) were designed based on the EST sequence. The Marathon™-ready cDNA library was prepared according to manufacturer's instructions
(Marathon™ cDNA Amplification Kit; Clontech, Palo Alto,
CA) using human stomach poly A+ RNA (Clontech) . The probe was amplified in a polymerase chain reaction as follows: 1 cycle at 94°C for 1 minute 30 seconds; 35 cycles of 94°C for 15 seconds and 64°C for 30 seconds, followed by 1 cycle at 72°C for 10 minutes. The resulting DNA fragment was electrophoresed on a 2% low melt agarose gel (SEA PLAQUE GTG low melt agarose, FMC Corp., Rockland, ME), the fragment was purified using the QIAquick™ method (Qiagen, Chatsworth, CA) , and the sequence was confirmed by sequence analysis. The probe was radioactively labeled using the random priming MULTIPRIME DNA labeling system (Amersham, Arlington Heights, IL) , according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene, La Jolla, CA) . ExpressHyb™
(Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C using 2 x IO6 cpm/ml of labeled probe. The blots were then washed at 25°C in 2X SSC, 0.05% SDS for 1 hour, then at 50°C in 0. IX SSC, 0.1% SDS for 1 hour. A transcript of approximately 2 kb was seen in stomach. A Human RNA Master Dot Blot (Clontech) that contained RNAs from various tissues that were normalized to 8 housekeeping genes was also probed and hybridized as described above. Expression was seen only in stomach.
Mouse Multiple Choice^ Tissue Northern Blots (OriGene Technologies, Inc., Rockville, MD) were probed to determine the tissue distribution of mouse ZSMF-3 expression. The 200 bp stomach derived probe (SEQ ID NO: 6) described above was used as a probe. Hybridization took place overnight at 65°C using 1 x 10^ cpm/ml of labeled probe. The blots were then washed at 25°C in 2X SSC, 0.1% SDS for 1 hour, then at 50°C in 0. IX SSC, 0.1% SDS for 1 hour. A transcript of approximately 1.5 kb was seen in all tissues, stomach, small intestine, muscle, lung, skin, brain, heart, kidney, spleen, thymus and liver.
A mouse RNA Master Blot™ (Clontech) was probed as described above. Expression was detected only in epididymus .
Example 3 Chromosomal Assignment and Placement of ZSMF-3
ZSMF-3 was mapped to chromosome 5 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) . The GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) . A publicly available WWW server (http : //www-genome .wi .mit . edu/cgi-bin/contig/ rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR" radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel .
For the mapping of ZSMF-3 with the GeneBridge 4 RH Panel, 20 μl reactions were set up in a 96-well microtiter plate (Stratagene) and used in a RoboCycler Gradient 96 thermal cycler (Stratagene) . Each of the 95 PCR reactions consisted of 2 μl 10X KlenTaq PCR reaction buffer (Clontech), 1.6 μl dNTPs mix (2.5 mM each, Perkin- Elmer, Foster City, CA) , 1 μl sense primer, 15,398 (SEQ ID N0:15), 1 μl antisense primer, ZC 15,397 (SEQ ID N0:16), 2 μl flediLoad (Research Genetics, Inc.), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech) , 25 ng of DNA from an individual hybrid clone or control and ddH20 for a total volume of 20 μl . The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 62°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) . The results showed that ZSMF-3 maps 384.88 cR_3000 from the top of the human chromosome 5 linkage group on the WICGR radiation hybrid map. The proximal and distal framework markers were D5S492 and WI-3394, respectively. The use of surrounding markers positions ZSMF-3 in the 5q23.1 - q23.2 region on the integrated LDB chromosome 5 map (The Genetic Location Database, University of Southhampton, WWW server :http : //cedar . genetics. soton.ac .uk/public_html/) .
Example 4 ZSMF-3 Adenovirus Expression Vector Construction
Production of adenovirus containing ZSMF-3 NF and
CF was done according to the procedure of He et al . , Proc .
Natl. Acad. Sci. USA 95: 2509-14, 1998. Briefly, the cDNA encoding zsmf3 was amplified by PCR using primers ZC 17960
(SEQ ID NO:38) and ZC 17961 (SEQ ID NO:39) . The oligos added two unique restriction sites, Fsel and Ascl. The reaction proceeded a 95°C for 5 minutes, followed by 25 cycles at 95°C for 1 minute, 58°C for 1 minute and 72°C for 1.5 minutes, with a 7 minute extension at 72°C. The PCR product was visualized by gel electrophoresis, excised and purified. The zsmf3 cDNA was digested with the restriction enzymes Fsel and Ascl, phenol/chloroform extracted, ethanol precipitated, and rehydrated in 20 μl TE (10 mM Tris HCl/l mM EDTA pH 8.0). The zsmf3 fragment was then ligated into the Fsel -Ascl sites of the transgenic vector pTG12-8 and transformed into DH10B competent cells by electroporation. Clones containing zsmf3 were identified by mini prep followed by digestion with Fsel-Ascl. Identity of the clone was confirmed by sequence analysis.
Preparation of DNA construct for generation of Adenovirus
A 966 base pair ZSMF-3 fragment (nucleotide 1-966 of SEQ ID NO:l) was released from the TG12-8 vector using Fsel and Ascl restriction enzymes. The cDNA was isolated by electrophoresis on a 1% low melt SeaPlaque GTG gel. A band of the appropriate size was excised from the gel and the DNA extracted.
The zsmf3 cDNA was cloned into the Fsel-Ascl sites of pAdTrack CMV vector (He, et al . , ibid. ) in which the native polylinker was replaced with Fsel, EcoRV, Ascl sites. Ligation was performed using the Fast-Link DNA ligation and screening kit by Epicentre Technologies (Madison, WI) . Five micrograms of the pAdTrack CMV zsmf3 plasmid was digested with Pmel to linearize the plasmid. One microgram of the linearized plasmid was co-transformed with 200 ng of supercoiled pAdEasy (He et al . , ibid. ) into BJ5183 cells (He et al . , ibid. ) by electroporation and plated onto plates coated with LB containing 25 μg/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin and recombinant adenovirus DNA identified by standard mini prep procedures. Digestion of the recombinant adenovirus DNA with Fsel -Ascl confirmed the presence of ZSMF-3. The recombinant adenovirus mini prep DNA was transformed into DHIOB competent cells and DNA prepared using a Plasmid Maxi Kit (Qiagen) according to manufacturer's instructions.
Transfection of 293a Cells with Recombinant DNA
Five micrograms of recombinant adenoviral DNA was digested with Pad enzyme (NEBiolabs, Beverly, MA) for 3 hours at 37°C in a reaction volume of 100 μl containing 20- 30U of Pad. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitate with ethanol. The DNA pellet was resuspended in 10 μl distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc . Canada) set up the day before which were 60-70% confluent were transfected with the Pad digested DNA. The Pad-digested DNA was diluted up to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES) . In a separate tube, 20 μl DOTAP
(Boehringer Mannheim, 1 μg/μl) was diluted to a total volume of 100 μl with HBS. The DNA was added to the DOTAP, mixed gently by pipetting up and down, and left at room temperature for 15 minutes . The media was removed from the 293A cells and washed with 5 mis serum-free MEMalpha (GIBCO BRL, Gaithersburg, MD) containing 1 mM Sodium Pyruvate (GIBCO BRL), 0.1 mM MEM non-essential amino acids (GIBCO BRL) and 25 mM HEPES buffer (GIBCO BRL) . Five milliliters of serum-free MEM was added to the 293A cells and held at 37°C. The DNA/lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours . After 4 hours the media containing the DNA/lipid mixture was aspirated off and replaced with 5 mis complete MEM containing 5% fetal bovine serum. The transfected cells were monitored for Green Fluorescent Protein (GFP) expression and formation of viral plaques.
Crude rAdV Lysate
Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells expressed the GFP protein and started to form foci . These foci are viral "plaques" and the crude viral lysate was collected by using a cell scraper to collect all of the 293A cells. The lysate was transferred to a 50 ml conical tube. To release most of the virus particles from the cells, three freeze/thaw cycles were done in a dry ice/ethanol bath and a 37° waterbath.
Primary (1°) Amplification of rAdV
The crude lysate was amplified to obtain a working "stock" of rAdV lysate. Ten, 10 cm plates of nearly confluent (80-90%) 293A cells were set up 20 hours previously, 200 μl of crude rAdV lysate added to each 10 cm plate and monitored for 48 to 72 hours looking for CPE
(Cytopathic Effect) under the white light microscope and expression of GFP under the fluorescent microscope. When all of the 293A cells showed CPE this 1° stock lysate was collected and freeze/thaw cycles performed as described under Crude rAdV Lysate.
Secondary (2°) Amplification of rAdV
Twenty, 15 cm tissue culture dishes of 293A cells were prepared so that the cells were 80-90% confluent. All but 20 mis of 5% MEM media was removed and each dish was inoculated with 300-500 μl 1° amplified rAdv lysate. After 48 hours the 293A cells were lysed from virus production and this lysate was collected into 250 ml polypropylene centrifuge bottles and the rAdV purified.
AdV/cDNA Purification
NP-40 detergent was added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells. Bottles were placed on a rotating platform for 10 minutes agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatant was transferred to 250 ml polycarbonate centrifuge bottles and 0.5 volumes of 20% PEG8000/2.5M NaCl solution added. The supernatant containing bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes and supernatant discarded. The white precipitate (precipitated virus/PEG) was collected using a sterile cell scraper and the precipitate from 2 bottles was resuspended in 2.5 mis PBS. The virus solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G in the microfuge for 10 minutes to remove any additional cell debris. The supernatant from the 2 ml microcentrifuge tubes was transferred into a 15 ml polypropylene snapcap tube and adjusted to a density of 1.34g/ml with Cesium Chloride (CsCl) . The solution was transferred polycarbonate thick-walled centrifuge tubes 3.2 ml (Beckman Instruments, Palo Alto, CA) and spun at 80,000 rpm (348,000 X G) for 3-4 hours at 25°C in a Beckman Optima TLX micro ultracentrifuge with a TLA-100.4 rotor. The virus formed a white band. Using wide bore pipette tips virus band was collected.
Desalting the Virus Pharmacia PD-10 columns prepacked with Sephadex G-25M (Pharmacia) were used to desalt the virus preparation. The column was equilibrated with 20 mis of PBS. The virus was loaded and allow it to run into the column. Five milliliters of PBS was added to the column and fractions of 8-10 drop collected. The optical densities of 1:50 dilutions of each fraction was determined at 260 nm on a spectrophotometer . Those fractions having virus were pooled and the optical density of a 1:50 dilution determined.
TCID 50 Viral Titration Assay
Recombinant virus infectivity was measured according to the method of Quantum Biotechnologies, Inc.
Briefly, two 96-well tissue culture plates were seeded with
1 X IO4 293A cells per well in MEM containing 2% fetal bovine serum for each recombinant virus to be assayed.
After 24 hours 10-fold dilutions of each virus from 1 X 10" 2 to l X 10~14 were made in MEM containing 2% fetal bovine serum. One hundred microliters of each dilution was placed in each of 20 wells. After 5 days at 37°C, wells were read either positive or negative for Cytopathic Effect
(CPE) and a value for "Plaque Forming Units/ml" was calculated
The zsmf3 adenovirus had a titer of 8.0 X 1011 pfu/ml .
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS What is claimed is:
1. An isolated polypeptide comprising a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321, wherein said polypeptide is a semaphorin.
2. An isolated polypeptide according to claim 1, wherein any difference between said amino acid sequence of said isolated polypeptide and said corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution .
3. An isolated polypeptide according to claim 1 wherein said polypeptide comprises amino acid residues 20 through 321 of SEQ ID NO : 2.
4. An isolated polypeptide according to claim 1, further comprising a heterologous affinity tag or binding domain .
5. An isolated polypeptide according to claim 4, wherein said affinity tag is a Glu-Glu tag.
6. An isolated polypeptide having the amino acid sequence of SEQ ID NO: 2.
7. An isolated polypeptide comprising the amino acid sequence of amino acid residue 1 to amino acid residue 19 of SEQ ID NO: 2.
8. An isolated polypeptide consisting of a semaphorin domain, a transmembrane domain and a cytoplasmic domain, wherein said semaphorin domain comprises the amino acid sequence of SEQ ID NO : 2.
9. A fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion comprising a polypeptide wherein said polypeptide comprises a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO : 2 from Phe, residue 20, through Arg, residue 321; wherein said polypeptide is a semaphorin; and said second portion comprising another polypeptide.
10. A fusion protein according to claim 9, wherein said second polypeptide is a transmembrane domain of a semaphorin protein.
11. A fusion protein according to claim 10, wherein said second polypeptide further comprises a cytoplasmic domain of a semaphorin protein.
12. A fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1 to 19 of SEQ ID NO : 2 , wherein said secretory signal sequence is operably linked to an additional polypeptide.
13. An isolated polynucleotide selected from the group consisting of : a) a polynucleotide encoding a polypeptide comprising a sequence of amino acids that is 96% identical to the amino acid residues as shown in SEQ ID NO: 2 from Phe, residue 20, through Arg, residue 321; b) degenerate polynucleotide sequences of a) ; and c) a polynucleotide sequence complementary to a) or b); wherein said polynucleotide encodes a polypeptide which is a semaphorin.
14. An isolated polynucleotide according to claim 13 wherein said polypeptide comprises residues 20 through 321 of SEQ ID NO: 2.
15. An isolated polynucleotide according to claim 13 wherein said protein comprises residues 1 through 321 of SEQ ID NO: 2.
16. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator.
17. /An expression vector according to claim 16 further comprising a secretory signal sequence operably linked to said DNA segment .
18. An expression vector according to claim 16 further comprising an affinity tag operably linked to said DNA segment .
19. A cultured cell containing an expression vector according to claim 16 wherein said cell expresses said' DNA segment .
20. A cultured cell according to claim 19 wherein the expression vector further comprises a secretory signal sequence operably linked to said DNA segment and the cell secretes said polypeptide.
21. A cultured cell according to claim 19 wherein the expression vector further comprises an affinity tag operably linked to said DNA segment.
22. A method of making a semaphorin protein comprising:
(a) culturing a host cell containing an expression vector according to claim 16; and
(b) recovering said protein encoded by said DNA segment .
23. A method according to claim 22 wherein the expression vector further comprises a secretory signal sequence operably linked to said DNA segment, the cell secretes the protein into a culture medium, and the protein is recovered from the medium.
24. A method according to claim 22 wherein the expression vector further comprises an affinity tag operably linked to said DNA segment.
25. An antibody or antibody fragment that specifically binds to a polypeptide according to claim 1.
26. An antibody according to claim 25, wherein said antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.
27. An antibody fragment according to claim 25, wherein said antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.
28. An anti-idiotype antibody that specifically binds to said antibody of claim 25.
29. A binding protein that specifically binds to a polypeptide according to claim 1.
30. A pharmaceutical composition comprising a polypeptide according to Claim 1, in combination with a pharmaceutically acceptable vehicle.
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US6635742B1 (en) 2000-01-25 2003-10-21 Nuvelo, Inc. Antibodies specific for semaphorin-like polypeptides

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