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WO2006101000A1 - Anticorps de sulfate d’anti-dermatan et oligosaccharide sulfate fonctionnel - Google Patents

Anticorps de sulfate d’anti-dermatan et oligosaccharide sulfate fonctionnel Download PDF

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Publication number
WO2006101000A1
WO2006101000A1 PCT/JP2006/305229 JP2006305229W WO2006101000A1 WO 2006101000 A1 WO2006101000 A1 WO 2006101000A1 JP 2006305229 W JP2006305229 W JP 2006305229W WO 2006101000 A1 WO2006101000 A1 WO 2006101000A1
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Prior art keywords
antibody
dermatan sulfate
functional
derived
brain
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PCT/JP2006/305229
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English (en)
Japanese (ja)
Inventor
Kazuyuki Sugahara
Xingfeng Bao
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Japan Science And Technology Agency
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Priority to JP2007509226A priority Critical patent/JP4884373B2/ja
Publication of WO2006101000A1 publication Critical patent/WO2006101000A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof

Definitions

  • the present invention relates to an anti-dermatan sulfate antibody that specifically recognizes and detects dermatan sulfate having a specific structure, and a function that is recognized by the antibody and that is involved in regulation of activities such as growth factors and neurotrophic factors.
  • This relates to sulfated oligosaccharides.
  • the present invention provides a technique useful particularly in the research and analysis of the structure and function of sugar chains and in the field of application to medicine and the like.
  • chondroitin sulfate (CS) and dermatan sulfate (DS) are a type of glycosaminodarlican (GAG), which is proteoglycan (PG) covalently bound to the core protein.
  • GAG glycosaminodarlican
  • PG proteoglycan
  • the skeletal structures of CS and DS are [-GlcUA-GalNAc-], [-IdoUA-GalNAc-] (GlcUA, GalNAc, and IdoUA represent glucuronic acid, N-acetylethylgalatatosamine, and iduronic acid, respectively. ) Disaccharide repeating structure power. In addition, it has these structures in various proportions. There is also an hybrid sugar chain (Non-patent Document 9). Among these disaccharide units, GlcUA / IdoUA 2-position and GalNAc 4-position and 6-position are sulfated modified in various combinations. With these modifications, CS, DS, and CS / DS hybrid sugar chains have a characteristic sulfate pattern and a vast diversity of structures.
  • Non-Patent Document 10-12 the abundance ratio of GlcUA / IdoUA and sulfated pattern of CS / DS in the brain changed with ontogeny (Non-Patent Document 10-12), and differed in neurite production. It has been shown that the sub-population of the sugar chain of the structure plays a different role! (Non-patent Documents 13-15). Furthermore, polysulfated CS / DS chains derived from various marine animals have mouse neurite outgrowth promoting activity of mouse hippocampus-euron in vitro (Non-patent Documents 16-19).
  • CS / DS chain of the brain has a small but significant polysaccharide sulfated disaccharide structure (Non-Patent Documents 11, 14, and 20).
  • CS / DS (E-CS / DS) chains from porcine fetal brain contain 1.7% D-unit [GlcUA (2S) -GalNAc (6S)] or iD-unit DdoUA (2S) -GalNAc (6S) ]
  • E-unit [GlcUA-GalNAc (4S, 6S)] (2S, 4S, 6S represent 2-0-sulfuric acid, 4-0-sulfuric acid, 6-0-sulfuric acid, respectively) (Non-patent document 11).
  • Non-Patent Documents 11-15 Furthermore, it has been reported that the CS chain of amyloid precursor protein abican derived from cultured glioma cells has 14.3% of E-units (Non-patent Document 21). This suggests that PG in the brain may retain clusters of polysulfate and CS disaccharide structures! / However, the existence of a polysulfate ⁇ DS structure in the mammalian brain has not been studied to date.
  • DS chain (An-DS) derived from ascidian nigra is composed of iD-disaccharide unit [IdoUA (2S) -GalN Ac (6S)] (; ⁇ 80%) IC-disaccharide unit [IdoUA-GalNAc (6S)] ( ⁇ 20%) (Non-patent Document 22).
  • the structure containing the iD-unit has not been found in rare mammalian tissues.
  • An-DS has the ability to bind to hepatocyte growth factor (HGF) (Non-patent Document 23) and exhibits neurite outgrowth promoting activity of hippocampal neurons in vitro (Non-patent Document 18).
  • HGF hepatocyte growth factor
  • Non-patent literature 1 The Biocnemistry of Glycoproteins and Proteoglycans (Lennarz, W. J.
  • Non-Patent Document 2 Annu. Rev. Biochem. 47, 385-417 (1991)
  • Non-Patent Document 3 Trends Glycosci. Glycotechnol. 12, 321-349 (2000)
  • Non-Patent Document 4 Persp. Dev. Neurol. 3, 319-330 (1996)
  • Non-Patent Document 5 Physiol. Rev. 80, 1267-1290 (2000)
  • Non-Patent Document 6 Arch. Biochem. Biophys. 374, 24-34 (2000)
  • Non-Patent Document 7 Curr. Opin. Struct. Biol. 13, 612-620 (2003)
  • Non-Patent Document 8 Glycoconj. J. 21, 329-341 (2004)
  • Non-Patent Document 9 IUBMB Life 54, 177-180 (2002)
  • Non-Patent Document 10 J. Biol. Chem. 272, 31377-31381 (1997)
  • Non-Patent Document 11 J. Biol. Chem. 279, 9765-9776 (2004)
  • Non-Patent Document 12 J. Biol. Chem. 278, 35805-35811 (2003)
  • Non-Patent Document 13 J. Cell Biol. 126, 783-799 (1994)
  • Non-Patent Document 14 J. Biol. Chem. 273, 28444-28453 (1998)
  • Non-Patent Document 15 J. Biol. Chem. 280, 9180-9191 (2005)
  • Non-Patent Document 16 J. Biol. Chem. 273, 3296-3307 (1998)
  • Non-Patent Document 17 Neurosci. Lett. 269, 125-128 (1999)
  • Non-Patent Document 18 J. Biol. Chem. 278, 43744-43754 (2003)
  • Non-Patent Document 19 J. Biol. Chem. 280, 4058-4069 (2005)
  • Non-Patent Document 20 J. Biol. Chem. 275, 37407-37413 (2000)
  • Non-Patent Document 21 J. Biol. Chem. 276, 37155-37160 (2001)
  • Non-Patent Document 22 J. Biol. Chem. 270, 31027-31036 (1995)
  • Non-Patent Document 23 Biochem. Soc. Trans. 31, 352-353 (2003)
  • the first object of the present invention is to provide an anti-dermatan sulfate antibody that specifically recognizes and detects dermatan sulfate useful as a research reagent.
  • Several antibodies against chondroitin sulfate are commercially available, but antibodies that show specificity for dermatan sulfate have not been known so far.
  • the first object of the present invention is to provide an antibody that specifically detects dermatan sulfate containing an iD-unit, which is considered to play an important role in the development of the nervous system.
  • a second object of the present invention is to provide a dermatan sulfate-derived functional saccharide oligosaccharide containing such an iD-unit.
  • oligosaccharides that bind to Henoline Kofactor II and have blood coagulation inhibitory activity
  • sulfated oligosaccharides with a clear structure derived from dermatan sulfate have not been known.
  • an object of the present invention is to provide functional sulfated oligosaccharides involved in the regulation of growth factor and neurotrophic factor activities.
  • An-DS which is a dermatan sulfate derived from a marine animal sea squirt, is not only hepatocyte growth factor (HGF) but also fibroblast growth factor-2 (FGF-2), It has been shown to bind to various growth factors such as prioro fin (PTN), mitocaine (MK) and heparin-binding epidermal growth factor (HB-EGF). Since An-DS also exhibits hippocampal-euron neurite outgrowth-promoting activity, it is possible that the DS structure of An-DS may play an important physiological function during the development of the mammalian brain. It was.
  • mAb 2A12 against squirt-derived An-DS was prepared and used for immune tissue.
  • Various analyzes such as chemical analysis described below were performed.
  • the epitope recognized by the antibody 2A12 was specifically detected in the hippocampus and cerebellum of the fetal mouse brain 7 days after birth and disappeared in the cerebellum of the adult mouse. It has been clarified that it inhibits neurite outgrowth of Euron.
  • the present invention provides an antibody that specifically recognizes or detects dermatan sulfate.
  • antibodies that specifically recognize or detect dermatan sulfate containing iD-units are functional dermatan containing iD-units that are expressed in the mammalian brain (particularly the neonatal brain until about 10 days after birth). It is useful for the detection of sulphate sulfate.
  • iD-put is [IdoUA (2S) ⁇ 1-3GalNAc (6S)], that is, [iduronic acid (2-sulfuric acid) ⁇ 1-3N-acetylgalatatosamine (6-sulfuric acid)].
  • a dermatan sulfate containing a disaccharide structure and containing an iD-unit means a dermatan sulfate having one or more such disaccharide structures in a sugar chain.
  • the DS structure containing the iD-unit was also expressed in the hippocampus-euron derived from the mouse fetal brain, and the 2A12 antibody of the present invention significantly inhibited neurite outgrowth of cultured hippocampus-euron. .
  • the epitope recognized by antibody 2A12 is combined with the results specifically found in the hippocampus and cerebellum of the fetal mouse brain at 7 days after birth, and these results show the DS structure containing iD-units. Has a specific role in the formation and development of the central nervous system.
  • the antibody of the present invention is a particularly useful tool in research on the structure and function of dermatan sulfate containing an iD-unit, which is considered to play an important role in the formation and development of the central nervous system. It will be.
  • the use of the present invention is not limited to this, and has various uses as an antibody that specifically binds to dermatan sulfate.
  • the antibody of the present invention inhibited neurite formation / elongation, it can be used as an inhibitor. Is possible.
  • the antibody of the present invention can also be used for developing diagnostic methods for diseases including cancer.
  • the dermatan sulfate recognized by the antibody of the present invention binds to growth factors and the like, is involved in cell proliferation and sorting, and is considered to be involved in various life activities.
  • the antibody of the present invention can be prepared using dermatan sulfate An-DS derived from ascidian nigra as an antigen, as shown in the Examples below.
  • the An-DS chain has a unique iD-put disaccharide repeat structure.
  • the monoclonal antibody 2A12 is prepared by immunizing mice with this An-DS as an antigen, and specifically recognizes the decasaccharides (10-a and 10-b) described later, which are rich in iD-units, as a minimal structure. It is an antibody.
  • the method for producing the antibody of the present invention is not limited to the above method, and various modifications can be made.
  • an antigen other than An-DS may be used, or a synthesized sugar chain may be used as an antigen in addition to a sugar chain prepared from a natural product.
  • the antibody to be produced may be a polyclonal antibody.
  • the column is Harlow et al. “Antibodies: A laboratory manual” (Cold bpnng Harbor Laboratory, New York (1988)), Iwasaki et al. Kodansha (1 991) "etc. can be mentioned.
  • the antibody means a protein that recognizes and binds to a specific antigen, and its type (for example, IgG, IgM, IgA, IgD, IgE, etc.) and the animal used for production. Regardless of the origin (eg, rabbit, mouse, rat, goat, hidge, camel, etc.). In addition, antibodies can be monovalent or polyvalent.
  • the antibody of the present invention may be labeled with a fluorescent substance, radioactivity, enzyme, or other protein (for example, avidin), or the second antibody that recognizes and binds to the antibody of the present invention.
  • a fluorescent substance for example, radioactivity, enzyme, or other protein (for example, avidin)
  • avidin for example, avidin
  • the target dermatan sulfate may be detected without labeling the antibody of the present invention.
  • the present invention provides a functional sulfated oligosaccharide derived from dermatan sulfate containing an iD-unit.
  • the functional sulfate oligosaccharide of the present invention has an iD-tube in its sugar chain. However, it may have a plurality of continuous or discontinuous iD-unit structures. Further, the length of the sugar chain is not particularly limited, but is preferably 10 sugars or more.
  • the functional sulfate oligosaccharide of the present invention includes an oligosaccharide having the following decasaccharide structure (a) or (b).
  • oligosaccharides 10-a and 10-b described later are fragments of dermatan sulfate An-DS derived from sea squirt (Ascidian nigra) by enzymatic treatment, and further, the antibodies of the present invention It was isolated and purified using the reactivity with 2A12 as an index.
  • these decasaccharides 10-a and 10-b are recognized by the antibody 2A12 of the present invention, and inhibit the binding of the antibody 2A12 and its antigen, An-DS.
  • these decasaccharides 10-a and 10-b interact with various growth factors' neurotrophic factors, as shown in Examples below, and these growth factors and neurotrophic factors are derived from porcine fetal brain.
  • the growth factors and neurotrophic factors used in the examples are fibroblast growth factor 2 (FGF2), pleiomouth fin (PTN), mitodocaine (MK), glial cell-derived neurotrophic factor (GDNF), and brain origin. It was a neurotrophic factor (BDNF), and V-deviation was also derived from recombinant humans.
  • FGF2 fibroblast growth factor 2
  • PTN pleiomouth fin
  • MK mitodocaine
  • GDNF glial cell-derived neurotrophic factor
  • BDNF neurotrophic factor
  • V-deviation was also derived from recombinant humans.
  • the sulfated oligosaccharide power of the present invention is a growth factor such as FGF2, PTN, MK and other functional proteins such as neurotrophic factors such as GDNF and BDNF, or even cytodynamic force-in. It shows that it can be used for functional regulation of, for example, it can be used as an activity-regulating binding agent for functional proteins when mixed with these proteins.
  • the sulfated oligosaccharide of the present invention can be produced by enzymatic treatment of dermatan sulfate An_DS derived from ascidian an nigra
  • the method for producing the sulfated oligosaccharide of the present invention is not limited to this method.
  • a natural product-derived sugar chain other than An-DS may be isolated and purified, or chemically synthesized using a sugar chain synthesis technique, or a chemically synthesized sugar chain or a natural product-derived sugar chain.
  • the sulfated oligosaccharide of the present invention may be produced by chemically sulphating the salt.
  • FIG. 1 is a diagram showing the results of electrophoresis using a cellulose acetate membrane. Chondroitinase ABC (third lane from the right), AC-I (second lane from the right) and B (the rightmost lane) are spotted on the cellulose acetate membrane and subjected to electrophoresis. went. After electrophoresis, GAG was detected by staining with Alcian blue. The control of the interactive An-DS (second lane from the left) shows the electrophoresis behavior of standard products HA, HS, CS-A, CS-B and heparin (each 1 ⁇ g) at the far left. Shown in the lane.
  • FIG. 2 AC are graphs showing the results of examining the substrate specificity of the antibody mAb 2A12 of the present invention.
  • A The reactivity of various GAGs with mAb 2A12 was examined by ELISA with Hr-DS, which is a DS containing a main unit of iB prepared from various standard GAG and ascidian Halo cynthia roretzi. Pyotinylated GAG (2 g each) was immobilized on streptavidin-coated plastic plate wells, reacted with primary antibody mAb 2A12 (diluted 400-fold), then goat anti-mouse bound with alkaline phosphatase. Reaction with Ig (G + M) (5,000-fold dilution).
  • FIG. 3 Diagram showing immunological and histological detection and localization of 2A12 epitopes in mouse brain It is. Sagittal brain sections from P7 (panel A) and adult (panel E) mice were stained with mAb 2A12 as described in Experimental methods. Anti-CS mAb CS-56 was used for comparison (panels B and F). When P7 mouse tissue sections were pretreated with chondroitinase ABC (panel C) or chondroitinase B (panel D), there was little staining for mAb 2A12. The removal of epitopes by these CS-degrading enzymes was also detected in adult mouse brain tissue. The black arrow indicates the site stained with mAb 2A12.
  • FIG. 4 AC are diagrams showing the results of staining of hippocampus-euron with mAb 2A12.
  • E15.5 mouse-derived hippocampus-euron was cultured for 36-48 hours on P-ORN-coated coverslips.
  • Cells were fixed and stained with (A) neuronal markers (anti-MAP2 and anti-NF) or (B) mAb 2A12.
  • (C) shows the superposed staining pattern of both.
  • mAb 2A12 specifically stained cell bodies with neurites and multiple neurites. Scale: 50 m.
  • AD are the results of examining the effect of mAb 2A12 on neurite outgrowth of cultured hippocampus-euron.
  • E16.5 mouse hippocampal cells were seeded on the P-ORN layer, and 2 hours later, mAb 2A12 was added to the culture medium at various concentrations. After 60 hours of culture, the cells were fixed and stained with anti-MAP2 and anti-NF.
  • A 100 ⁇ g / ml mAb 2A12! /
  • Representative images of cell morphology in the presence (B) were shown.
  • C and D The effect of 2A12 on neurite outgrowth was measured as the ratio of neurite-producing cells (C) and total neurite length per cell (D).
  • Mouse IgM (100 g / ml) was used as a control. Three experiments were performed, and the mean value standard error of the obtained values was shown. *: 0.01 ⁇ p ⁇ 0.05, **: 0.001 ⁇ p ⁇ 0.01, showing a significant difference from the value obtained from the experiment without exogenous antibody. Scale: 50 m.
  • FIG. 6 A—D are the results of examining the minimum An-DS size recognized by mAb 2A12.
  • B Using an equal amount (0.5 g) of each fraction obtained by gel filtration, the inhibitory activity against the reaction between mAb 2A12 and immobilized An-DS was determined by the ELISA method.
  • the decasaccharide fraction was the smallest size that showed significant inhibitory activity.
  • C Anion exchange HPLC was performed on the decasaccharide fraction according to the method described in the experimental method to obtain two major decasaccharide fractions 10-a and 10-b. *: Baseline shift.
  • D Two kinds of purified decasaccharides were subjected to an inhibition experiment by ELISA as described above. 10-a and 10-b showed similar inhibitory activity. B and D values are expressed as mean standard error obtained from two independent experiments.
  • FIG. 7 ⁇ ⁇ ⁇ are graphs showing the results of enzymatic chemical analysis of An-DS decasaccharide 10-a.
  • Decasaccharide 10-a was fluorescently labeled with 2AB, and treated with chondroitinase ABC and ⁇ -hexuronic acid-2-sulfatase.
  • A Co-chromatography of 2AB labeled standard and 2AB labeled 10-a chondroitinase ABC degradation product (peak b).
  • FIG. 8 The results of investigating the effects of An-DS decasaccharides 10-a and 10-b recognized by mAb 2A12 on the binding of immobilized E-CS / DS to growth factors and neurotrophic factors. It is a graph to show. Inhibitory effects of An-DS 10-a and 10-b on the binding of growth factors (FGF2, PTN and MK) and neurotrophic factors (GDNF and BDNF) to E-CS / DS chain using BIAcore system was analyzed. Each growth factor or neurotrophic factor and decasaccharide 10-a or 10-b (1.5 g / ml) were mixed and injected onto the surface of an E-CS / DS-immobilized sensor chip.
  • FGF2 growth factors
  • PTN and MK neurotrophic factors
  • GDNF and BDNF neurotrophic factors
  • FIG. 9 is a photograph (magnification x200) showing the expression of 2A12 epitopes in hair follicles. The left side shows the results of staining mouse follicles with antibody 2A12, and the right side shows the results of staining cell nuclei with DAPI.
  • FIG. 10 is a photograph (magnification x400) showing the expression of 2A12 epitopes in hair follicles. The left side shows the results of staining mouse follicles with antibody 2A12, and the right side shows the results of staining cell nuclei with DAPI.
  • the plate was washed with phosphate buffered saline (PBS), and the operation described below was performed. Substrate para-tro-lluic acid was added and the absorbance was recorded 2 hours later.
  • PBS phosphate buffered saline
  • [0032] Antibody production and selection Using a DS-peptide obtained from A. nigra, BALB / cA Jol mice were immunized to produce monoclonal antibodies. Every two weeks, 200 g of cocoons were injected subcutaneously in the back of the mice. After three injections, the serum was screened by ELISA based on the reactivity to highly purified An-DS. After culturing the fused hyperpridoma cells, screening was carried out by ELISA using the culture supernatant using piotinylated An-DS. Four positive clones (2A12, 3G11, 4B5 and 5F4) were selected.
  • An amount of 2A12 was purified using a mouse IgM purification kit.
  • the purified antibody was quantified by BCA protein assay and used for the inhibition of neurite outgrowth of cultured fetal hippocampus-euron.
  • mice P7 and adult ddY mice were anesthetized, brains were removed, immediately frozen on dry ice, and stored at -80 ° C. Within 2-4 days after excision, frozen brain sections were prepared at a thickness of 12 m, dehydrated by heating at 60 ° C for 1 hour, stored at 80 ° C, and used. For immunohistochemical staining of antibodies, brain sections were fixed with acetone-methanol (1: 1) and rehydrated with distilled water. The sections were treated with the following solutions.
  • Hippocampal cells were cultured using 15.5 day (E 15.5) or 16.5 day old (E16.5) fetal mouse brain according to the method described in J. Cell Biol. 126, 783-799 (1994). Hippocampus was obtained by microdissection and trypsinized for several minutes to separate cells. The separated cells were suspended in Eagle's minimal essential medium (EMEM) containing N2 sub-lement, seeded on P-ORN-coated force burst, and cultured at 37 ° C under humidified 5% CO.
  • EMEM Eagle's minimal essential medium
  • E16.5 mouse brain hippocampus-euron neurite outgrowth was measured using a method slightly modified from the method described in J. Cell Biol. 126, 783-799 (1994) and the like.
  • the isolated hippocampal cells were seeded at 10,000, 25,000 or 50,000 cells / cm 2 on a cover glass precoated with P-ORN and cultured for 2 hours. After preculture, purified mAb 2A12 (10-200 g / ml) was added to the culture. Mouse IgM was added as a control.
  • [0039] Matrix-assisted laser desorption ionic time-of-flight mass spectrometry (MALDI-TOF MS) Drying oligosaccharides (purified decasaccharide 5-10 pmol and unpurified oligosaccharide mixture 30 pmol) Then mix with 1-2 ⁇ 1 (Arg-Gly) (10 pmol) and add 1 ⁇ 1 gentisic acid solution (1
  • a 4,5 HexUA a 1-3GalNAc ( ⁇ ⁇ unit) is produced from GlcUA j8 1-3GalNAc (O unit).
  • HexUA a l-3GalNAc (6S) (AC unit) is GlcUA j8 1-3GalNAc (6S) (C unit) Alternatively, it is produced from IdoUA a 1-3GalNAc (6S) (iC unit).
  • ⁇ 4,5 HexUA a 1-3GalNAc (4S) ( ⁇ A unit) is produced from GlcUA j8 1-3GalNAc (4S) (A unit) or IdoUA a 1-3GalNAc (4S) (iA unit).
  • a 4,5 HexUA (2S) a l-3GalNAc (6S) (AD unit) is GlcUA (2S) j8 1— 3GalNAc (6S) (D unit) or IdoUA (2S) a 1— 3GalNAc (6S) (iD unit) ).
  • a 4,5 HexUA (2S) a 1— 3GalNAc (4S) ( ⁇ ⁇ unit) is GlcUA (2S) ⁇ 1— 3GalNAc (4S) (B unit) or IdoUA (2S) a 1— 3GalNAc (4S) (iB Unit).
  • ⁇ 4,5 HexUA a l-3GalNAc (4S, 6S) ( ⁇ ⁇ unit) is GlcUA j8 1—3GalNAc (4S, 6S) (E unit) or IdoUA a-3GalNAc (4S, 6S) (iE unit) Produced from.
  • a 4,5 HexUA (2S) a 1— 3GalNAc (4S, 6S) ( ⁇ ⁇ unit) is GlcUA (2S) ⁇ 1— 3GalNAc (4S, 6 S) (T unit) or IdoUA (2S) a 1-3GalNAc Produced from (4S, 6S) (iT unit).
  • polysaccharides or oligosaccharides (100-500 pmol as disaccharide) were treated with chondroitinase ABC of 5 mIU for 2 hours at 37 ° C.
  • the decomposition product was converted into 2-AB, and excess 2AB was removed with black mouth form.
  • decasaccharides (An-DS 10 0-a and 10-b) purified from An-DS were mixed with various chondroitinases, chondroitinase ABC (1 mIU), chondroitinase B (1 mlU) or chondroitin
  • Each enzyme was treated with a mixture of AC-1 (0.5 mlU) and AC-II (0.5 mlU) at 37 ° C for 1 hour, and the resolution of each enzyme was examined.
  • Half of the degradation product was subjected to anion exchange HPLC using a PA-03 column, and the other half was further treated with ⁇ -hexuronic acid 2-sulfatase (4 / z IU) for a total volume of 30 ⁇ m.
  • BIAcore system shows the inhibitory activity of solid-phased E-CS / DS on growth factor and neurotrophic factor binding by An-DS 10-a and 10-b decasaccharides. It investigated using.
  • Various growth factors or neurotrophic Factor 100 ng / per time was mixed with polysaccharide or oligosaccharide (1.5 g / ml) and injected onto the surface of the sensor chip. After performing the binding reaction for 2 minutes (bonding phase), the sensor was washed for at least 2 minutes (dissociation phase). Response curves were recorded, and the inhibitory effect was expressed as a relative ratio between the response when there was no sugar chain and the response when there was no sugar chain.
  • AD disaccharide (76.6 mol%) was the main disaccharide unit, and ⁇ ⁇ , AC, and ⁇ units were trace amounts of disaccharide units. It was.
  • DS Disaccharide composition of DS from ascidian nigra DS chain (An-DS) was isolated from adult Ascidian nigra, and was completely degraded to disaccharide with chondroitinase ABC. The obtained unsaturated disaccharides were identified and quantified by anion exchange HPLC as shown in the experimental method. The value indicates the average value obtained from two independent experimental forces.
  • Monoclonal antibodies against An-DS were prepared in mice and 4 types of antibodies were selected.
  • 2A12 showed a particularly high specificity for An-DS (described later). Therefore, in this example, the characteristics of mAb 2A12 were examined.
  • Piotinylated GAGs were immobilized on streptavidin-coated plates, and the reactivity of 2A12 against various GAG types was analyzed using ELISA ( Figure 2A).
  • 2A12 reacts specifically with An-DS, CS-A, CS-B, CS-C, CS-D, CS-E, CS-H, Hr-DS (iB-unit is the main disaccharide unit.
  • other GAGs such as heparin did not react.
  • 2A12 has An-DS containing iD [Ido UA (2S) -GalNAc (6S)], and has many D-units [GlcUA (2S) -GalNAc (6S)] (20-21%) CS-D This result shows that it is distinguished from other polysulfated CS and DS. This specificity was also confirmed by a significant inhibition of soluble An-DS against the binding of immobilized An-DS to 2A12 in an inhibition experiment by ELISA. In order to investigate whether 2A12 recognizes the intact An-DS disaccharide repeat region and recognizes the binding region between the oligosaccharide and the peptide, various An-DSs are used.
  • ELISA was performed using a Nunc Maxiso rp plastic plate that was treated with the appropriate chondroitinase and bound more strongly than the disaccharide repeat region. As shown in FIG. 2B, the reactivity of 2A12 to An-DS treated with chondroitinase ABC was completely lost. This indicates that 2A1 2 recognizes the structure in the disaccharide repeat region that is not in the An-DS binding region! Treatment of An-DS with chondroitinase AC-1, AC-II or B did not change the reactivity to 2A12. This is in support of the above results that An-DS is not cleaved by these CS-degrading enzymes ( Figure 1). mAb 2A12 was determined to be IgM because it did not bind to anti-mouse IgG or IgA but only to anti-mouse IgM in ELISA experiments.
  • 2A12 in the hippocampus, fetal mouse brain force was also isolated from hippocampus-eurone and double-stained using antibodies against neuronal markers (anti-MAP2 and anti-NF) and 2A12. As shown in FIG. 4, the cultured hippocampus-euron was positive for 2A12. 2A12 stained the cell body and most neurites. The expression of 2A12 epitope was particularly high in the neurite sprouting region than in other regions of the cell body.
  • Isolate hippocampus-euron on a substrate containing E-CS / DS subpopulation and P-ORN In culturing for 24 hours at a density of 10,000 cells / cm 2 , multiple neurites are formed.
  • the substrate without the E-CS / DS chain was Above, higher density (2.5-5 times) and longer culture periods (2-3 times) were required (Figure 5A). Therefore, 2A12 was added to the hippocampal-euron culture to examine whether 2A12 epitopes present on the hippocampal neuronal cell surface are involved in neurite formation and proliferation.
  • AC and AD represent A HexUA-GalNAc (6S) and ⁇ HexUA (2S) -GalNAc (6S), respectively.
  • iC and iD represent IdoUA-GalNAc (6S) and IdoUA (2S) -GalNAc (6S), respectively.
  • Both An-DS 10-a and 10-b were decomposed by chondroitinase ABC.
  • the chondroitinase AC-I, AC-II or B did not decompose.
  • AD was digested with chondroitinase ABC, only AD was detected.
  • An-DS 10-a In order to confirm the presence of the iC unit in the sequence, the reducing end of the oligosaccharide is labeled with 2AB, the labeled oligosaccharide is treated with chondroitinase ABC, and then ⁇ -hexuronic acid-2- Treated with sulfatase. Chondroitinase ABC cleaves 2AB-modified oligosaccharides to produce 2AB-modified tetrasaccharides regardless of the sulfated structure of the original oligosaccharide.
  • ⁇ Hexuronic acid-2-sulfatase is It is known to remove the sulfate group from the C-2 position of ⁇ HexUA on the non-reducing end side.
  • treatment of 2AB-modified An-DS 10-a chondroitinase ABC with ⁇ -hexuronic acid-2-sulfatase shifted the elution position of 2AB-unsaturated tetrasaccharides forward. . This indicates that the non-reducing terminal force sulfate group of the tetrasaccharide has been removed. This shift was also observed in the 2AB unsaturated tetrasaccharides derived from An-DS 10-b.
  • ⁇ Hexuronic acid-2-sulfatase is not 2AB-modified such as ⁇ AD-2AB [ ⁇ HexUA-GalNAc (4S) -GlcUA (2S) -GalNAc (6S) -2AB] used as a negative control. It did not affect the sulfate group at the C-2 position of ⁇ HexUA at the reducing end of the saturated tetrasaccharide.
  • An-DS 10-a consists of four consecutive iD units and one iC unit that appears to be present at the reducing end.
  • E-CS / DS chains and PTN in vitro show the hippocampal-euron neurite outgrowth promoting activity (J. Biol. Chem. 280 , 9180-9191 (2005)).
  • the polysulfated disaccharide unit of the E-CS / DS chain and the disaccharide unit containing IdoUA bind to PTN and are essential for neurite outgrowth.
  • An-DS 10-a and 10 for the interaction between immobilized E-CS / DS and growth factors and neurotrophic factors were investigated.
  • Inhibition by -b was analyzed by BIAcore system. As shown in Figure 8, in addition to growth factors, neurotrophic factors The offspring GDNF (transforming growth factor-j8 superfamily) and BDNF (
  • the K values for the binding of BDNF and GDNF to E-CS / DS are 102.1 and 28.9 n, respectively.
  • the affinity of E-CS / DS which is comparable to the binding affinity of GDNF and HS, is similar to that of HS, where the CS / DS chain acts as a coreceptor for GDNF in the brain. It seems to suggest that it is functioning.
  • an antibody against DS derived from ascidian nigra was prepared, and the characteristics of the novel IgM antibody 2A12 that recognizes the unique polysulfate-DS epitope structure present in the mouse brain were revealed.
  • the ability of the 2A12 antibody to react specifically with An-DS was shown by ELISA that it does not react with other typical CS / DS variants or heparin ( Figure 2A).
  • This consists of 2-0-sulfuric acid whose essential residues constitute the structure of ⁇ 4IdoUA (2S) al ⁇ 3GalNAc (6S) ⁇ 1 ⁇ and ⁇ 3GalNAc (6S) ⁇ l ⁇ 4IdoUA (2S) a 1 ⁇ A-IdoUA and 6-0-sulfated j8-GalNAc.
  • An-DS decasaccharide is the smallest oligosaccharide size required to inhibit the binding of mAb 2A12 to An-DS, and is longer! The oligosaccharide inhibits more strongly (Figure 6B). This indicates that the size of the sugar chain other than the iD disaccharide unit alone is also important for the recognition of this antibody.
  • Mouse brain, pig brain, DSD-1-PG / CS-PG derived from neuronal cells such as phosphacan and versican, D or iD is found in a small amount (typically 1.5-5%) but a significant amount is found. Yes.
  • mAb 2A12 reacts with E-CS / DS (Fig. 2C) and recognizes specific regions of the mouse brain (Fig. 3) and hippocampal neurons (Fig. 4). This indicates that the DS structure containing iD is expressed in the mammalian brain.
  • 2A12 staining was significantly reduced in mouse brain sections pretreated with chondroitinase ABC or B ( Figures 3C and 3D).
  • 2A12 epitope is thought to contain a series of iD units such as An-DS 10a and 10b decasaccharide isolated from An-DS, but An-DS and its decasaccharide are chondro Considering the result ( Figure 1 and Table 2) that it is not cleaved by itinase B, the minimum of 2A12
  • an epitope is a decasaccharide in which a core iD unit and other disaccharide structures are mixed, or a decasaccharide in which a plurality of iD units are dispersed. Isolating structures that respond to brain-derived CS / DS chain strength 2A12 will help define the structure of this rare epitope.
  • Immunohistochemical staining with mAb 2A12 revealed a temporally and spatially regulated expression of 2A12 epitopes specifically in the mouse brain. While there was a report that the expression of CS-56 epitopes was strong and widespread (described above), the expression of 2A12 epitopes was confined to the cerebellum and hippocampus early in life. The difference between these two distinct staining patterns is probably due to the different specificities of these antibodies.
  • CS-56 epitope is an octasaccharide containing A- D [GlcUA-GalNAc (4S) -GlcUA (2S) -GalANc (6S)] tetrasaccharide, whereas 2A12 epitope gives iD as described above. This is because the structure is included. It is noteworthy that CS-56 strongly stains the boundary between the CA1 region and the dentate gyrus while the 2A12 epitopes do not exist in either the developing mouse brain or the adult mouse brain. is there.
  • the expression of CS-56 epitope also inhibits CA1 region force from axon extension to the dentate gyrus, so 2A12 epitope plays a different role in CS guidance than the CS-56 epitope. It is thought that. Furthermore, the temporal-spatial expression of 2A12 epitopes present in the developing cerebellum and hippocampus appears to play an important role in the development of these two regions, particularly in the central nervous system. This supports the result that the cultured fetal hippocampus-euron is strongly stained with 2A12 and that neurite outgrowth of the hippocampus-euron cultured in the culture medium supplemented with this antibody is inhibited.
  • the biosynthesis of the iD unit would occur in the order of GalNAc 6-0-sulfation, D-GlcUA isomerization to L-IdoUA, and IdoUA 2-0-sulfation. This is because GalNAc's 6-0-sulfate reaction occurs before the isomeric and 2-0-sulfate reactions, and the 2-0-sulfotransferase transfers sulfate groups to both GlcUA and IdoUA. Because it does. Regulation of iD unit expression by 6-0-sulfotransferase, 2-0-sulfotransferase and C-5 epimerase plays an important role in the development of the hippocampus and cerebellum. It must be.
  • CS / DS chains like HS, have various growth factors such as PTN, MK, HGF and FGF7. It has been reported to be involved in the regulation of offspring signals.
  • the present inventor recently requires that the CS / DS chain cooperates with PTN to promote neurite outgrowth, and that a unit containing polysulfinyl disaccharide units and IdoUA is required for the interaction between PTN and CS / DS chain. It was made clear that.
  • neurotrophic factors such as GDNF and BDNF have been reported to be involved in the suppression of myelination and neurodegeneration in Parkinson's disease, and HS is required for GDNF signaling.
  • CS-PG The functions of CS-PG and their sugar chains in brain development have not yet been elucidated. So far, polysulfated CS, DS and their hybrid sugar chains have been shown to promote neurite outgrowth. In contrast, brain CS chains have traditionally been considered to be endogenous inhibitors in various -Euron axon outgrowths and projections. That is, CS chain removal by CS-degrading enzyme is axon blockage of nigrostriatal tract and spinal cord injury after spinal cord injury Enables playback. The fact that this seemingly contradictory phenomenon of neurite outgrowth promoting activity is observed together is the structural change of the CS / DS chain at the developmental stage and other factors that are regulated by CS / DS chains with different structures.
  • the antibodies and sulfated oligosaccharides of the present invention are useful tools for analysis of dynamic structural and functional changes accompanying the development of the central nervous system.
  • the antibodies and sulfated oligosaccharides of the present invention may be useful in investigating and analyzing what PG power has 2A12 epitopes containing this unusual iD unit, and in the development of the central nervous system. It will provide valuable insights into further understanding the functions of CS / DS-PG.
  • the antibody of the present invention can be used as a useful reagent applicable to various studies and diagnoses.
  • the sulfated oligosaccharide of the present invention interacts with various growth factors and neurotrophic factors, and also has the possibility of interacting with other functional proteins such as cyto force-in, it has various physiological activities. It can be used as a useful bioactive agent that exerts a regulatory function. For example, in order to regulate the function of these functional proteins, they can be mixed with these proteins and used as an activity-regulating binding agent for functional proteins.

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Abstract

L’invention porte sur un nouvel anticorps monoclonal 2A12 contre le sulfate de dermatan An-DS dérivé de l’ascidie (Ascidian nigra). Un déterminant antigénique de sulfate de dermatan ayant une unité d’identification reconnue par l’anticorps 2A12 est spécifiquement détecté dans l’hippocampe et le cervelet dans le cerveau d’une souris nouveau née de sept jours, mais disparaît du cervelet dans le cerveau d’une souris adulte. De plus, deux types d’oligosaccharides sulfatés reconnus par l’anticorps 2A12 sont isolés. Les deux oligosaccharides sulfatés peuvent interagir avec divers facteurs de croissance et facteurs neurotrophiques pour réguler leurs fonctions, et de ce fait sont des oligosaccharides sulfatés fonctionnels utiles.
PCT/JP2006/305229 2005-03-18 2006-03-16 Anticorps de sulfate d’anti-dermatan et oligosaccharide sulfate fonctionnel WO2006101000A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007256217A (ja) * 2006-03-25 2007-10-04 Japan Science & Technology Agency 糖鎖マーカー認識プローブ、それを用いた神経突起伸長阻害剤、ニューロン染色剤、腫瘍細胞染色剤、免疫組織染色剤、薬学的組成物および医薬
EP2203477B1 (fr) * 2007-10-08 2014-05-28 Sanofi Pasteur Anticorps monoclonaux specifiques de l'hemagglutinine du virus grippal
WO2016159296A1 (fr) * 2015-03-31 2016-10-06 生化学工業株式会社 Procédé de sulfatation de glycosaminoglycane

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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JP5344785B2 (ja) * 2005-07-06 2013-11-20 独立行政法人科学技術振興機構 プレイオトロフィン結合性および非結合性のコンドロイチン硫酸／デルマタン硫酸オリゴ糖

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BAO X. ET AL.: "Heparin-binding growth factor, pleiotrophin, mediates neuritogenic activity of embryonic pig brain-derived chondroitin sulfate/dermatan sulfate hybrid chains", J. BIOL. CHEM., vol. 280, no. 10, 11 March 2005 (2005-03-11), pages 9180 - 9191, XP003001550 *
COUCHMAN J.R. ET AL.: "Proteoglycans and associated proteins of the mammalian hair follicle", J. INVEST. DERMATOL., vol. 104, no. 5, SUPPL., 1995, pages 40S - 41S, XP003001549 *
HIKINO M. ET AL.: "Oversulfated dermatan sulfate exhibits neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons: implications of dermatan sulfate in neuritogenesis in the brain", J. BIOL. CHEM., vol. 278, no. 44, 2003, pages 53744 - 43754, XP002993445 *
HIKINO M. ET AL.: "Taryusanka Dermatan Ryusan wa Shinkei Tokki Shincho Kassei o Yusuru", DAI 75 KAI CSJ: THE CHEMICAL SOCIETY OF JAPAN TAIKAI HAPPYO SHOROKUSHU, 2002, pages 734 (2P-124), XP002993446 *
NISHIMURA H. ET AL.: "Buta Taishino Yurai chondroitin Ryusan / Dermatan Ryusan no Shinkei Tokki Shincho Sokushin Kassei", DAI 123 KAI NIPPON YAKUGAKUKAI KOEN YOSHISHU, 2003, pages 91 (II-337), XP003001551 *
PAVAO M.S. ET AL.: "A unique dermatan sulfate-like glycosaminoglycan from ascidian. Its structure and the effect of its unusual sulfation pattern on anticoagulant activity", J. BIOL. CHEM., vol. 270, no. 52, 1995, pages 31027 - 31036, XP009061833 *
XINGFENG BAO ET AL.: "Neurite outgrowth promoting activity of embryonic pig brain chondroitin/dermatan sulfate (cs/ds) fraction with the IdoUA-containing epitopes", DAI 76 KAI CSJ: THE CHEMICAL SOCIETY OF JAPAN TAIKAI HAPPYO SHOROKUSHU, 2003, pages 908 (3P-047), XP003001552 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007256217A (ja) * 2006-03-25 2007-10-04 Japan Science & Technology Agency 糖鎖マーカー認識プローブ、それを用いた神経突起伸長阻害剤、ニューロン染色剤、腫瘍細胞染色剤、免疫組織染色剤、薬学的組成物および医薬
EP2203477B1 (fr) * 2007-10-08 2014-05-28 Sanofi Pasteur Anticorps monoclonaux specifiques de l'hemagglutinine du virus grippal
WO2016159296A1 (fr) * 2015-03-31 2016-10-06 生化学工業株式会社 Procédé de sulfatation de glycosaminoglycane
JP6063103B1 (ja) * 2015-03-31 2017-01-18 生化学工業株式会社 グリコサミノグリカンの硫酸化方法
JP2017048404A (ja) * 2015-03-31 2017-03-09 生化学工業株式会社 グリコサミノグリカンの硫酸化方法
EP3279220A4 (fr) * 2015-03-31 2018-12-19 Seikagaku Corporation Procédé de sulfatation de glycosaminoglycane
US10259889B2 (en) 2015-03-31 2019-04-16 Seikagaku Corporation Method for sulfating glycosaminoglycan

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