WO2017170896A1

WO2017170896A1 - Mannose-6-phosphate-group-containing glycoprotein production method, and method for detecting intracellular distribution of mannose-6-phosphate-group-containing glycoprotein capable of binding to fluorescent group

Info

Publication number: WO2017170896A1
Application number: PCT/JP2017/013322
Authority: WO
Inventors: 伊藤　孝司; 宗一郎西岡; 松崎　祐二; 健太飯野; 秀樹瀬筒; 小林　功
Original assignee: 国立大学法人徳島大学
Priority date: 2016-03-30
Filing date: 2017-03-30
Publication date: 2017-10-05
Also published as: JP2017184736A; JP6996676B2

Abstract

A mannose-6-phosphate-group (Man6P)-containing glycoprotein production method for producing a Man6P-containing glycoprotein by a transglycosylation reaction between a sugar chain donor that has a structure derived from an N-binding sugar chain having a Man6P bound to a non-reducing end thereof and a sugar chain acceptor represented by general formula (1) in the presence of an Endo-M mutant (a): an Endo-M mutant having an amino acid sequence represented by SEQ ID NO: 1 in which the 175th amino acid residue is replaced by a glutamine or alanine residue. In general formula (1), Y¹ represents an acylamino group containing a glycoprotein-derived structure.

Description

Method for producing mannose-6-phosphate group-containing glycoprotein and method for detecting intracellular distribution of fluorescent group-bound mannose-6-phosphate group-containing glycoprotein

The present disclosure relates to a method for producing a mannose-6-phosphate group-containing glycoprotein and a method for detecting the intracellular distribution of a fluorescent group-bound mannose-6-phosphate group-containing glycoprotein.

It is known that in a deficiency of a glycoprotein gene that functions in a cell, the function of the glycoprotein in the cell becomes defective, causing a serious disease. For example, lysosomal diseases are intractable diseases that develop due to genetic defects such as hydrolases (lysosomal enzymes) present in lysosomes, which are intracellular organelles. Current treatment methods include prepared enzymes (recombinant enzymes). Enzyme replacement therapy is known which is administered by intravenous administration. Enzyme replacement therapy is associated with binding of a non-reducing mannose-6-phosphate group (hereinafter referred to as Man6P) of an N-linked sugar chain of a glycoprotein to Man6P receptors present on the cell surface and lysosomes. It is a therapy that utilizes the phenomenon of uptake into cells by endocytosis, and is a treatment method that exerts a therapeutic effect by giving a lysosomal enzyme having Man6P from outside the cell to cells targeted by a lysosomal disease patient.
Here, when a target glycoprotein is to be introduced into a target cell or organelle with high efficiency, the glycoprotein in which Man6P is added to the non-reducing end of the N-linked sugar chain is used. It is important that enzyme replacement therapy with a high therapeutic effect is possible.

As a method for preparing a glycoprotein having Man6P, a method using a cell into which an individual glycoprotein gene has been introduced has been known (for example, see JP-T-2007-523648).

However, in the method of preparing using cells into which a glycoprotein gene has been introduced, the phosphate group is not necessarily added to the glycoprotein, and there is a problem that the content of Man6P cannot be increased depending on the glycoprotein. Another problem is that many glycoproteins that are not bound to Man6P are mixed into the resulting glycoprotein due to the influence of phosphatases from cells serving as hosts.

On the other hand, as an enzyme-chemical method for converting an N-linked sugar chain of glycoprotein, as described in JP-A-7-59587, endo-β-N-acetylglucosaminidase M derived from hair mold ( End M) reports a method in which a large oligosaccharide chain site on the non-reducing end side of an N-linked sugar chain, which is a sugar chain donor, is transferred to a sugar chain acceptor at one time. In addition, a mutant (glycosynthase) in which the amino acid of endo M is modified can efficiently transfer a sugar chain of a sugar chain donor containing a structure derived from a target N-linked sugar chain to a sugar chain acceptor. , Umekawa et al. [J. Biol. Chem., Vol. 285 (1), 511-521, (2010)].

Therefore, if the glycosynthase can transfer a sugar chain to a sugar chain acceptor using an N-linked sugar chain having Man6P as a sugar chain donor, the desired sugar having a high content of Man6P is consequently obtained. It can be expected that protein will be obtained.

However, since the sugar chain donor used in the above document uses an oxazolined compound that requires strong acidic conditions for its preparation, an oxazolineated sugar chain donor having Man6P is used. In the case of preparing, it has a drawback that it is necessary to oxazoline so as not to eliminate a phosphate group that is weak in acidic conditions. Moreover, since the oxazoline ring is unstable, it is difficult to introduce a phosphate group after oxazolinization.

In addition, in the sugar chain transfer reaction of the oxazolineized sugar chain donor, there is a concern that the sugar chain transfer also occurs on residues other than GlcNAc of the sugar chain acceptor, for example, amino acid residues.

Thus, as a method for easily preparing Man6P, a new production method using glycosynthase and using a sugar chain donor that is not oxazolated has been desired.

The present disclosure has been made in view of the above, and a production method capable of easily producing a Man6P-containing glycoprotein using a glycosyl transfer reaction, and a fluorescent group-bound mannose-6-phosphate group-containing glycoprotein cell It is an object to provide a method for detecting an internal distribution.

Specific means for solving the problems include the following forms.
<1> A sugar chain donor having a structure derived from an N-linked sugar chain in which a mannose-6-phosphate group is bound to a non-reducing end in the presence of the endo M mutant of the following (a) or (b): A mannose-6-phosphate group-containing glycoprotein is produced by producing a mannose-6-phosphate group-containing glycoprotein by a sugar chain transfer reaction between a sugar chain receptor represented by the following general formula (1): Production method:
(A) Endo M mutant having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine (b) Other than the 175th amino acid residue of the amino acid sequence of (a) And having the amino acid sequence modified within 80% or more homology with the amino acid sequence of (a) by deletion, addition or substitution of one or more amino acid residues An endo M mutant having an activity of catalyzing a chain transfer reaction.

(In general formula (1), Y ¹ represents an acylamino group containing a structure derived from a glycoprotein. GlcNAc represents an N-acetylglucosaminyl group.)

<2> The method for producing a mannose-6-phosphate group-containing glycoprotein according to <1>, wherein Y ¹ in the general formula (1) is an acylamino group containing a structure derived from a lysosomal enzyme.

<3> The method for producing a mannose-6-phosphate group-containing glycoprotein according to <1> or <2>, wherein the sugar chain donor is represented by the following general formula (2).

(In General Formula (2), X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a hydrogen atom or a saccharide-derived group, and X ¹ , X ² , X ³ , X ^4, at least one of ^{X 5} and ^{X 6,} the non-reducing end is a group derived from a saccharide having a mannose-6-phosphate group .Z ¹ represents a hydrogen atom or a GlcNAc, ^{Z 1} is GlcNAc In this case, the GlcNAc is bound to Man linked to GlcNAc at β1-4 by β1-4, Y ² represents a monovalent substituent, and GlcNAc represents an N-acetylglucosaminyl group. , Β1-4 represents a β glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β glycoside bond between position 1 of Man and position 4 of GlcNAc, Man represents a mannosyl group, α1-6 represents Man 1st and Man 6th It represents α-glycosidic bond, [alpha] 1-3 represents a α-glycosidic bond between the 1- and 3-position of Man of Man.)

<4> The method for producing a mannose-6-phosphate group-containing glycoprotein according to any one of <1> to <3>, wherein the sugar chain donor is represented by the following general formula (3).

(In the general formula (3), X ⁷ , X ⁸ and X ⁹ each independently represent a hydrogen atom, Man, Manα1-2Man, Man6P, Man6Pα1-2Man or Man6Pα1-6Man. Man represents a mannosyl group. .Man6P represents a mannosyl group phosphoric acid group is bonded to the 6-position. ^{also, X} 7 -6 indicates the ^{X 7} bound to 6-position of ^{Man, X} 8 -3 is attached to the 3-position of Man and showed ^{X ^8,} ^X 9 -2 at least one ^.X 7, ^{X 8} and ^{X 9} showing the ^{X 9} attached to the 2-position of Man is Man6P, either Man6Pα1-2Man and Man6Pα1-6Man GlcNAc represents an N-acetylglucosaminyl group, and α1-6 is an α-glycoside bond between position 1 of Man and position 6 of Man or position 1 of Man6P and M n represents the α-glycoside bond at the 6-position, α1-3 represents the α-glycoside bond between the 1-position of Man and the 3-position of Man, and α1-2 represents the α-glycoside between Man's 1-position and Man's 2-position Represents an α-glycoside bond between position 1 of Man6P and position 2 of Man, β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β position between position 1 of Man and position 4 of GlcNAc Represents a glycosidic bond, Y ³ represents a monovalent substituent.)

<5> Fluorescent group-bound mannose obtained by further introducing a fluorescent group into the mannose-6-phosphate group-containing glycoprotein obtained by the production method according to any one of <1> to <4> A method for detecting intracellular distribution of a fluorescent group-bound mannose-6-phosphate group-containing glycoprotein by applying a -6-phosphate group-containing glycoprotein to cells.

According to the present disclosure, a production method capable of easily producing a Man6P-containing glycoprotein using a glycosyl transfer reaction, and a method for detecting intracellular distribution of a fluorescent group-bound mannose-6-phosphate group-containing glycoprotein. Can be provided.

FIG. 1 is a diagram showing the IDUA activity of an extract obtained from a middle silk gland produced by a transgenic silkworm introduced with a human α-iduronidase (IDUA) gene. FIG. 2 is a diagram showing the results of Western blotting using an anti-IDUA antibody on an extract obtained from a middle silk gland produced by a transgenic silkworm into which an IDUA gene has been introduced. FIG. 3 is a diagram showing the results of SDS-PAGE of a solution obtained by purifying the extract obtained from the middle silk gland of a transgenic silkworm introduced with the IDUA gene by each chromatography operation. FIG. 4 is a diagram showing a ¹ H-NMR spectrum of Compound 12, which is a sugar chain donor in the present disclosure. FIG. 5 is an enlarged view of the ¹ H-NMR spectrum of FIG. FIG. 6 is a diagram showing a mass spectrum of Compound 12 obtained by MALDI-TOF MS measurement. FIG. 7A is a graph showing the results of SDS-PAGE of a sample after sugar chain transfer to a sugar chain acceptor (IDUA after endoenzyme treatment (GlcNAc-IDUA)) of a sugar chain donor (compound 12). It is. Lane 1 is a sample containing IDUA obtained from Chinese hamster (CHO) cells into which IDUA gene has been introduced, Lane 2 is a sample containing IDUA before endo-enzyme treatment, and Lane 3 is a sugar chain receptor (endo). A sample containing IDUA (GlcNAc-IDUA)) after enzyme treatment, and lane 4 is a sample after sugar chain transfer of a sugar chain donor (compound 12) to a sugar chain acceptor (GlcNAc-IDUA). FIG. 7B is a view showing the results of lectin blotting with the same sample as FIG. 7A using a lectin (Dom9-His) that specifically binds to Man6P. FIG. 8A is a graph showing an activity recovery ratio in intracellular IDUA activity after adding M6P-containing IDUA to mucopolysaccharidosis type 1 (MPS-1) patient skin fibroblasts and the like and incubating for 24 hours. FIG. 8B shows 4-methylumbelliferyl (intracellular solution (enzyme source)) of 4-methylumbelliferyl obtained after adding M6P-containing IDUA to mucopolysaccharidosis type 1 (MPS-1) patient skin fibroblasts and the like. FIG. 4 is a graph showing the measurement of β-hexosaminidase (Hex) (control enzyme) activity against 4MU) -N-acetyl-β-D-glucopyranoside (MUG). FIG. 9 is a schematic diagram showing a method for modifying a fluorescent group to a Man6P-containing compound (Man6P-Man5-AFO). FIG. 10 is a diagram showing a MALDI-TOF MS spectrum of a fluorescent group-modified Man6P-containing compound (Man6P-Man5-AFO). FIG. 11 shows mucopolysaccharidosis type 1 (MPS-1) patient skin fibroblasts observed with a fluorescence microscope after addition of a fluorescent group-modified Man6P-containing compound (Man6P-Man5-AFO) and incubation for 24 hours. It is a figure which shows a result. FIG. 12 shows that mucopolysaccharidosis type 1 (MPS-1) patient skin fibroblasts after addition of fluorescent group-modified Man6P-containing IDUA (Man6P-IDUA-AFO) and incubation for 24 hours were observed with a fluorescence microscope. It is a figure which shows a result. FIG. 13A is a graph showing the results of SDS-PAGE of a sample after sugar chain transfer to a sugar chain acceptor (CTSA after endoenzyme treatment (GlcNAc-CTSA)) of a sugar chain donor (compound 12). It is. Lane 1 is a sample containing CTSA obtained from Chinese hamster (CHO) cells into which a CTSA gene has been introduced, and lane 2 is a sample containing a sugar chain receptor (CTSA after endoenzyme treatment (GlcNAc-CTSA)). Yes, lane 3 is a sample after the sugar chain transfer of the sugar chain donor (compound 12) to the sugar chain acceptor (GlcNAc-CTSA). FIG. 13B is a diagram showing the result of lectin blotting with the same sample as FIG. 13A using a lectin (Dom9-His) that specifically binds to Man6P.

Terms used throughout this specification and claims will be explained.
“˜” representing a numerical range represents a range including upper and lower numerical values.

“Endo M” is a kind of “Endoenzyme”, and “Endoenzyme” has the same meaning as “Endo-β-N-acetylglucosaminidase” and extends the end of the arrow in the following general formula (4). Means an enzyme that hydrolyzes the glycosidic bond between GlcNAc and GlcNAc. An “endoenzyme variant” is one in which one or more amino acid residues of an endoenzyme amino acid have been deleted, added or substituted. The substance to be subjected to sugar chain transfer refers to a sugar chain donor and a sugar chain acceptor in a sugar chain transfer reaction.

In the general formula (4), X represents an oligosaccharide that binds to the 4-position of GlcNAc on the non-reducing end side of the reducing end chitobiosyl residue (GlcNAcβ1-4GlcNAc) in the N-linked sugar chain, and R represents a polypeptide Indicates. The arrow represents the position where the endoenzyme or endoenzyme variant is hydrolyzed.

“Transglycosylation” means a part of a sugar chain structure of a sugar chain donor, for example, in the case of the general formula (4), a part on the left side from the position where the tip of the arrow is extended (β1-4 glycoside bond part). , Refers to binding (transfer) to a sugar chain receptor. The sugar chain receptor in the present disclosure refers to a sugar-containing substance having GlcNAc represented by the general formula (1) at the non-reducing end.
“Transglycosylation activity” refers to the ability of Endo M or Endo M mutant to transfer a sugar chain donor to a sugar chain acceptor to generate a new product (transfer to a sugar chain).

“Transglycosylation yield” is a product generated after the transglycosylation reaction, and is transferred to the glycan acceptor from the position where the arrow in the above general formula (4) is extended to the glycan acceptor. The amount of glycoprotein produced by transferring the part. The product may be referred to as a product after sugar chain transfer.

Here, the “endo M mutant” refers to an endo enzyme mutant in which the amino acid of the amino acid sequence of endo M (see SEQ ID NO: 1) has been deleted, added, or substituted. “End M” means an endoenzyme derived from the hair mold Mucor Himaris (GenBank Accession No. BAB43869), and “End D” means an endoenzyme derived from Streptococcus pneumoniae (GenBank Accession No. BAB62042.1). .

“Homology” refers to residues in a protein amino acid sequence variant that are identical after aligning the sequence by introducing gaps, if necessary, to achieve maximum homology (percent). Defined as a percentage. Methods and computer programs for alignment are well known in the art and use ClustralW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) herein. .
In addition, as a method of describing amino acid residues, it is expressed by either one of three letters or one letter.

Also, regarding the notation indicating a specific amino acid residue in the amino acid sequence of the protein, for example, in the amino acid sequence of endo M, the 175th asparagine residue is indicated as N175. In addition, regarding the notation of an amino acid residue substituted, that is, an amino acid residue substituted with another amino acid, for example, a residue obtained by substituting the 175th asparagine residue of endo M with a glutamine residue (or alanine) is N175Q ( Alternatively, End M, which is designated N175A) and contains it, may be referred to as the N175Q variant of Endo M, or simply the N175Q variant.

In addition, among the endo M mutants, a mutant having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine is referred to as “endo M mutant of the present disclosure”. There is. Further, among the endo M mutants of the present disclosure, the amino acid sequence of 175 of the amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence of glutamine or alanine, and 175 of the amino acid sequence represented by SEQ ID NO: 1 Amino acids modified within a range of homology of 80% or more to the amino acid sequence of the endo-M mutant by deletion, addition or substitution of one or more amino acid residues other than the amino acid residue From the viewpoint of distinguishing the endo-M mutant having a sequence and transglycosylation activity from the above-described N175Q mutant or N175A mutant, it may be particularly referred to as “the endo-M mutant homolog of the present disclosure”. is there.

The transglycosylation activity by the endo M mutant can be confirmed by measuring the Man6P-containing glycoprotein produced in the solution after the reaction, for example, by SDS-PAGE, lectin blotting and MALDI-TOF MS.

Examples of the MALDI-TOF MS include the following method.
That is, a certain amount of acetone was added to the solution after the reaction, the dissolved portion was dried, and then a certain amount of DHBA solution (20 mg / mL 2,5-dihydroxybenzoic acid dissolved in 50% aqueous methanol solution) was added. Dissolve. Thereafter, a part of the dissolved solution is spotted on a plate for MALDI-TOF MS analysis, dried, and measured with an autoflex speed-tko1 reflector system (manufactured by Bruker Daltonics) under the following conditions: The mass of the product after the reaction can be confirmed.
<Condition>
Measurement mode: positive ion mode or negative ion mode and reflector mode or linear mode
・ Measurement voltage: 1.5Kv ~ 2.5Kv
Measurement molecular weight range: 0 to 10,000 (m / z)
・ Total number of times: 500-10000

In addition, as a lectin blot, for example, a lectin against Man6P can be used, and the lectin described in the literature by Akeboshi et al. [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 73, 4805-4812, (2007)] Man6PR (sometimes referred to as Dom9-His).

The product after sugar chain transfer can be identified by, for example, measurement by SDS-PAGE, lectin blot and MALDI-TOF MS as described above, and the sugar obtained by SDS-PAGE and lectin blot From the comparison of the density of the band of the product after chain transfer, it is possible to qualitatively confirm the magnitude of the yield of the product after sugar chain transfer (yield of sugar chain transfer). Moreover, from this result, it is possible to qualitatively confirm the size of the endo-M mutant for the transglycosylation activity.

In this specification and the like, the carbohydrate portion is described with reference to the nomenclature usually used for describing oligosaccharides. These nomenclatures are found, for example, in Hubbard et al. [Ann. Rev. Biochem., Vol. 50, 555, (1981)]. According to this method, Mannose is represented by Man, 2-N-acetylglucosamine is represented by GlcNAc, galactose is Gal, fucose is Fuc, and glucose is Glc. Sialic acid is represented by the abbreviated notation NeuAc for 5-N-acetylneuraminic acid and NeuGc for 5-glycolylneuraminic acid. Further, the N-acetylglucosaminyl group may be referred to as a GlcNAc residue, and the mannosyl group may be referred to as a Man residue.

The monosaccharide means only the above-mentioned sugars such as Man and GlcNAc itself.
The position of the carbon that forms the sugar is represented by the 1st position of the reducing end and the 2nd position of the adjacent carbon atom. When expressed as an atom and a glycosidic bond, it is simply expressed as “glycoside bond between the 1-position of GlcNAc and the 4-position of GlcNAc”. A combination of these two or more monosaccharides is called an oligosaccharide, and its derivative is called an oligosaccharide derivative. That is, the N-linked sugar chain is an oligosaccharide.

“Glycoside bond” refers to a bond in which the hydroxyl group at the 1-position of a sugar unit in a sugar chain and the hydroxyl group of another sugar are dehydrated and bonded together via an oxygen atom. For example, an α1-6 glycoside bond The term “sugar” refers to a glycosidic bond in which the 1-position (carbon) of a sugar and the 6-position (oxygen atom at the 6-position) of another sugar are bonded in an α-type. In addition, the hydroxyl group at the 1-position of the sugar has α type and β type.
According to the above, for example, with respect to the general formula (4), the glucosaminyl group (GlcNAc) on the non-reducing end side of the chitobiosyl site (-GlcNAcβ1-4GlcNAc-) does not contain the oxygen atom at the 1-position of the GlcNAc, and It indicates that it contains a 4-position oxygen atom. GlcNAc that binds to Y on the reducing end side does not include the oxygen atom at the 1-position and includes an oxygen atom at the 4-position.
In Man and GlcNAc, the 2nd, 3rd, 4th and 6th carbons that are not glycosidic bonded indicate that a hydroxyl group is bonded.
In addition, as an expression for explaining a sugar chain, a sugar chain including three monosaccharides may be referred to as three sugars, and a five sugar chain including five sugars.

The “core sugar chain” refers to the sugar chain part represented by C-1 below in the N-linked sugar chain, and “trimannosyl” refers to the three mannose parts in the core sugar chain. . Further, as shown in the broken line portion on the left side of C-1, among the trimannosyl portion, Man binding at α1-6 on the non-reducing end side is Man2, and Man binding at α1-3 on the non-reducing end side is Man3. And Man on the reducing end side is referred to as Man1.

In addition, the oligosaccharide as shown on the left side of the position where the tip of the arrow in the general formula (4) is extended may be referred to as a trimannosyl GlcNAc-containing sugar chain.

In the present specification, “protein” means containing a peptide. That is, the glycoprotein includes a glycopeptide. In addition, the term “glycopeptide” refers to a glycoprotein having 50 or less amino acid residues. In the present specification, “protein” includes both glycoproteins and non-glycoproteins unless otherwise specified.
“Glycoprotein” indicates that at least one or more N-linked sugar chains are present in the peptide or polypeptide portion. Unless otherwise specified, the type of the plurality of N-linked sugar chains is 1 Species and two or more types are included.

Also, “lysosomal enzyme” refers to a glycoprotein that functions as an enzyme in lysosomes in eukaryotic cells.

In the present disclosure, a stable natural-type substrate can be used in the preparation of a chemo-enzymatic Man6P-containing glycoprotein, and a Man6P-containing glycoprotein that cannot be achieved by the prior art can be produced.
When an N-linked sugar chain derivative having a chitobiosyl skeleton is used as a sugar chain donor in the sugar chain transfer reaction of endo M, the reactivity in the sugar chain transfer is due to the sugar chain structure on the non-reducing terminal side of endo M. It was known to change significantly. In particular, an N-linked sugar chain having a large polar residue such as a phosphate group is expected to be unsuitable as a sugar chain donor for a sugar chain transfer reaction by an endo M mutant. It was shown that unexpectedly high transglycosylation yields can be achieved.
The reason why the above-mentioned high transglycosylation yield can be achieved is not clear, but the inventor thinks as follows. That is, the endo M mutant recognizes an N-linked sugar chain having Man6P at the non-reducing end, but recognizes it as a sugar chain donor, and the sugar chain acceptor represented by the general formula (1) is water. It is considered that the transglycosylation reaction proceeds by preferentially binding to the glycan donor rather than the glycan donor, and once the product after the transglycosylation is formed, the endo M mutant is re-linked to the saccharide. Since it is difficult to recognize the product after chain transfer, it is presumed that the yield of the product after sugar chain transfer increases as a result.

In addition, the sugar chain donor having no oxazoline skeleton used in the above-mentioned sugar chain transfer reaction has an advantage that it can be synthesized by a method in which the phosphate group is not exposed to acidic conditions, and is stable even in a buffer solution. Can exist. In addition, the sugar chain donor having a chitobiosyl skeleton in the present disclosure is extremely unlikely to bind to a group other than GlcNAc of the sugar chain acceptor, like an oxazolineized sugar chain donor.
For this reason, the sugar chain transfer reaction in the present disclosure is a reaction that facilitates the production of a Man6P-containing glycoprotein that could not be easily produced by conventional oxazolineated sugar chain donors. Application to large-scale preparation can also be considered.

Hereinafter, a method for producing the Man6P-containing glycoprotein of the present disclosure will be described.

<< Manufacturing method of Man6P-containing glycoprotein >>
The method for producing a Man6P-containing glycoprotein of the present disclosure includes a sugar chain having a structure derived from an N-linked sugar chain in which Man6P is bound to a non-reducing end in the presence of the endo M mutant of (a) or (b) below: This is a method for producing a Man6P-containing glycoprotein by producing a Man6P-containing glycoprotein by a sugar chain transfer reaction between a donor and the sugar chain acceptor represented by the general formula (1).
(A) Endo M mutant having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine (b) Other than the 175th amino acid residue of the amino acid sequence of (a) And having the amino acid sequence modified within 80% or more homology with the amino acid sequence of (a) by deletion, addition or substitution of one or more amino acid residues An endo M mutant having an activity of catalyzing a chain transfer reaction.

(Man6P-containing glycoprotein)
Man6P-containing glycoproteins produced by the production method of the present disclosure include those found in nature and those synthesized. In addition, it is known that the second amino acid residue from the asparagine is necessarily a threonine residue or a serine residue on the C-terminal side of the asparagine to which the N-linked sugar chain binds in a naturally occurring glycoprotein. However, the same applies to the present disclosure.

The glycoprotein of the Man6P-containing glycoprotein produced by the production method of the present disclosure is not particularly limited. For example, if derived from animal cells, glycoproteins secreted inside or outside the cell or on the cell surface and having an N-linked sugar chain are exemplified. Examples include various enzymes, various hormones, cell adhesion factors, various receptors, and the like. Examples include various cytokines.
Among these, from the viewpoint of functioning in animal cells, various enzymes are preferable. Examples of various enzymes include enzymes that function in cells, and among them, lysosomal enzymes are preferable. As lysosomal enzymes, β-hexosaminidase (βN-acetylglucosaminidase, βN-acetylgalactosaminidase), αN-acetylglucosaminidase, βgalactosidase, αgalactosidase, αglucosidase, αiduronidase, αiduronic acid 2 sulfatase, βglucuronidase, β-glucocerebrosidase, β-galactocerebrosidase, αN-acetylgalactosaminidase, αfucosidase, cathepsin A, cathepsin B, cathepsin D, cathepsin H, cathepsin L, aspartyl glucosaminidase, endo β-galactosidase, endo βN-acetylglucosaminidase, β Mannosidase, α-mannosidase, allylsulfatase, hyaluronidase, acid lipase, acid ceramidase, acid sphingomye Nase, α neuraminidase 1, α neuraminidase 4, N-acetylglucosamine-1-phosphotransferase, N-acetylglucosamine-1-phosphodiester αN-acetylglucosaminidase, tripeptidylpeptidase, sialin, heparan N-sulfatase, acetyl-CoAαN- Acetylglucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, galactose-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, thioesterase, cathepsin K, prosaposin, saposin A, saposin B, saposin C, saposin D, GM2 activating protein, NPC1 protein, and lipoprotein lipper Etc. The.
The origin of the glycoprotein is not particularly limited as long as it does not impair the effect of the method of the present disclosure. However, from the viewpoint of introducing and functioning in an animal cell, it is preferably derived from an animal cell, and further to humans. From the viewpoint of enhancing the effect of enzyme replacement therapy, it is more preferably derived from a human.

<End M mutant>
The endo M variant in the present disclosure is (a) an endo M variant having an amino acid sequence in which the amino acid residue at position 175 of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine, or (b) Modification within 80% or more homology with the amino acid sequence of (a) above by deletion, addition or substitution of one or more amino acid residues other than the 175th amino acid residue of the amino acid sequence And an endo-M mutant (endo-M mutant homologue) having an amino acid sequence and an activity of catalyzing a sugar chain transfer reaction. Here, the sugar chain transfer reaction refers to a sugar chain donor having a structure derived from an N-linked sugar chain in which Man6P is bonded to the non-reducing end, and a sugar chain acceptor represented by the above general formula (1): This refers to the transglycosylation reaction.

The endo M mutant of the present disclosure is a mutant in which a mutation is introduced into the amino acid of endo M. The endoenzyme represented by SEQ ID NO: 1 is an endo β-N acetylglucosaminidase (GenBank Accession No. BAB43869) derived from Mucor Himalis.
In the present disclosure, the endo M mutant is N175Q or N175A, so that hydrolysis of the product after the transglycosylation reaction can be sufficiently suppressed. Further, from the viewpoint of sugar chain transfer yield, N175Q mutant is preferable.

The endo M mutant of the present disclosure can be prepared by a normal genetic engineering technique, and can be prepared using various types of hosts and corresponding appropriate protein expression vectors. Examples of the host include Escherichia coli, Brevibacillus, cyanobacteria, lactic acid bacteria, yeast, insect cells and animal cells. Among these, from the viewpoint of ease of preparation and expression level, it is preferable to prepare by a method using E. coli as a host or a method using yeast as a host. The specific production method is described in detail in the literature of Umekawa et al. [J. Biol. Chem., Vol.285 (1), 511-521, 大腸菌 (2010)] for Escherichia coli. This is described in detail in JP-A-11-332568.

The endo-M mutant and endo-M mutant homologue of the present disclosure are fused endo-M mutants (or fused forms) fused with other peptides or proteins on the C-terminal side or the N-terminal side in a transglycosylation reaction. Endo M mutant homologue). The peptide or protein that can be fused is not particularly limited as long as it does not inhibit the transglycosylation reaction. For example, hexahistidine peptide (amino acid sequence is from N-terminal to HHHHHH), flag peptide (amino acid sequence is N-terminal) To DYKDDDDK), influenza HA polypeptide (amino acid sequence is YPYDVPDYA from the N-terminus), glutathione-S-transferase, luciferase, avidin, chitin-binding protein, c-myc, thioredoxin, disulfide isomerase (DsbA), maltose-binding protein ( MBP) and green fluorescent protein (GFP). Among these, hexahistidine peptides and flag peptides are particularly preferable from the viewpoint of ease of preparation of endo M mutant or endo M mutant homolog.

In the above-mentioned fusion-type endo M mutant or fusion-type endo M mutant homolog, there is a gap between the polypeptide part of the peptide or protein to be fused and the polypeptide part of the endo-M mutant or endo-M mutant homolog. Contains a peptide moiety as a linker region of several to 50 residues. In addition, it does not specifically limit as a kind of amino acid residue which comprises a linker region. The linker region may include an amino acid sequence portion (protease site) that is hydrolyzed by a protease. Although it does not specifically limit as a protease site, For example, a factor Xa site, a thrombin site, an enterokinase site, a precision protease site is mentioned.

As shown in the publicly known information (http://www.cazy.org/), saccharide hydrolase found in nature has a family of hundreds of glycosylhydrases from the homology of amino acid sequences. (GH family). Endo M is a glycosyl hydrolase belonging to the GH family 85, and other proteins belonging to the GH family 85 are known to be widely distributed from humans to bacteria. When a homology search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) that can be generally used is performed based on the amino acid sequence of End M (SEQ ID NO: 1), End M and other GH families 85 It can be seen that the protein belonging to the group has high homology within the region including the catalytic region from the amino terminal side to the vicinity of the 500th amino acid.
For this reason, if the endoenzyme mutant homologue of the present disclosure has an amino acid sequence length of about 500 residues including the catalytic region of endo M, it can be presumed to have transglycosylation activity as endo M. .
The endo M mutant homologue of the present disclosure may be any amino acid other than the 175th amino acid residue as long as it has 80% homology with the N175Q mutant or N175A mutant and has transglycosylation activity. Those prepared by substituting, deleting or adding residues to other amino acids may be used.

From the above viewpoint, in the present disclosure, an endo M mutation caused by deletion, addition, or substitution of one or more amino acid residues other than the 175th amino acid residue of the amino acid sequence of the endo M mutant of the present disclosure If the homology of the amino acid sequence of the body homologue to the amino acid sequence of the endo M mutant is 80% or more, the transglycosylation activity can be sufficiently maintained. Moreover, if it is 90% or more, it is preferable from the sugar-transfer yield easily improving, More preferably, it is 95% or more, Especially preferably, it is 98% or more, Most preferably, it is 99% or more.

In the present disclosure, the concentration of the endo M mutant or endo M mutant homolog in the reaction solution is preferably 0.1 U / ml to 1 U / ml. When the amount is 0.1 U / ml or more, the sugar chain transfer yield can be increased, and when the amount is 1 U / ml or less, the hydrolysis reaction of the product after the sugar chain transfer reaction can be further suppressed. Here, 1 unit (U) indicates the amount of enzyme that transfers 1 μmol of sialoglycopeptide (hereinafter referred to as SGP) to β-paranitrophenyl GlcNAc (GlcNAcβ1-O-pNP) in 1 minute. Further, the concentration is more preferably 0.1 U / ml to 1 U / ml, and particularly preferably 0.2 U / ml to 0.5 U / ml.

The purity of the endo M mutant or endo M mutant homolog used in the reaction is 50% from the viewpoint of shortening the reaction time and the yield of sugar chain transfer from the results of SDS-polyacrylamide electrophoresis. % Or more, more preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more. When the degree of purification is 50% or more, the reaction time can be shortened and the sugar chain transfer yield can be easily increased.

(Sugar chain donor)
As the sugar chain donor, it is sufficient that Man6P has a structure derived from an N-linked sugar chain bonded to the non-reducing end, and the sugar chain can be transferred to the sugar chain acceptor represented by the general formula (1). If it is a thing, it will not specifically limit. The structure derived from the N-linked sugar chain means that the N-linked sugar chain itself and a derivative of the N-linked sugar chain are included.

Further, from the viewpoint of further increasing the sugar chain transfer yield, a sugar chain donor represented by the following general formula (2) is preferable.

In the general formula (2), X ¹ to X ⁶ each independently represents a hydrogen atom or a saccharide-derived group, and at least one of X ¹ to X ⁶ is derived from a saccharide having Man6P at the non-reducing end. It is a group. Z ¹ represents a hydrogen atom or GlcNAc, and when Z ¹ is GlcNAc, the GlcNAc is bonded to Man linked to GlcNAc at β1-4 at β1-4. Y ² represents a monovalent substituent.

In general formula (2), X ¹ to X ⁶ may be a hydrogen atom or a saccharide-derived group, and at least one of X ¹ to X ⁶ may be a saccharide-derived group having Man6P at the non-reducing end. The saccharide-derived group is not particularly limited as long as it does not impair the effect of the method of the present disclosure. In addition, the saccharide-derived group having Man6P includes Man6P itself.

In the general formula (2), among X ¹ to X ⁶ , groups other than the saccharide-derived group having Man6P at the non-reducing end include, for example, GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2, NeuGcα2-6Galβ1-4GlcNAcβ1-2 and NeuGcα2-3Galβ1-4GlcNAcβ1-2, heterologous antigens: Galc1-4GlcNAcβ1-2 4GlcNAcβ1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), keratan sulfate _{[Galβ1-4GlcNAc (6SO 3) β1-3]} nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number) _{[Gal (6SO 3) β1-4GlcNAc (} 6SO 3) β1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, sulfated LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, terminal sulfate Modified (3SO ₃ ) Galβ1-4GlcNAcβ1-2, Lewis sugar chains: Galβ1-4 (Fucα1-3) GlcNAcβ1-2, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAcβ1-2, Galβ1-3 (Fucα1-4) GlcNAcβ1 -2, Fucα1-2Galβ1-3 (Fucα1-4) GlcNAcβ1-2, blood group antigens: Fucα1-2Galβ1-4GlcNAcβ1-2, Galα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1 -2, GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-2, HNK1 antigen: (3SO ₃ ) GlcAβ1-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2 and GlcNAcAβ1-2.

Among X ¹ to X ^6, the saccharide-derived groups having Man6P at the non-reducing end include GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, [Galβ1-4GlcNAcβ1-3] nGalβ1-4GlcNAcβ1-2 (n is Any number), [Galβ1-4GlcNAc (6SO ₃ ) β1-3] nGalβ1-4GlcNAcβ1-2 (where n is an arbitrary number), etc. Things.

In addition, among X ¹ to X ⁶ , a group other than a saccharide-derived group having Man6P at the non-reducing end may have any of a structure derived from a high mannose-type sugar chain in addition to a hydrogen atom. Examples of X ¹ to X ⁶ include Manα1-6, Manα1-3, Manα1-2Manα1-3, Manα1-2Manα1-6, Manα1-2, Manα1-2Manα1-2, and the like.
In addition, among X ¹ to X ⁶ , saccharide-derived groups having Man6P at the non-reducing end include, for example, Man6Pα1-6, Man6Pα1-3, Man6Pα1-2Manα1-3, Man6Pα1-2Manα1-6, Man6Pα1-2 , Man6Pα1-2Manα1-2 and the like.

When X ¹ to X ⁶ are as described above, Z ¹ may be a hydrogen atom or GlcNAcβ1-4.

In the general formula (2), examples of Y ² include a substituent containing a structure in which an oxygen atom, a nitrogen atom, a carbon atom, or a sulfur atom is directly bonded to the 1-position carbon of GlcNAc.
Y ² is not particularly limited as long as it does not reduce the transglycosylation activity. For example, hydroxyl group, alkoxy group, aryloxy group, alkenyloxy group, acyloxy group, alkyl group, alkenyl group, alkynyl group, aralkyl group, alkyl ester group, aryl ester group, amino group, acylamino group, imide group, azide group , Carboxyl group, alkylsulfinyl group, arylsulfinyl group, alkylsulfonyl group, arylsulfonyl group, alkylsulfonyloxy group, or halogen group. Among these groups, alkoxy group, acylamino group, aryloxy group, alkenyl Examples thereof include an oxy group, an acyloxy group, an alkylsulfinyl group, an arylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, an alkylsulfonyloxy group, and an arylsulfonyloxy group. These substituents may further have a substituent.

Among these, for ease of preparation, Y ² is a hydroxyl group, an alkoxy group having 1 to 30 carbon atoms which may have a substituent, or an alkyl group having 6 to 30 carbon atoms which may have a substituent. An aryloxy group, an optionally substituted alkenyloxy group having 1 to 30 carbon atoms and an optionally substituted acylamino group are preferred.

Furthermore, Y ² is an alkoxy group optionally having a substituent having 1 to 10 carbon atoms and a substitution having 2 to 10 carbon atoms from the viewpoint of ease of preparation as a sugar chain donor and a yield of sugar chain transfer. An alkenyloxy group which may have a group, an aryloxy group which may have a substituent having 6 to 24 carbon atoms, and an acylamino group which may have a substituent are preferable, and more preferably 1 to An alkoxy group optionally having 8 substituents, an alkenyloxy group optionally having 2 to 6 carbon atoms, and an aryloxy optionally having 6 to 12 carbon atoms An acylamino group which may have a group and a substituent is particularly preferred.
Specific examples include a methoxy group, an ethoxy group, a phenoxy group, a paramethoxyphenoxy group, and a paranitrophenoxy group.

The acylamino group which may have a substituent is preferably an acylamino group to which the amino group of the side chain of the asparagine of the peptide or protein is bonded, and the solubility in the reaction solution and the transglycosylation yield. From the viewpoint, an acylamino group to which the amino group of the side chain of the asparagine of the peptide is bonded is more preferable.

In addition, the above-described substituents which may be substituted include an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms, a halogen atom, a cyano group, a nitro group, When it is at least one selected from an amino group, a carboxyl group and a pyridyl group and there are two or more substituents, each substituent may be the same or different.

The method for preparing the sugar chain donor represented by the general formula (2) is not particularly limited, but for the ease of the preparation method, for example, an N-linked sugar chain is prepared and Man6P is chemically introduced. A method is mentioned.
Examples of N-linked sugar chains include high mannose sugar chains and complex sugar chains. The high mannose sugar chains can be prepared from natural products, even by chemical synthesis. There may be. As a method by chemical synthesis, for example, as described in the literature of Matsuo et al. [Tetrahedron, Vol. 62, 8262-8277, (2006)], As described in Japanese Patent Application Publication No. 2007-297429, a method combining a chemical synthesis method and mannosidase can be mentioned. Examples of the method of preparing from natural products include a method of extracting from egg white albumin as described in Japanese Patent No. 3776952 and JP-A-10-251304.
Among the methods for preparing the above-described high mannose sugar chain, a synthetic preparation method is preferred from the viewpoint of introducing Man6P into the non-reducing end.

The method for preparing the complex type sugar chain may be a method by chemical synthesis or a method of preparing from a natural product. As a method by chemical synthesis, for example, it can be prepared by the method described in Wang et al. [J. Am. Chem. Soc., Vol. 134 (29), 12308, (2012)]. Examples of the method of preparing from a natural product include a method of extracting from egg yolk (for example, International Publication No. 962255 and Japanese Patent Application Laid-Open No. 2011-231293).
Among the preparation methods of the above complex type sugar chains, those obtained by synthesis are preferable from the viewpoint of introducing Man6P into the non-reducing end.

Also, as a method for introducing Man6P into the non-reducing end of the N-linked sugar chain obtained above, a chemical introduction method can be mentioned. As a chemical introduction method, any synthetic route may be used as long as Man6P can be introduced into the non-reducing end of the N-linked sugar chain, and it is appropriately selected depending on the structure of the target sugar chain donor. The method for introducing a phosphate group into mannose is not particularly limited. For example, the phosphate group can be introduced by reacting a phosphorylating reagent with the 6-position hydroxyl group of the mannose residue at the non-reducing end of an N-linked sugar chain. . Examples of the phosphorylating reagent include tetrabenzyl pyrophosphate, cyclic phosphite amide, phosphite, phosphate triester, and amidite. When introducing a phosphate group, from the viewpoint of introducing a phosphate group only to the 6-position hydroxyl group of the mannose residue at the non-reducing end of the N-linked sugar chain, other than the hydroxyl group is protected by a protecting group. It is preferable. In addition, protection means that the hydroxyl group is converted into a group that is not reactive with the phosphorylating reagent.

Further, from the sugar chain transfer yield and the efficiency of introducing the product after sugar chain transfer into cells, a sugar chain donor represented by the following general formula (3) is preferable.

In the general formula (3), X ⁷ , X ⁸ and X ⁹ each independently represent a hydrogen atom, Man, Manα1-2Man, Man6P, Man6Pα1-2Man or Man6Pα1-6Man. Man represents a mannosyl group. Man6P represents a mannosyl group having a phosphate group bonded to the 6-position. ^{Further, X} 7 -6 indicates the ^{X 7} bound to 6-position of ^{Man, X} 8 -3 shows the ^{X 8} attached to the 3-position of ^{Man, X} 9 -2 is attached to the 2-position of Man X ⁹ is shown. At least one of X ⁷ , X ⁸ and X ⁹ represents any of Man6P, Man6Pα1-2Man and Man6Pα1-6Man. GlcNAc represents an N-acetylglucosaminyl group. α1-6 represents an α-glycosidic bond between the 1-position of Man and the 6-position of Man or an α-glycosidic bond between the 1-position of Man6P and the 6-position of Man, and α1-3 represents Α1-2 represents an α-glycoside bond between the 1-position of Man and the 2-position of Man, or an α-glycoside bond between the 1-position of Man6P and the 2-position of Man. β1-4 represents a β glycosidic bond between position 1 of GlcNAc and position 4 of GlcNAc or β glycoside bond between position 1 of Man and position 4 of GlcNAc. Y ³ represents a monovalent substituent.

In the general formula (3), when at least one of X ⁷ , X ⁸ and X ⁹ is any one of Man6P, Man6Pα1-2Man or Man6Pα1-6Man, the other is a hydrogen atom, Man, Manα1-2Man, Man6P, Man6Pα1 -2Man or Man6Pα1-6Man. In the general formula (3), the one having Man6P at the non-reducing end may be any of X ⁷ to X ⁹ and is appropriately adjusted depending on the type of the target peptide or glycoprotein to be transferred to the sugar chain. It is preferable. However, in view of the efficiency of introducing the product after sugar chain transfer into the cell, X ⁷ is more preferably any of Man6P, Man6Pα1-2Man, or Man6Pα1-6Man. Further, the number of Man6Ps among X ⁷ to X ⁹ is more preferably 2 or more, and particularly preferably 3.

Of the sugar chain donors represented by the general formula (3), from the viewpoint of being a high mannose type sugar chain already found in natural glycoproteins, the following general formulas (3-1) to (3) Those represented by the general formula (3-10) are preferable, and among them, the general formula 3-3 is more preferable.

In the above general formulas (3-1) to (3-10), Y ³ represents a monovalent substituent. Man6P represents a mannosyl group having a phosphate group bonded to the 6-position.
Y ³ has the same meaning as ^{Y 2} in the general formula (2), and the preferred range is also the same.

The compound represented by the general formula (3) can be prepared by appropriately combining, for example, a known chemical glycosylation method and the above-described method for introducing a phosphate group or the like.

(Sugar chain receptor)
The sugar chain receptor is a GlcNAc-containing glycoprotein represented by the following general formula (1). The sugar chain receptor is not particularly limited as long as it is represented by the general formula (1) as long as it does not inhibit the sugar chain transfer activity of the endo M mutant.

In general formula (1), Y ¹ represents an acylamino group containing a structure derived from a glycoprotein. GlcNAc represents an N-acetylglucosaminyl group.

In Y ^1, as the glycoprotein, for example, various enzymes, various hormones, cell adhesion factors, and various receptors and various cytokines and the like. Among them, various enzymes are preferable from the viewpoint of functioning in cells, and lysosomal enzymes are preferable from the viewpoint of functioning in lysosomes using the Man6P receptor on the lysosomal membrane. As lysosomal enzymes, β-hexosaminidase (βN-acetylglucosaminidase, βN-acetylgalactosaminidase), αN-acetylglucosaminidase, βgalactosidase, αgalactosidase, αglucosidase, αiduronidase, αiduronic acid 2 sulfatase, βglucuronidase, β-glucocerebrosidase, β-galactocerebrosidase, αN-acetylgalactosaminidase, αfucosidase, cathepsin A, cathepsin B, cathepsin D, cathepsin H, cathepsin L, aspartyl glucosaminidase, endo β-galactosidase, endo βN-acetylglucosaminidase, β Mannosidase, α-mannosidase, allylsulfatase, hyaluronidase, acid lipase, acid ceramidase, acid sphingomye Nase, α neuraminidase 1, α neuraminidase 4, N-acetylglucosamine-1-phosphotransferase, N-acetylglucosamine-1-phosphodiester αN-acetylglucosaminidase, tripeptidylpeptidase, sialin, heparan N-sulfatase, acetyl-CoAαN- Acetylglucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, galactose-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, thioesterase, cathepsin K, prosaposin, saposin A, saposin B, saposin C, saposin D, GM2 activating protein, NPC1 protein, and lipoprotein lipper Etc. The.
The origin of the glycoprotein is not particularly limited as long as it does not impair the effect of the method of the present disclosure. However, from the viewpoint of introducing and functioning in an animal cell, it is preferably derived from an animal cell, and further to humans. From the viewpoint of enhancing the effect of enzyme replacement therapy, it is more preferably derived from a human.

Among the sugar chain receptors represented by the general formula (1), those in which Y ¹ is an acylamino group containing a structure derived from a glycoprotein having 30 or less amino acid residues are disclosed in, for example, Huang et al. [Chembiochem , Vol.12 (6), 932-941, (2011)], a chemical method or a chemistry including a peptide extension step as shown in a patent document (Japanese Patent Laid-Open No. 10-45788). Can be prepared by standard synthetic methods. Furthermore, a glycopeptide or N-linked sugar chain derivative obtained by a reagent manufacturer can be easily prepared by hydrolysis with the endo M mutant of the present disclosure or a commercially available endo enzyme.
Y ¹ is an acylamino group containing a structure derived from a glycoprotein is a glycoprotein obtained by purchasing a commercially available reagent or food, a glycoprotein prepared by a genetic engineering technique, or a food or natural product. Can be easily prepared by hydrolyzing the glycoprotein obtained by a general extraction / separation method with the endo-M mutant of the present disclosure or a commercially available endo-enzyme.

The commercially available endoenzymes include End H (manufactured by New England Biolabs), End S (manufactured by Sigma-Aldrich), End D (manufactured by Cosmo Bio), End M (manufactured by Tokyo Chemical Industry Co., Ltd.), End F1 ~ And F3 (manufactured by Sigma-Aldrich). Non-commercial enzymes include glycosyl hydrolases belonging to GH family 85, or enzymes that belong to GH family 18 and have been shown to hydrolyze N-linked sugar chains. Can do.

In the preparation of glycoprotein by the above-mentioned genetic engineering technique, a gene encoding a target glycoprotein is introduced (transgenic) into a host cell or individual to prepare a desired glycoprotein. it can. Although it does not specifically limit as a host, From a viewpoint of preparing glycoprotein easily, yeast, an insect cell, the cell derived from various animals, animals, such as an insect which is an individual, an amphibian, and a mammal, are mentioned. Among these hosts, from the viewpoint of economy in preparing sugar chain receptors, insect cells or insects that are individuals are more preferred, silkworm cells or silkworms are particularly preferred, and silkworms that are individuals are most preferred.
As for silkworms, for example, as shown in the literature of Ito et al. [Abstracts of Annual Meeting of the Carbohydrate Society of Japan (2011), 30th, p. 54], silkworms introduced with foreign genes (transgenic silkworms) It is known that foreign proteins are expressed in large quantities in silk glands. In addition, the expressed sugar chain of the protein contains a human-type-like partial structure, and has an advantage that an insect-specific sugar chain structure is not added.
In addition, since glycoproteins prepared from transgenic silkworms, for example, as shown in JP-A-2015-208260, N-linked glycoprotein sugar chains are mainly high mannose sugar chains, many endoenzymes are used. This is advantageous for the preparation of sugar chain receptors.
For this reason, it can be said that the silkworm which can be raised in large quantities and cheaply is economically advantageous for preparation of said glycoprotein.

(reaction)
The reaction means a sugar chain transfer reaction, and the sugar chain transfer reaction is performed in a solution in which an endo M mutant, a sugar chain donor, and a sugar chain acceptor are dissolved. The solution used for the reaction is not particularly limited as long as it does not inhibit the transglycosylation activity of the endo M mutant. For example, phosphate buffer, citrate buffer, carbonate buffer, Tris-HCl buffer, MES Examples include a buffer solution, a MOPS buffer solution, a HEPES buffer solution, a borate buffer solution, and a tartrate buffer solution. These buffers may be used alone or in combination.
Among these, MES buffer solution, MOPS buffer solution, HEPES buffer solution and phosphate buffer solution are preferable from the viewpoint of glycosyltransferase activity of endo M mutant or endo M mutant homolog.
The concentration of the phosphate buffer is preferably 10 mM to 250 mM, more preferably 20 mM to 150 mM, and particularly preferably 50 mM to 100 mM, from the viewpoint of glycosyl transfer activity of the endo M mutant or endo M mutant homolog. . By setting the concentration to 10 mM or more, the buffering capacity is increased, and by setting the concentration to 250 mM or less, the sugar chain transfer activity of the endo M mutant or the endo M mutant homolog can be increased and the sugar chain transfer yield can be increased.

Further, the pH of the solution in the reaction is preferably 5.5 to 8.5, more preferably 6.0 to 8.0, and particularly preferably 6.5 to 7.5 from the viewpoint of increasing the sugar chain transfer yield.

The temperature in the reaction is 4 from the viewpoint of transglycosylation activity of the endo M mutant or endo M mutant homolog, from the viewpoint of increasing the transglycosylation yield, and from the viewpoint of the stability of the sugar chain receptor, endo M mutant, etc. It is preferably from 40 ° C to 40 ° C, more preferably from 20 ° C to 40 ° C, and particularly preferably from 25 ° C to 35 ° C. By setting the temperature to 4 ° C. or higher, the transglycosylation activity of the endo M mutant can be increased, and by setting the temperature to 40 ° C. or lower, the stability of the endo M mutant or the endo M mutant homolog can be increased. .

In the sugar chain transfer reaction, the concentration of the endo M mutant (or endo M mutant homolog) in the reaction solution is preferably 0.005 μg / μL to 0.5 μg / μL. When it is 0.005 μg / μL or more, the transglycosylation activity is improved, and when it is 0.5 μg / μL or less, the product of the endo-M mutant (or endo-M mutant homolog) after the transglycosylation Rehydrolysis can be suppressed, and the sugar chain transfer yield can be improved.

In the sugar chain transfer reaction, the concentration of the sugar chain receptor in the reaction solution is not particularly limited as long as it does not inhibit the sugar chain transfer reaction of the End M mutant or End M mutant homolog. It is adjusted as appropriate in consideration of the sugar chain transfer yield and the solubility of the sugar chain receptor in the reaction solution. However, when the sugar chain receptor represented by the general formula (1) contains a protein, 1.0 mg / mL to 20 mg / mL is preferable. By being in this range, a higher sugar chain transfer yield can be obtained. Of these, 2.0 mg / mL to 10 mg / mL is more preferable.

In the sugar chain transfer reaction, the molar ratio of the sugar chain donor to the sugar chain acceptor (number of moles of sugar chain donor / number of moles of sugar chain acceptor) is the type of sugar chain donor or sugar chain acceptor used. In addition, it can be appropriately set in consideration of recovery of the reaction after the reaction. However, from the viewpoint of transglycosylation yield, the molar ratio is preferably 10 to 3000, more preferably 100 to 2000, particularly preferably 200 to 1500, and further preferably 300 to 1000. Is most preferred. When the molar ratio is 10 or more, a more sufficient transglycosylation yield can be obtained, and when the molar ratio is 3000 or less, a higher transglycosylation yield can be obtained.

The reaction time in the transglycosylation reaction is appropriately set depending on the reaction temperature and the concentration of the endo M mutant or endo M mutant homolog in the solution, but is preferably 3 hours to 100 hours. By being 3 hours or longer, the sugar chain transfer yield can be further increased. By being 100 hours or shorter, the influence of hydrolysis of the product after the sugar chain transfer is further suppressed, and the sugar chain transfer yield is increased. be able to. Among the above reaction times, it is more preferably 10 hours to 50 hours.
In addition, as a reaction method, from the viewpoint of further increasing the sugar chain transfer yield of the reaction, after the reaction for a certain time, the sugar chain donor and the endo M mutant or the endo M mutant homolog are added to the reaction solution again. It is preferable to react for a certain time.

In the method for producing a Man6P-containing glycoprotein of the present disclosure, since no individual, animal tissue, cell, or the like is used, there is very little possibility of contamination from outside or a virus. For this reason, Man6P containing glycoprotein manufactured with said manufacturing method can be provided as a pharmaceutical composition and the chemical | medical component which maintained the function safely. In addition to the Man6P-containing glycoprotein produced by the production method of the present disclosure, the pharmaceutical composition includes a pharmaceutically acceptable stabilizer, buffer, excipient, binder, disintegrant, flavoring agent, A coloring agent, a fragrance | flavor, etc. can be added suitably and it can be set as dosage forms, such as an injection, a tablet, a capsule, a granule, a fine granule, and a powder.
Further, in the method for producing a Man6P-containing glycoprotein of the present disclosure, a protein having a high proportion of many types of Man6P-containing sugar chains can be obtained using many types of sugar chain donors. They are also useful as glycoprotein preparations.

≪Method for detecting intracellular distribution of fluorescent protein-binding Man6P-containing glycoprotein≫
The method for detecting the intracellular distribution of the fluorescent group-bound Man6P-containing glycoprotein of the present disclosure includes a fluorescent group-bound Man6P-containing product obtained by further introducing a fluorescent group into the Man6P-containing glycoprotein obtained by the above production method. This is a method for detecting the intracellular distribution of a glycoprotein by applying the glycoprotein to a cell. The fluorescent group binding type means that the fluorescent group is bound to the Man6P-containing glycoprotein by a covalent bond or an ionic bond.
That is, by introducing a fluorescent group into the Man6P-containing glycoprotein obtained by the above production method and imparting it to the target cells, the distribution of the Man6P-containing glycoprotein in the cells is distributed. Can be detected. In the above method, fluorescent group-bound Man6P-containing glycoproteins and the like easily enter the cell by the Man6P receptor, and thus the behavior of the fluorescent Man6P glycoprotein and the like after the entry in the cell is traced by the fluorescence emitted by the fluorescent group. can do.

Methods for introducing fluorescent groups are described, for example, in the literature of Asanuma et al. [Angew. Chem. Int. Ed. Engl., Vol. 53 (24) 6085-6089, (2014)] and the website of Goryika Chemical Co., Ltd. Referring to the literature (http://goryochemical.com/products/acidifluor/acidifluor-orange.html), appropriately introducing a desired fluorescent group into the Man6P-containing glycoprotein obtained by the above production method Can be prepared. A reagent for introducing a fluorescent group is commercially available. For example, AcidiFluor® ORANGE-NHS manufactured by Goryuka Kayaku Co., Ltd. can be used.

In the detection method, for example, by adding the fluorescent Man6P glycoprotein to a target cell or tissue, incubating at a constant atmosphere, temperature, and time, and observing with a fluorescence microscope or the like, The localization and distribution of the fluorescent Man6P glycoprotein in the cell can be known. The amount, temperature, and time of addition of the fluorescent Man6P glycoprotein to the cells are appropriately adjusted depending on the type of cells, the function and properties of the protein in the fluorescent Man6P glycoprotein, and the like.

Hereinafter, the method of the present disclosure will be described in detail by way of examples. However, the scope of the method of the present disclosure is not limited to the embodiments described in these examples.
Hereinafter, human α-iduronidase is referred to as IDUA. Unless otherwise specified, IDUA refers to an enzyme having activity as an enzyme.
“GlcNAc-IDUA” is a protein obtained by hydrolyzing an IDUA N-linked sugar chain with an endoenzyme, and at least one GlcNAc is bound to a polypeptide constituting IDUA. It shows that.
“Man6P-IDUA” refers to a Man6P-containing glycoprotein, and unless otherwise specified, is obtained from a transgenic silkworm (also referred to as TG silkworm) described later and then purified by three-stage chromatography.
Further, human IDUA usually has 6 N-linked sugar chains, and IDUA prepared by TG silkworm hereinafter is a mixture of 1 to 6 N-linked sugar chains.

[Example 1]
<Preparation of sugar chain receptor (GlcNAc-IDUA)>

(Preparation of TG silkworm with IDUA gene introduced)
Takahashi et al. [Proc Natl Acad Sci USA., Vol. 104, 8941-8946, (2007)] and Kobayashi et al. [Arch Insect Biochem Physiol., Vol. 76 (4), 195-210, (2011). ] With reference to the piggyBac transposon vector, the transcription factor GAL4 downstream of the silkworm middle silk gland (tissue) specific transcription promoter (serine 1 promoter), and the human α-iduronidase (IDUA) downstream of its recognition sequence UAS A gene and a kynurenine oxidase gene for selection of transgenic individuals were inserted and injected into silkworm eggs. By this method, transgenic silkworms that highly express IDUA specifically in the middle silk gland of transgenic silkworm larvae were produced.

(Expression and purification of α-iduronidase by silkworm)
First, various measurement methods for confirming that IDUA is obtained from the TG silkworm obtained above will be described, and next, a method for extracting and separating and purifying IDUA from TG silkworm will be described.
<Various measurement methods>
~ SDS-PAGE / CBB staining ~
For SDS-PAGE, a slab electrophoresis apparatus (manufactured by Biocraft) was used, and electrophoresis was performed using a 10% polyacrylamide uniform gel. Detection of protein in the gel after SDS-PAGE was performed by shaking in CBB staining solution [10% CBB-R350 (manufactured by GE), 27% methanol, 9% acetic acid, 0.1% CuSO ₄ ] for 1 hour or longer. And then decoloring for 6 hours or more using a decolorizing solution (10% acetic acid).

~ Western blotting ~
After electrophoresis of the sample with 10% SDS-PAGE, transfer of the protein to the PVDF membrane using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (manufactured by Bio-Rad) was performed at a constant voltage of 15 V for 1 hour. went. As a transfer buffer, a mixed solution of 48 mM Tris, 39 mM glycine, and 20% methanol was used. The transferred PVDF membrane was blocked by shaking in 50% Blocking one (Nacalai Tesque) / TBS at room temperature for 1.5 hours.
1 ^st IDUA antibodies as probes (Anti-IDUA, sheep IgG polyclonal antibody, (R & D Co., Ltd.)) was used to shake the PVDF membrane in 50% Blocking one / TBS (4 ℃, O / N). Thereafter, the PVDF membrane was washed 5 times at room temperature in 0.1% Tween20 / TBS (TBST) 5 times, and further washed in TBS at room temperature for 5 minutes. Use as 2 ^nd probe anti-sheep antibodies (Biotin-conjugated anti-sheep antibody (Vector Co.)), in 50% Blocking one / TBS, was shaken for 1 hour PVDF membranes at room temperature. Thereafter, the PVDF membrane in TBST was repeatedly washed 5 times at room temperature for 5 minutes, and further washed in TBS at room temperature for 5 minutes. 3 ^rd using HRP-conjugated anti-biotin antibody as a probe (Cell Signaling Inc.), the PVDF membrane was shaken for 1 hour at room temperature in 50% Blocking one / TBS. Thereafter, the PVDF membrane in TBST was washed 5 times at room temperature for 5 minutes, and further washed in TBS at room temperature for 5 minutes. The PVDF film was subjected to chemiluminescence using Western lighting Chemiluminescence Reagent Ultra / Plus (manufactured by Perkin Elmer), and signal (band) detection was performed using an image analyzer [LAS-4000miniEPUV (manufactured by Fuji Film)].

-Measurement method of enzyme activity using artificial fluorescent substrate-
Human α-iduronidase (IDUA) activity is sodium citrate buffer using 4-methylumbelliferyl (4MU) -α-L-idpyranoside (manufactured by Toronto Research Chemicals) (hereinafter sometimes referred to as 4MU-Ido) as a substrate. After incubating at 37 ° C. for 30 minutes in (pH 4.5), the fluorescence intensity (Ex: 355 nm, Em: 460 nm) of the released 4MU was measured as an index. The enzyme activity using 4MU-Ido as a substrate may be referred to as IDUA activity.

<How to collect the middle silk gland>
The middle silk gland from silkworms was collected by a known general method. Break the outer skin (by hand with surgical gloves) of the TG silkworm from the 5th to the mature stage, and pull out the silk gland that is pushed out from the cracked part. It was. In physiological saline, the front and rear silk glands and the attached fat body tissue were removed from the whole collected silk gland tissue, and only the middle silk gland was fractionated and collected. After washing twice with physiological saline, 50 heads were packed in a conical tube and stored frozen at −20 to −30 ° C.

<IDUA purification from middle silk gland>
IDUA was obtained by purifying by performing the three-stage chromatography operation (affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography) in combination with the above IDUA activity measurement method.
First, the middle silk gland collected from the above TG silkworm was thawed and then shredded and sonicated in 20 mM sodium acetate buffer (pH 4.5) containing 0.5 M NaCl to prepare an extract A. did.
FIG. 1 shows the results of measuring the enzyme activity (IDUA activity) against 4MU-Ido for the obtained extract A. In FIG. 1, the term “not introduced” means that the IDUA activity was measured for the extract B of the middle silk gland obtained from the silkworm not introduced with the IDUA gene in the same manner as described above.
From the results, the extract A obtained from the TG silkworm into which the IDUA gene was introduced showed significantly higher IDUA activity than the extract B.
Further, when Western blotting using human IDUA antibody was performed on the obtained extract, as shown in

lanes

1 and 2 in FIG. 2, a protein-containing solution extracted from silkworms into which no IDUA gene had been introduced, While no band was obtained, as shown in

lanes

3 and 4, a strong band was found around 80 kDa in the protein-containing solution extracted from the TG silkworm into which the IDUA gene was introduced.
From the above results, an extract A containing IDUA having IDUA activity was obtained from a TG silkworm into which a human IDUA gene was introduced.

The extract A obtained above was centrifuged by centrifugation [20,000 × g, 4 ° C., 20 minutes (Beckman Coulter, Avanti J-E, rotor: JRA 16.250)], and the supernatant was collected. First, CaCl ₂ and MnCl ₂ were added to the resulting supernatant to a final concentration of 1 mM, and a binding buffer [20 mM sodium acetate buffer, 500 mM NaCl, 1 mM CaCl ₂ and 1 mM MnCl ₂ (pH 4) was previously added. .5)] in the centrifuge tube containing Con A resin (Con A Sepharose 4B, manufactured by GE), sealed with a lid, and gently stirred with a rotator (RT-50 manufactured by Taitec), and then in extract A Was adsorbed onto ConA resin (4 ° C., O / N). The supernatant after centrifugation was removed, and the ConA resin was washed with a ConA resin washing buffer [containing 20 mM sodium acetate buffer, 1 M NaCl, 1 mM CaCl ₂ and 1 mM MnCl ₂ (pH 4.5)]. Went. Thereafter, elution was performed with an elution buffer [20 mM sodium acetate buffer, 500 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl ₂ , and 0.5 M methyl-α-D-mannopyranoside (pH 4.5)]. . The obtained elution fraction (hereinafter referred to as ConA elution fraction) was concentrated using an Amicon ultra filter unit (fractional molecular weight 30 kDa) (Millipore), and then a cation exchange column binding buffer [10 mM acetic acid. Buffer exchange was performed using sodium buffer, 150 mM NaCl (pH 6.0)]. Then, using an 0.22 μm filter (Millipore), the insoluble matter in the elution fraction was removed, and then used for cation exchange chromatography equipped with AKTA purifier UPC-10 (GE). Column (HiTrap SP HP (manufactured by GE)), and purification operation was performed under the condition that the NaCl concentration was continuously increased from 150 mM to 500 mM. The elution fraction showing IDUA activity (hereinafter referred to as SP elution fraction) was concentrated using an Amicon ultra filter unit (fractional molecular weight 30 kDa) (Millipore), and then a butyl-Sepharose column binding buffer [50 mM acetic acid. Buffer exchange with sodium, 1M ammonium sulfate, 150 mM NaCl (pH 4.5)] was performed. Thereafter, the mixture was filtered through a 0.22 μm filter, and applied to a hydrophobic interaction chromatography column (HiTrap Butyl FF, manufactured by GE) equipped with AKTA purifier UPC-10, and the ammonium sulfate concentration was changed from 1M to 0M. A purification operation was performed under the condition of continuously decreasing, the IDUA activity of each fractionated fraction was measured, and fractions having activity (hereinafter referred to as Butyl-eluting fractions) were collected. Table 1 shows the results of the specific activities of the fractions obtained by the respective purification methods. Further, FIG. 3 shows the results of SDS-PAGE of the extract, the ConA elution fraction, and the SP elution fraction. In Table 1 and FIG. 3, Extract indicates the extract A, ConA elute indicates the Con A elution fraction, SP elute indicates the SP elution fraction, and Butyl elate indicates the Butyl. Represents the eluted fraction. SDS-PAGE was performed by applying the same amount (10 μg) of protein to each lane.

From the results, the specific activity for 4MU-Ido of the Butyl elution fraction obtained by the above purification method is improved to 30 times or more the specific activity of Extract A (Extract) for 4MU-Ido. Highly purified IDUA could be obtained from TG silkworm.
Further, from the results of SDS-PAGE in FIG. 3, the staining concentration of bands other than the band around 80 kDa of the SP elution fraction and the Butyl elution fraction is significantly thinner than that of the extract, It was shown that IDUA present in the obtained SP elution fraction and Butyl elution fraction was highly purified. Hereinafter, IDUA present in the Butyl elution fraction is referred to as TG-IDUA.
The fraction containing IDUA obtained by the above purification operation was used for the preparation of the sugar chain receptor shown below.

<Preparation of GlcNAc-IDUA (sugar chain receptor) by endoenzyme>
As described below, the sugar chain receptor is obtained by converting the TG-IDUA obtained above into an N-linked sugar chain having a mannose residue number of 5 or less using an endoenzyme (Endo-D, New England Biolabs). Prepared by hydrolysis.
4 mg of TG-IDUA and 1000 U of Endo-D were dissolved in 800 μL of 50 mM sodium phosphate buffer (pH 7.5) (containing 500 mM NaCl) and incubated at 37 ° C. for 24 hours. After the incubation, Endo-D was removed by adsorption using Chitin resin (manufactured by New England Biolab). Specifically, the solution after incubation was added to 100 μL of Chitin resin equilibrated with 50 mM sodium phosphate buffer (pH 7.5) (containing 500 mM NaCl), and gently stirred at 4 ° C. for 2 hours. Thereafter, the sample was added to a Muromac column (manufactured by Muromachi Technos), and the flow-through fraction was collected. This fraction was concentrated using an Amicon ultrafilter unit (fractional molecular weight 30 kDa) (Millipore), and further diluted with 50 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.0). Buffer exchange was performed. The obtained fraction was designated as GlcNAc-IDUA (sugar chain receptor).

<Preparation of sugar chain donor>
(Reagent and measurement method)
Unless otherwise stated, the reagents used were those from Tokyo Chemical Industry Co., Ltd. For NMR, ECA-400 manufactured by JEOL Ltd. was used. The MALDI-TOF MS measurement was performed using an autoflex speed-tko1 reflector system manufactured by Bruker Daltonics. Thin layer chromatography (hereinafter referred to as TLC) was developed using Merck 60F 254 and colored with 10% ethanol sulfate.

<Various measurement methods>
~ MALDI-TOF MS ~
A solution obtained by dissolving the reaction solution as it is or after purification in a solvent (for example, methylene chloride, ethyl acetate, methanol, toluene, acetone, water) and a DHBA solution (20 mg / ml 2,5-dihydroxybenzoic acid in 50% methanol) 0.5 to 2 μL each of the solution dissolved in an aqueous solution was spotted on a plate for MALDI-TOF MS analysis and dried, and MALDI-TOF MS analysis was performed using an autoflex speed-tko1 reflector system (manufactured by Bruker Daltonics). ) And the following measurement conditions were used.
Measurement mode: positive ion mode or negative ion mode and reflector mode or linear mode
・ Measurement voltage: 1.5Kv ~ 2.5Kv
Measurement molecular weight range: 0 to 10,000 (m / z)
・ Total number of times: 500-10000
Hereinafter, a specific method for preparing a sugar chain donor will be described.

(Synthesis of Compound 12 (Sugar Chain Donor))
Compound 12 was synthesized as a sugar chain donor by the route shown below.

As shown in the above route, a hexasaccharide derivative (Compound 6) was synthesized by a known glycosylation method. Thereafter, the paramethoxyphenyl group at the reducing end is converted to a fluoro group which is a leaving group, and glycosylation is performed under the glycosylation conditions described below to synthesize compound 9 which is a heptasaccharide derivative, Deprotection of two protecting groups at the position where an acid group is introduced, followed by introduction of a phosphate group, followed by deprotection of the protecting group, a parasaccharide phenyl derivative of 7 sugars having Man6P at the terminal (sugar chain Donor) was obtained.

~ Synthesis of Compound 6 ~
Man [2Bz, 3All, 46Bzd] β (1-4) GlcNPhth [36Bn] -β-MP (product code: M2442) manufactured by Tokyo Chemical Industry Co., Ltd. and compound 1 (2.9 g, 3.1 mmol) and Compound 2 (4.7 g, 4.6 mol) synthesized according to the literature [Tetrahedron Lett., Vol. 51, 4323-4327, (2010)] was dissolved in 150 ml of toluene, and 4-15 g of molecular sieves was added and stirred at room temperature for 1 hour. The reaction solution was cooled to −30 ° C., 1.6 g (7.0 mmol) of N-iodosuccinimide and 61 μL (0.7 mmol) of trifluoromethanesulfonic acid were added, and the mixture was stirred for 3 hours. After confirming the reaction by TLC, 210 μL (1.5 mmol) of triethylamine was added to the reaction solution to stop the reaction, followed by filtration through Celite. The reaction mixture was diluted with ethyl acetate, washed successively with saturated aqueous sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 11: 1) to obtain Compound 3 (4.5 g, 2.5 mmol) in a yield of 76%.
Next, 0.91 g (3.5 mmol) of silver trifluoromethanesulfonate, 0.67 g (1.8 mmol) of hafnocene dichloride, and 4.2 g of molecular sieves are placed in an eggplant flask to remove the benzylidene acetal protecting group of Compound 3. Then, a solution of compound 4 (1.3 g, 0.7 mmol) obtained in 21 ml of toluene was added and stirred for 1.5 hours. After the reaction solution was cooled to −30 ° C., O- (6-O-acetyl-2,3,4-tri-O— synthesized according to the literature [Tetrahedron Lett., Vol.40, 8049-8053, (1999)]. Benzyl-α-D-mannopyranosyl)-(1 → 6) -2,3,4-tri-O-benzyl-α-D-mannopyranose prepared from compound 5 (0.82 g, 0.88 mmol) in toluene A 21 mL solution was added to the reaction solution and stirred for 1 hour. The solution was filtered through celite, diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 8: 1 → 5: 1) to obtain 1.7 g of a product. The product was subjected to NMR measurement, and the following results were obtained. ¹ H-NMR (CDCl ₃ , 400 MHz) δ (ppm): 5.54 (d, 1H, J = 8.7 Hz, H-1a) 67 (brs, 1H, H-1b), 4.33 (m, 1H, H-2a), 4.18 (m, 1H, H-3a), 4.06 (m, 1H, H-4a), 3.42 (m, 1H, H-5a), 1.93 (s, 3H, Ac), 1.88 (s, 3H, Ac).
¹³ C-NMR (CDCl ₃ , 100 MHz) δ: 170.92, 170.73, 155.34, 150.87, 137.90-139.10 (arom.), 126.86-128.51 (arom. ), 118.72, 114.30, 101.99, 101.49, 99.28, 98.43, 98.00, 97.62, 83.15, 79.84, 79.67, 79.40, 79.06, 78.60, 75.36, 75.17, 75.10, 74.97, 74.82, 74.71, 74.57, 74.41, 74.12, 73.47, 73. 35, 72.70, 72.54, 72.20, 72.01, 71.36, 71.05, 70.58, 69.97, 68.12, 66.68, 65.61, 63.57, 63.48, 55.57, 2 .84.
From the above measurement results, it was shown that Compound 6 (0.61 mmol), which is a sugar chain donor, was obtained in a yield of 87%.

-Synthesis of Compound 9-
Compound 6 (1.0 g, 0.36 mmol) was dissolved in 20 mL of acetonitrile, 15 mL of toluene, and 10 mL of water. After cooling to 0 ° C., 2.0 g (3.6 mmol) of ammonium cerium (IV) nitrate was added and stirred for 2 hours. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed successively with water, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 3: 1) to obtain 0.81 g (0.31 mmol) of a 1-OH compound in which the reducing end was deprotected at a yield of 84%. . 0.81 g (0.31 mmol) of the obtained 1-OH compound was dissolved in 8.1 mL of methylene chloride and then cooled to −20 ° C., and 0.16 mL (1.2 mmol) of (diethylamino) sulfur trifluoride was added. Stir for 5 hours. After confirming the reaction by TLC, methanol (0.1 mL, 3.1 mmol) was added to the reaction solution to stop the reaction and diluted with methylene chloride. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (toluene: ethyl acetate = 5: 1) to obtain Compound 7 (0.72 g, 0.27 mmol).
Next, 0.21 g (0.82 mmol) of silver trifluoromethanesulfonate, 0.28 g (1.6 mmol) of hafnocene dichloride, and 2.2 g of molecular sieves were added to the eggplant flask [Reference: Reference 1 (Tetrahedron Lett., Vol. 30, 4853-4856, (1989)) and Reference 2 (Carbohydr Lett., Vol. 295, 25-39, (1996))], and compound 8 (product code: M1615) manufactured by Tokyo Chemical Industry Co., Ltd. (0.24 g, 0.41 mmol) was added to a toluene 11 mL solution and stirred for 2 hours under an argon atmosphere. After cooling the reaction solution to −30 ° C., a solution of compound 7 (0.72 g, 0.27 mmol) in toluene (11 mL) was added and stirred for 13 hours. After confirming the reaction by TLC, the solution was filtered through Celite, diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to obtain Compound 9 (0.70 g, 0.22 mmol) with a yield of 72% based on Compound 6.

~ Synthesis of Compound 10 ~
Compound 9 (0.52 g, 0.16 mmol) was dissolved in 10 mL of n-butanol, 0.54 mL (8.1 mmol) of ethylenediamine was added, and the mixture was stirred at 110 ° C. for 6 hours, then stirred at room temperature for 10 hours, and further raised to 140 ° C. Warmed and stirred for 10 hours. After confirming the reaction by TLC, the reaction solution was concentrated under reduced pressure and subjected to toluene azeotropy. To the obtained residue, pyridine (5 mL) was added and dissolved, acetic anhydride (0.24 mL, 2.58 mmol) and 4-dimethylaminopyridine 5.0 mg (0.041 mmol) were added, and the mixture was stirred at room temperature for 4 hours, and further stirred. Stir at 0 ° C. for 3 hours. After confirming the reaction by TLC, the reaction solution was concentrated under reduced pressure, azeotroped with toluene, and diluted with ethyl acetate. The organic layer was washed successively with 2M hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (methylene chloride: methanol = 100: 1 → 80: 1) to obtain 0.35 g (0.11 mmol) of an acetylated compound in a yield of 71%. 0.34 g (0.12 mmol) of the obtained compound was dissolved in a mixed solvent of 1.7 mL of tetrahydrofuran and 1.7 mL of methanol, sodium methoxide (2.5 μL, 0.012 mmol) was added, and the mixture was stirred for 2.5 hours. . Thereafter, sodium methoxide (5.0 μL, 0.023 mmol) was further added and stirred for 17 hours. DOWEX (50WX8 (200-400H ⁺ )) was added for neutralization, and after filtration, the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (dichloromethane: methanol = 70: 1) to obtain Compound 10 (0.24 g, 0.085 mmol) in a yield of 73%.

~ Synthesis of Compound 11 ~
Phosphorylation was performed according to the literature [Bull.Chem.Soc.Jpn., Vol.81, No.7, 796-819, (2008)]. Compound 10 (0.10 g, 0.034 mmol) was dissolved in 3 mL of tetrahydrofuran, 0.18 g (0.34 mmol) of tetrabenzyl pyrophosphate was added under an argon atmosphere, and the mixture was cooled to −60 ° C. Thereafter, 67 μL (0.088 mmol) of lithium bis (trimethylsilyl) amide (about 26% tetrahydrofuran solution, about 1.3 mol / L) was added and stirred for 1.5 hours. Further, after 2 hours and 3.5 hours, 67 μL (0.088 mmol) of lithium bis (trimethylsilyl) amide (about 26% tetrahydrofuran solution, about 1.3 mol / L) was added and stirred for 0.5 hours. After confirming the reaction by TLC, ethyl acetate and saturated aqueous sodium hydrogen carbonate solution were added to the reaction solution to stop the reaction. After dilution with ethyl acetate, the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was roughly purified by gel filtration column chromatography. Subsequently, it was purified by silica gel column chromatography (toluene: acetone = 8: 1 → 7: 1) to obtain Compound 11 (90 mg, 0.026 mmol) in a yield of 76%.

~ Synthesis of Compound 12 ~
Compound 11 (0.085 g, 0.024 mmol) was dissolved in a mixed solution of 15 mL of tetrahydrofuran, 6 mL of ethanol, and 6 mL of water, palladium on carbon (0.34 g) was added, and the mixture was stirred under a hydrogen stream for 22 hours. After confirming the reaction by TLC, the reaction solution was filtered through Celite and concentrated under reduced pressure. The resulting residue was converted to a sodium salt with DOWEX (Na ⁺ ) and then purified by gel filtration column chromatography to obtain 0.035 g of product. The product was subjected to NMR measurement and the following results were obtained. Measurement values obtained by NMR measurement are shown below. The obtained ¹ H-NMR spectrum is shown in FIG. 4, and the enlarged spectrum is shown in FIG. Further, the measurement values obtained by MALDI-TOF MS measurement of the product are shown below, and the obtained mass spectrum is shown in FIG.
¹ H-NMR (D ₂ O, 400 MHz) δ (ppm): 6.94-7.03 (m, 4H), 5.39 (brs, 1H), 5.01 to 5.03 (m, 2H) 4.90 (m, 2H), 4.81 (brs, 1H), 4.62 (d, 1H, H-1, J _1,2 = 7.78 Hz), 3.62-4.22 (m 44H), 2.08 (s, 3H, Ac), 2.02 (s, 3H, Ac).
¹³ C-NMR (D ₂ O, 100 MHz) δ (ppm): 175.53, 175.44, 155.53, 151.73, 118.94, 115.76, 103.15, 102.08, 101. 61, 101.15, 100.51, 100.19, 80.43, 79.75, 75.43, 75.09, 72.85, 72.64, 71.39, 71.35, 71.01, 70.87, 70.63, 67.20, 66.68, 60.64, 56.48, 55.63, 22.92, 22.81.
³¹ P-NMR (D ₂ O, 162 MHz) δ (ppm): 3.03 (brs, 2P).
^{MALDI-TOF / MS calcd for [} M-H] - 1499.40; found, 1499.96.
The above measurement results showed that Compound 12 as a sugar chain donor was obtained in a yield of 86%.

<End M mutant>
The N175Q mutant (manufactured by Tokyo Chemical Industry Co., Ltd., glycosynthase recombinant) was used as the endo M mutant used in this example.

(Glycosyl transfer by endo M mutant)
<Preparation method of Man6PR (Dom9-His)>
A lectin (CI-Man6PR) for detecting Man6P was prepared as follows.
That is, a plasmid vector for expression of CI-Man6PR (Dom9-His) prepared by referring to Akeboshi et al. [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 73, 4805-4812, (2007)] was used as a methanol-assimilating yeast strain. (Pichia pastoris GS11) was introduced into 5 strains by electroporation. After culturing in BMGY medium containing 2% methanol for 5 days, the whole culture solution containing yeast after culturing is centrifuged (6000 rpm, 4 ° C., 1 h, KUBOTA 6800 (rotor: RA-1500)), and the culture supernatant is recovered. did. The culture supernatant was filtered using a 0.22 μm filter (Millipore) and concentrated by ultrafiltration [Labscale TFF System, Pellicon XL5K (Millipore)].
The concentrated culture supernatant was bound to a His tag purification resin (Ni-Sepharose 6 Fast Flow, manufactured by GE), and then washed with a washing buffer [20 mM sodium phosphate according to the protocol attached to the resin. , 0.5 M NaCl (pH 8.0)] and elution with elution buffer [20 mM sodium phosphate, 0.5 M NaCl, 50 mM imidazole (pH 8.0)] A solution containing CI-Man6PR (Dom9-His) was obtained. The obtained solution was appropriately diluted and used for detection of Man6P in the product in the solution after the transglycosylation reaction described later.

<Glycosylation reaction by endo M mutant>
GlcNAc-IDUA as a sugar chain acceptor and the above compound 12 as a sugar chain donor are dissolved in 10 μL of 50 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.0). The sugar chain transfer reaction was performed under the following conditions.
<Condition>
Sugar chain donor: Compound 12 ... 200 nmol
Sugar chain receptor: GlcNAc-IDUA ... 200pmol
・ End M mutant: N175Q ... 2mU
-Molar ratio (D / A) of sugar chain donor (D) to sugar chain acceptor (A): 1000
-Reaction temperature: 30 ° C
・ Reaction time: 24 hours

<Lectin blot>
Samples (solution after sugar chain transfer reaction, etc.) were subjected to electrophoresis with 7.5% SDS-PAGE, and then transferred to a PVDF membrane using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The protein was transferred at a constant voltage of 15 V for 1 hour. As a transfer buffer, a mixed solution of 48 mM Tris, 39 mM glycine, and 20% methanol was used. The transferred PVDF membrane was blocked by shaking in 50% Blocking one (Nacalai Tesque) / TBS at room temperature for 1.5 hours.
1 using the above Dom9-His as ^st probes were shaken PVDF membranes in 50% Blocking one / TBS (4 ℃, O / N). Thereafter, the PVDF membrane was washed 5 times at room temperature in 0.1% Tween20 / TBS (TBST) 5 times, and further washed in TBS at room temperature for 5 minutes. Use as 2 ^nd probe anti-His antibody (Anti-His tag-mouse IgG (QIAGEN Inc.)), in 50% Blocking one / TBS, was shaken for 1 hour PVDF membranes at room temperature. Thereafter, the PVDF membrane in TBST was repeatedly washed 5 times at room temperature for 5 minutes, and further washed in TBS at room temperature for 5 minutes. 3 ^rd using HRP-conjugated anti-mouse IgG as a probe (Biosource, Inc.), the PVDF membrane was shaken for 1 hour at room temperature in 50% Blocking one / TBS. Thereafter, the PVDF membrane in TBST was washed 5 times at room temperature for 5 minutes, and further washed in TBS at room temperature for 5 minutes. The PVDF film was chemiluminescent using Western lightning Chemiluminescence Reagent Ultra / Plus (manufactured by Perkin Elmer), and using an image analyzer [LAS-4000miniEPUV (manufactured by Fujifilm)] with high exposure conditions with an exposure time of 1 second. The signal (band) was detected at.

FIG. 7A shows the result of SDS-PAGE for the solution after the above reaction, and FIG. 7B shows the result of lectin blotting. In FIG. 7A and FIG. 7B, lane 1 is a sample containing IDUA (hereinafter referred to as CHO-IDUA) obtained by culturing after culturing CHO cells (K1 strain, RIKEN Cell Bank) into which IDUA gene has been introduced. Lane 2 is the result of the sample containing TG-IDUA, Lane 3 is the result of the sample after treating TG-IDUA with End D, and Lane 4 is the result after the transglycosylation reaction. It is a result.
From FIG. 7A, as shown in lane 3, since the band of lane 3 appears on the lower molecular weight side than the band of lane 2, TG-IDUA to GlcNAc-IDUA are processed by the above processing of End D. It was shown that it was obtained. Next, as shown in Lane 4, after the transglycosylation reaction, the GlcNAc-IDUA band in the vicinity of 80 kDa disappeared in Lane 3, and a band appeared on the high molecular weight side instead. Therefore, sugar chain transfer occurred in GlcNAc-IDUA, which is a sugar chain receptor, and a product after sugar chain transfer in which 2 to 12 Man6P were bound to 1 to 6 N-linked sugar chains was obtained. It was shown that.
Further, from the result of FIG. 7B, a strong luminescence signal is obtained only in the band corresponding to the band of lane 4 in lanes 2 to 4 of FIG. 7B, suggesting that the product after sugar chain transfer has Man6P. From these results, it was shown that the sugar chain containing Man6P in the sugar chain donor was transferred to the sugar chain acceptor. From the above results, it was suggested that almost all the products after sugar chain transfer have at least one M6P-containing sugar chain.
Thus, it was shown that the sugar chain transfer reaction using the sugar chain donor having Man6P described above is effective for the preparation of a Man6P-containing glycoprotein.

<Preparation of Man6P-containing glycoprotein (Man6P-IDUA)>
The solution after the transglycosylation reaction of Example 1 was subjected to ultrafiltration using an Amicon ultra filter unit (fractionated molecular weight of 30 kDa) (manufactured by Millipore) to remove unreacted sugar chain donors. Further, the buffer solution was replaced with a phosphate physiological buffer solution (hereinafter referred to as PBS) to obtain Man6P-IDUA (10.5 μg). The obtained Man6P-IDUA was used for examining the supplementary effect on the following mucopolysaccharidosis type 1 patient fibroblasts (MPS1 patient-derived fibroblasts) and the like.
Hereinafter, TG-IDUA, GlcNAc-IDUA, and Man6P-IDUA may be collectively referred to as supplementary enzymes.

(Supplementary effect on MPS1 patient-derived fibroblasts)
The replenishment effect was examined by comparing the activity of the intracellular enzyme (enzyme source) obtained after each supplemental enzyme was added to various cells as follows.

<Various cells>
MPS1 patient-derived fibroblasts are present in Ham's F-10 medium (+ 10% Fetal bovine serum) from oral mucosal tissues after approval by Tokushima University Hospital Small Ethics Committee and informed consent to patients It was obtained by inducing under and establishing by adhesion culture and subculture. “Normal fibro” (Hs68), a healthy human fibroblast, was obtained by purchasing an ATCC product (product number: CRL-1635), and similarly cultured and subcultured.

<Each enzyme source>
Each enzyme source was prepared as follows.
To each 1 mL of culture solution having 5 × 10 ⁴ cells / well of MPS1 patient-derived cells or healthy human fibroblasts (HS68) cultured in a collagen-coated 24-well plate (Collagen-coated microplate 24well, manufactured by IWAKI) TG-IDUA (endoenzyme-free IDUA) was added at 2000 nmol / h (2.5 μg), GlcNAc-IDUA was added at 2000 nmol / h (2.85 μg), and Man6P-IDUA 2000 nmol / h (4.6 μg) was added. The cells were cultured for 24 hours at 37 ° C. in a 5% CO ₂ atmosphere. After culturing, the cells were washed twice with 1 mL of PBS, and further 0.2 mL of 0.5% trypsin EDTA solution was added, and the cells were detached from the wells by incubation for 5 minutes. After adding 1 mL of the culture solution to stop the trypsin reaction, the cells were collected by centrifugation at 4 ° C. and 200 × g for 5 minutes. 100 μL of 50 mM sodium acetate buffer (pH 4.5) (containing 500 mM NaCl, 1% NP40, 1 μM pepstatin A, 20 μM leupeptin and 2 mM EDTA) is added to the obtained cells, and an ultrasonic generator Cells were disrupted with (MUS-10, Tokyo Rika Kikai Co., Ltd.). Thereafter, the mixture was centrifuged at 17,900 × g for 15 minutes at 4 ° C., and the obtained supernatant was used for the next enzyme activity measurement as an enzyme source. Moreover, the same extraction process was performed also on the healthy person's fibroblast, and the obtained enzyme source was used for the following enzyme activity measurement.
In addition, after incubating MPS1 patient-derived fibroblasts without adding any of the supplementary enzymes, the enzyme source obtained by the above preparation method is referred to as MPS-E, and the healthy human fibroblasts (HS68) The enzyme source obtained by the above preparation method after incubation without adding any of the supplemental enzymes is referred to as NF-E. In addition, after TG-IDUA was added to MPS1 patient-derived fibroblasts and incubated, the enzyme source obtained by the above preparation method was referred to as MPS-CE, and GlcNAc-IDUA was added to MPS1 patient-derived fibroblasts. The enzyme source obtained by the above preparation method is called MPS-GI-E, and Man6P-IDUA is added to MPS1 patient-derived fibroblasts and incubated, and then the above preparation method The enzyme source obtained by this is referred to as MPS-MI-E.

<Enzyme activity measurement>
For IDUA activity, 4-methylumbelliferyl (4MU) -α-L-idpyranoside (manufactured by Toronto Research Chemicals) (hereinafter sometimes referred to as 4MU-Ido) as a substrate in a sodium citrate buffer (pH 4. After incubating at 37 ° C. for 30 minutes in 5), the fluorescence intensity (Ex: 355 nm, Em: 460 nm) of the released 4MU was measured as an index. The enzyme activity using 4MU-Ido as a substrate may be referred to as IDUA activity.
In addition, MUG degradation activity, which is β-hexosaminidase activity, is sodium citrate buffer (pH 4) using 4-methylumbelliferyl (4MU) -N-acetyl-β-D-glucosaminide (manufactured by SIGMA) as a substrate. .5) was incubated at 37 ° C. for 30 minutes, and the fluorescence intensity (Ex: 355 nm, Em: 460 nm) of the released 4MU was measured as an index.

<Evaluation>
The supplementation effect on MPS1 patient-derived fibroblasts was evaluated as follows. That is, the activity recovery ratio of each IDUA activity of each enzyme source (MPS-E, NF-E, MPS-CE, MPS-GI-E, and MPS-MI-E) is determined by the MUG degradation activity of each enzyme source. Was calculated by the following formula 1 as the control enzyme activity.
Each activity recovery ratio = (IDUA activity of each enzyme source / MUG decomposition activity of each enzyme source) / IDUA activity of MPS-E Formula 1

FIG. 8A shows the activity recovery ratio in intracellular IDUA activity after adding each of the above-mentioned supplementary enzymes to MPS1 patient-derived fibroblasts and incubating for 24 hours, and FIG. 8B shows the MUG of each of the above enzyme sources. Degradation activity is shown. Further, the numbers on each column in FIG. 8A indicate the value of the activity recovery ratio, and the numbers on each column in FIG. 8B indicate the value of the MUG decomposition activity.
From the results, the activity recovery ratio of other enzyme sources other than MPS-E showed a very high value of 23 to 52 times that of MPS-E. In addition, the activity recovery ratio of MPS-MI-E was shown to be the highest in the activity recovery ratio compared to the activity recovery ratios of other enzyme sources. Therefore, it was shown that when Man6P-IDUA was added to cells having no IDUA activity (MPS1 patient-derived fibroblasts), the IDUA activity in the cells was remarkably improved. Thus, it was shown that by adding Man6P-IDUA to cells, an enzyme having IDUA activity was supplemented in the cells, and the activity was further improved.

(Detection of intracellular distribution of a fluorescent group-modified Man6P-containing compound)
As described below, a fluorescent group-modified Man6P-containing compound (Man6P-Man5-AFO) and a fluorescent group-modified Man6P-IDUA were prepared and used to produce the Man6P-containing compound and Man6P-IDUA in cells. The localization and distribution of was investigated.

<Synthesis of fluorescent group-modified Man6P-containing compound>
Compounds containing Man6P at the non-reducing end can be obtained from Asanuma et al. [Angew. Chem. Int. Ed. Engl., Vol. 53 (24), 6085-6089, (2014)] and the website of Goryuka Kagaku Co., Ltd. (Http://goryochemical.com/products/acidifluor/acidifluor-orange.html). That is, the compound 7 obtained above and 3- (Carbobenzoxyamino) -1-propyl 3,6-di-O-benzoyl synthesized according to the literature [Tetrahedron Lett., Vol. 61, 4313-4321, (2005)]. A compound having an n-propoxy group having an amino group at the 3-position as shown in FIG. 9 by using a method similar to the method of synthesizing compound 12 using -2-deoxy-2-phthalimido-β-D-glucopyranoside AF was synthesized. Further, compound AF1 was prepared by binding with a fluorescent molecule (AcidiFluor ORANGE-NHS, manufactured by Goryuka Kagaku Co., Ltd.) that is activated at acidic pH. FIG. 10 shows MALDI-TOF MS spectra of the obtained compound AF1 and compound AF. From the results, it was shown that the Man6P addition compound (Man6P-Man5-AFO) modified with a fluorescent group was obtained from the compound AF which is a raw material compound. AFO means a fluorescent compound (AcidiFluor ORANGE).

Next, it was evaluated whether or not Man6P-Man5-AFO, which is a derivative of 6-saccharide oligosaccharide, was taken into cells through binding to the Man6P receptor on the surface of MPS1 patient-derived fibroblasts. Man6P-Man5-AFO was added to a culture solution containing MPS1 patient-derived fibroblasts cultured in a 35 mm collagen-coated dish (35 mm / Collagen-coated dish, manufactured by IWAKI) at a final concentration of 50 nM, and 24 hours later, The fluorescence of Man6P-Man5-AFO was observed with a fluorescence microscope (BZ-9000, manufactured by KEYENCE). The results are shown in FIG.
From the results, the AFO reagent itself was not taken up into the cells, whereas Man6P-Man5-AFO was taken up into the cell name, and fluorescence was observed. On the other hand, since intracellular fluorescence was not observed in the presence of 5 mM mannose 6-phosphate (M6P), which is a competitive inhibitor in M6P receptor uptake, Man6P-Man5-AFO is mediated by M6P receptor on the cell surface. It has been shown to be taken up and transported to late endosomes / lysosomes, intracellular organelles with acidic pH. Thus, Man6P-Man5-AFO has been shown to function as a tag for delivery of molecules bound to Man6P to intracellular organelles (lysosomes).

<Synthesis of fluorescent group-modified Man6P-IDUA>
30 μg of Man6P-IDUA obtained as described above was dissolved in 300 μL of 0.1 M sodium bicarbonate buffer (pH 8.3), and then 60 μg of AcidiFluor ORANGE-NHS (manufactured by Goryuka Chemical Co., Ltd.) dissolved in DMSO (DMSO amount: 6 μL) ) Were added, mixed in a tube, and stirred at room temperature for 2 hours while being protected from light. Fluorescent group-modified (fluorescent group binding type) Man6P-IDUA (ultrafluorescent group binding type) by ultrafiltration with an Amicon ultra filter unit (fractional molecular weight 30 kDa) (manufactured by Millipore) and removing unreacted AcidiFluor ORANGE-NHS Man6P-IDUA-AFO) was obtained. The obtained Man6P-IDUA-AFO was stored at −80 ° C. until just before use.

<Detection of intracellular distribution of Man6P-IDUA-AFO>
The Man6P-IDUA-AFO obtained above was added to a culture solution of MPS1 patient-derived fibroblasts cultured on an 8-well chamber slide (manufactured by Nunc) at a final concentration of 70 nM. After a period of time, the cells were washed with PBS, and then the fluorescence of AFO was observed with a fluorescence microscope (BZ-9000, manufactured by KEYENCE). The results are shown in FIG.
From the results, it was taken up into cells and fluorescence was observed in acidic pH organelles (late endosome / lysosome). On the other hand, intracellular fluorescence was significantly reduced in the presence of 5 mM mannose 6-phosphate (M6P), which is a competitive inhibitor in M6P receptor uptake. Further, in this case, Man6P-IDUA-AFO was not taken up into the cells, and fluorescence with an acidic pH organelle was not observed. Therefore, Man6P-IDUA-AFO was taken up through the M6P receptor on the surface of fibroblasts and was shown to be transported to the late endosome / lysosome, which is an acidic organelle.
Thus, since the localization and distribution of the target Man6P-containing glycoprotein in the cell can be easily detected by using the Man6P-containing glycoprotein introduced with a fluorescent group, detection using the Man6P-containing glycoprotein is possible. The effectiveness of the method was demonstrated.

[Example 2]
<Preparation of Man6P-containing CTSA>

(Purification of CTSA from culture supernatant of CTSA gene constitutive expression CHO cell line)
A three-step purification method (affinity chromatography → hydrophobic interaction chromatography) of a CTSA protein from a CHO cell line (CHO / CTSA strain) constitutively expressing a recombinant human CTSA (recombinant human cathepsin A; CTSA) gene Chromatography → affinity chromatography).

First, affinity chromatography using ConA Sepharose 4B (manufactured by GE Healthcare) was performed on the culture supernatant of the CHO / CTSA strain to purify the CTSA protein. Specifically, the culture supernatant was collected, and CaCl ₂ and MnCl ₂ were added to the culture supernatant so that the final concentration was 1 mM. Next, ConA Sepharose was equilibrated with a binding buffer [20 mM MES buffer, 150 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl ₂ (pH 5.5)], and the culture supernatant was applied thereto for adsorption to Con A. It was. After removing the flow-through fraction, ConA Sepharose was washed with ConA washing buffer [20 mM MES buffer, 500 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl ₂ (pH 5.5)]. Thereafter, elution was performed with an elution buffer [20 mM MES buffer, 150 mM NaCl, 1 mM CaCl ₂ , 1 mM MnCl _2, 0.5 M methyl-α-D-mannopyranoside (pH 5.5)].

Next, the ConA column elution fraction obtained was concentrated with an Amicon ultrafilter unit 30 kDa cut, and the buffer solution was exchanged with a buffer solution for butyl binding [50 mM MES, 1 M ammonium sulfate (pH 5.5)]. Thereafter, filtration was performed with a 0.22 μm filter, and purification was performed by hydrophobic interaction chromatography using a HiTrap Butyl HP column (GE Healthcare). For purification, AKTA purifier UPC-10 was used, and elution was performed while the ammonium sulfate concentration was continuously changed from 1 M to 0 M after adsorption of CTSA protein to the column. The resulting Butyl column elution fraction was buffered in Phos-tag (registered trademark) binding buffer [100 mM Tris-acetate buffer solution (pH 7.5)] using an Amicon ultra filter unit 30 kDa cut (manufactured by Millipore). The liquid was changed.

Thereafter, purification was performed by affinity chromatography using Phos-tag (registered trademark) agarose (manufactured by Wako). Phos-tag® agarose was equilibrated with a binding buffer [100 mM ［Tris-acetate buffer (pH 7.5)], and a sample was applied thereto for adsorption to Phos-tag ™ agarose. It was. After removing the flow-through fraction, the Phos-tag (registered trademark) agarose was washed with Phos-tag (registered trademark) washing buffer [100 mM Tris-acetate buffer solution (pH 7.5)]. Thereafter, CTSA was eluted with an elution buffer [20 mM sodium phosphate buffer (pH 6.5)].

(Preparation of GlcNAc-CTSA (sugar chain receptor) by endoenzyme)
The sugar chain receptor hydrolyzes the chitobiose structure of an N-linked sugar chain having a mannose number of 5 or more using the endoenzyme (Endo-Hf, manufactured by New England Biolabs) for the CTSA obtained as described above. It was prepared by. 60 μg CTSA and 120 U Endo-Hf were dissolved in 200 μL of 50 mM MES buffer (pH 6.0) and incubated at 4 ° C. for 48 h. After the reaction, Endo-Hf was removed by adsorption using an MBP trap column (manufactured by GE Healthcare). Specifically, the reaction solution was replaced with 20 mM Tris-HCl buffer (pH 7.0) and 200 mM NaCl using an Amicon ultra filter unit 10 kDa cut (manufactured by Millipore). Thereafter, filtration with a 0.22 μm filter (Millipore) was performed, and the reaction solution was added to 1 mL of an MBP trap column equilibrated with 20 mM Tris-HCl buffer (pH 7.0) and 200 mM NaCl. Hf was adsorbed on the MBP trap column. The flow-through fraction was collected, and 20 mM Tris-HCl buffer (pH 7.0) and 2OmL of 200 mM NaCl were added to the MBP trap column to wash out the flow-through fraction. The flow-through fraction was concentrated using an Amicon ultra filter unit 10 kDa cut (manufactured by Millipore), and the buffer solution was exchanged with 50 mM MES buffer solution (pH 6.0). The obtained fraction was designated as GlcNAc-CTSA (sugar chain receptor).

(Glycosyltransferase reaction by endo M mutant)
GlcNAc-CTSA as the sugar chain acceptor, Compound 12 as the sugar chain donor, Endo M mutant (Glycosynthase (Endo-M-N175Q), manufactured by Tokyo Chemical Industry Co., Ltd.) as the glycosyltransferase; The product code G0365) was dissolved in 60 μL of 50 mM MES buffer (pH 6.0) and 150 mM NaCl, and a sugar chain transfer reaction was performed under the following conditions.
<conditions>
Sugar chain donor: Compound 12 ... 200 nmol
Sugar chain receptor: GlcNAc-CTSA ... 200pmol
・ Glycosyltransferase: Glycosynthase (Endo-M-N175Q) 2 mU
-Molar ratio (D / A) of sugar chain donor (D) to sugar chain acceptor (A): 1000
-Reaction temperature: 30 ° C
・ Reaction time: 24h

For the solution after the above reaction, the result of CBB staining of the gel after SDS-PAGE is shown in FIG. 13A, and the lectin (Dom9-His (Akeboshi H. et al., Glycobiology (2009) 19 (9)): FIG. 13B shows the result of lectin blotting using 1002-1009))) as the primary probe. In FIGS. 13A and 13B, lane 1 is the result of the sample containing CTSA, lane 2 is the result of the sample containing GlcNAc-CTSA, and lane 3 is the result after the transglycosylation reaction.
The result of FIG. 13A shows that GlcNAc-CTSA is obtained from CTSA by the above Endo-Hf treatment because the band of lane 2 appears on the lower molecular weight side than the band of lane 1. It was. Next, as shown in lane 3, after the transglycosylation reaction using the endo M mutant (Endo-M (N175Q)), the lane 3 band appeared on the higher molecular weight side than the lane 2 band. Therefore, it was shown that a glycosyltransferase reaction occurred in GlcNAc-CTSA, which is a sugar chain receptor, and a post-glycosylation product in which one or two terminal Man6P-containing N-type sugar chains were linked was obtained.
Further, from the result of FIG. 13B, since the luminescence signal disappeared in lane 2 of FIG. 13B is newly obtained in lane 3, the sugar chain containing Man6P in the sugar chain donor is added to the sugar chain acceptor. Showed metastasis.

The disclosure of Japanese Patent Application No. 2016-069437 filed on Mar. 30, 2016 is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually described to be incorporated by reference, Incorporated herein by reference.

Claims

A sugar chain donor having a structure derived from an N-linked sugar chain in which a mannose-6-phosphate group is bonded to a non-reducing end in the presence of an endo M mutant of the following (a) or (b); A method for producing a mannose-6-phosphate group-containing glycoprotein comprising producing a mannose-6-phosphate group-containing glycoprotein by a sugar chain transfer reaction between the sugar chain receptor represented by formula (1):
(A) Endo M mutant having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine (b) One other than the 175th amino acid residue of (a) Alternatively, the transglycosylation reaction has an amino acid sequence modified within the range of homology of 80% or more with respect to the amino acid sequence of (a) by deletion, addition or substitution of a plurality of amino acid residues. An endo-M mutant having an activity of catalyzing.

(In general formula (1), Y 1 represents an acylamino group containing a structure derived from a glycoprotein. GlcNAc represents an N-acetylglucosaminyl group.)
The method for producing a mannose-6-phosphate group-containing glycoprotein according to claim 1, wherein Y 1 in the general formula (1) is an acylamino group containing a structure derived from a lysosomal enzyme.
The method for producing a mannose-6-phosphate group-containing glycoprotein according to claim 1 or 2, wherein the sugar chain donor is represented by the following general formula (2).

(In General Formula (2), X 1 , X 2 , X 3 , X 4 , X 5 and X 6 each independently represent a hydrogen atom or a saccharide-derived group, and X 1 , X 2 , X 3 , X 4, at least one of X 5 and X 6, the non-reducing end is a group derived from a saccharide having a mannose-6-phosphate group .Z 1 represents a hydrogen atom or a GlcNAc, Z 1 is GlcNAc In this case, the GlcNAc is bound to Man linked to GlcNAc at β1-4 by β1-4, Y 2 represents a monovalent substituent, and GlcNAc represents an N-acetylglucosaminyl group. , Β1-4 represents a β glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β glycoside bond between position 1 of Man and position 4 of GlcNAc, Man represents a mannosyl group, α1-6 represents Man 1st and Man 6th It represents α-glycosidic bond, [alpha] 1-3 represents a α-glycosidic bond between the 1- and 3-position of Man of Man.)
The method for producing a mannose-6-phosphate group-containing glycoprotein according to any one of claims 1 to 3, wherein the sugar chain donor is represented by the following general formula (3).

(In the general formula (3), X 7 , X 8 and X 9 each independently represent a hydrogen atom, Man, Manα1-2Man, Man6P, Man6Pα1-2Man or Man6Pα1-6Man. Man represents a mannosyl group. .Man6P represents a mannosyl group phosphoric acid group is bonded to the 6-position. also, X 7 -6 indicates the X 7 bound to 6-position of Man, X 8 -3 is attached to the 3-position of Man and showed X 8, X 9 -2 at least one .X 7, X 8 and X 9 showing the X 9 attached to the 2-position of Man is Man6P, either Man6Pα1-2Man and Man6Pα1-6Man GlcNAc represents an N-acetylglucosaminyl group, and α1-6 is an α-glycoside bond between position 1 of Man and position 6 of Man or position 1 of Man6P and M n represents the α-glycoside bond at the 6-position, α1-3 represents the α-glycoside bond between the 1-position of Man and the 3-position of Man, and α1-2 represents the α-glycoside between Man's 1-position and Man's 2-position Represents an α-glycoside bond between position 1 of Man6P and position 2 of Man, β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β position between position 1 of Man and position 4 of GlcNAc Represents a glycosidic bond, Y 3 represents a monovalent substituent.)
A fluorescent group-bound mannose-6-phosphate group obtained by further introducing a fluorescent group into the mannose-6-phosphate group-containing glycoprotein obtained by the production method according to any one of claims 1 to 4. A method for detecting the intracellular distribution of the fluorescent group-bound mannose-6-phosphate group-containing glycoprotein by applying the contained glycoprotein to cells.