JPH0759587A - Production of saccharide and complex saccharide - Google Patents

Abstract

PURPOSE:To obtain a saccharide or a complex saccharide having improved physicochemical or physiological properties such as stability and cell recognition property by carrying out the rearrangement reaction of sugar chains in the presence of endo-beta-N-acetylglucosaminidase M. CONSTITUTION:Rearrangement reaction expressed by the formula A-GlcNAc-C+ D A-GlcNAc-D+C (A is saccharide; GlcNAc is N-acetylglucosamine; C and D are independently saccharide, complex saccharide) is carried out in the presence of endo-beta-N-acetylglucosaminidase M. This process enables the rearrangement of any of the high-mannose type, mixed type and complex type chain in the case of asparagine-bonded sugar chain.

Description

Detailed Description of the Invention

[0001]

The present invention utilizes the glycosyl transfer activity of endo-β-N-acetylglucosaminidase M to transfer sugar chains such as asparagine-linked sugar chains of glycoproteins to sugars or glycoconjugates. , A method for remodeling carbohydrates or glycoconjugates.

[0002]

BACKGROUND OF THE INVENTION Carbohydrates and glycoconjugates (glycoproteins, glycolipids, proteoglycans) are found in a wide range of organisms such as animals, plants, insects and microorganisms. Carbohydrates are cytoskeletal substances and energy sources, glycoconjugates are cell adhesion,
It is a substance involved in cell recognition and cell-cell information transfer, and is known to have a very important function in living organisms.

On the other hand, recently, it is becoming clear that the sugar chain structure greatly affects the pharmacokinetics in many glycoprotein drugs such as erythropoietin and interferon. Currently, some glycoprotein drugs are mass-produced using microorganisms and animal cells, but they have problems in pharmacokinetics and stability due to different sugar chain structures, which may lead to large doses and side effects. There is a cause. Therefore, by changing the sugar chain structure, it is expected to produce a glycoprotein drug with improved pharmacokinetics and stability.

An asparagine-linked sugar chain is a sugar chain bonded to an asparagine residue of a polypeptide and has a complex structure in which several to several tens of various sugars are variously bonded. This sugar chain is roughly classified into high-mannose type, mixed type, and complex type. In particular, the complex type sugar chain is a sugar chain structure often found in animals and has important physiological functions in vivo. Are known. Regarding the types of asparagine-linked sugar chains, Fig. 1 shows high-mannose type, Fig. 2 shows mixed type, Fig. 3 shows complex 1-branch type, Fig. 4 shows complex 2-branched type, and Fig. 5 shows complex type. Three-branching type, and FIG. 5 shows a composite four-branching type.

Since asparagine-linked sugar chains are composed of various sugars, if organic synthetic means are used to obtain these sugars, it is necessary to carry out a reaction to sequentially bond sugars. It is complicated. Therefore, a method for converting (remodeling) the sugar chain itself by a simple method is required. As such a method, RB Trimble et al. Have described Flavobacterium meningosepticum (Flavobacteri
um meningosepticum) -derived endo-β-N-acetylglucosaminidase (hereinafter referred to as “End F”) was reported [Journal of Biological Chemistry (J. Biol. Chem.), Vol. 12005 (1986)], K. Takegawa et al., Endo-β-N-acetylglucosaminidase derived from Arthrobacter protophormiae (hereinafter End A).
Has been reported (JP-A-5-6459).
No. 4).

[0006] In all of these methods, the sugar chain is transferred by utilizing the glycosyl transfer activity of endo-β-N-acetylglucosaminidase, which originally cleaves the asparagine-linked sugar chain of the glycoprotein. The transglycosylation reaction using endo-β-N-acetylglucosaminidase is extremely effective because it can transfer a sugar chain composed of many sugars at once. End-β-
The common enzymatic properties of N-acetylglucosaminidase are represented by the following formula [IV]:

[0007]

[Chemical 4]

Hydrolyzing the position of the arrow.
However, transglycosylation activity is not a general property of this enzyme, and for example, Streptomyces pricatus
It has not been found in endo-β-N-acetylglucosaminidase H derived from omyces plicatus). Furthermore, endo-β-N-acetylglucosaminidase capable of transferring the complex form of asparagine-linked sugar chain to a wide range of sugars or glycoconjugates has not been known so far.

For example, in the case of endo F, among the asparagine-linked sugar chains, high mannose type, bisecting N-
Hybrids without acetylglucosamine can undergo a double-stranded complex type transfer, but others cannot. Moreover, there is a problem that only glycerol can be used as an acceptor, and there is a limitation in use. Also, end A
In this case, various acceptors can be used, but since the original hydrolysis activity is limited to the high mannose type of asparagine-linked sugar chains and a part of mixed-type sugar chains [Applied And Environmental Microbiology (Appl. Environ. Microbiol.
), 55, 3107-3112 (1989)], the complex type sugar chain cannot be excised from the donor, and it is considered that the transfer reaction cannot be performed. Therefore, in the production of glycoprotein derived from animals, there are problems in use, such as limited use or difficulty in use.

On the other hand, endo-β-N-acetylglucosaminidase M is Mucor hiemalis SK-31 belonging to the genus Mucor.
It is an endo-β-N-acetylglucosaminidase produced by 4. Then, S. Kadowaki et al. Have reported a catalytic action of degrading the asparagine-linked sugar chains of glycoproteins in the high mannose type, the mixed type, and the complex type in the arrow portion of the formula [IV]. Agricultural and Biological Chemistry
l. Chem.), 52, pp. 2387-2389 (1988)].

However, it is not known that this enzyme has an activity of catalyzing the transfer reaction of sugar chain.

[0012]

In view of the above problems, a method for easily transferring a sugar chain to a sugar or a complex sugar, that is, a wide range of asparagine-linked sugar chains such as high mannose type, mixed molding, and complex type is used. It has been desired to develop a method for synthesizing sugars and glycoconjugates that transfer various kinds of sugar chains in a one-step reaction and have improved physicochemical or physiological properties such as stability and cell recognition.

[0013]

[Means for Solving the Problems] As a result of intensive studies on the above problems, the present inventors have found that endo-β-N-acetylglucosaminidase M catalyzes a glycosyl transfer reaction, and also endo-β. -By utilizing the action of N-acetylglucosaminidase M, it is possible to transfer a wide variety of sugar chains, and especially for asparagine-linked sugar chains, transfer of any of high mannose type, hybrid type and complex type. We have succeeded in developing a remodeling method for saccharides and glycoconjugates capable of producing saccharides, and completed the present invention.

That is, the present invention provides the following formula [I] in the presence of endo-β-N-acetylglucosaminidase M:

[0015]

[Chemical 5]

(Wherein A is a saccharide, GlcNAc is N-acetylglucosamine, and C and D are saccharides or complex saccharides), which is a saccharide or complex saccharide. Is a manufacturing method. The present invention also provides endo-β-N-
In the presence of acetylglucosaminidase M, the following formula [II]:

[0017]

[Chemical 6]

(Wherein A and B are saccharides, GlcNAc is N-acetylglucosamine, and C and E are saccharides or complex saccharides). It is a method for producing sugar. Furthermore, the present invention provides the following formula [III] in the presence of endo-β-N-acetylglucosaminidase M:

[0019]

[Chemical 7]

(Wherein A and B are saccharides, GlcNAc is N-acetylglucosamine, and C and E are saccharides or complex saccharides). It is a method for producing sugar. Examples of glycoconjugates include glycoproteins, glycolipids, or proteoglycans.

The sugar chain transferred to the sugar or glycoconjugate is of the asparagine-bonded type. Furthermore, the sugar chain transferred to a sugar or a complex sugar is an asparagine-bonded type, and the sugar chain is a mixed type or a complex type. Hereinafter, the present invention will be described in detail. The present invention can accept a wide variety of sugars or complex sugars, and can transfer various sugar chains. In particular, in the asparagine-linked sugar chain, a high mannose type, a mixed mold, It is characterized by a transglycosylation reaction system capable of transferring any of the complex types. In particular, the complex type is a sugar chain structure that is rich in structural changes and is often found in animals, and since it plays an important physiological function in vivo, it is extremely significant industrially to be able to transfer the complex type sugar chain. is there.

In the above-mentioned glycosyl transfer reaction of the present invention, in the formulas [I], [II] or [III], A and B represent sugars and are homologues composed of mannose, glucose, N-acetylglucosamine and the like. It is an oligomer or a hetero-oligomer composed of two or more components such as mannose, glucose, N-acetylglucosamine, galactose and sialic acid. Typical examples of these include asparagine-linked sugar chains of glycoproteins. The asparagine-linked sugar chain means a sugar chain that binds to an asparagine residue of a glycoprotein.

In the formula [I], [II] or [III],
C, D, and E are sugars or complex sugars, for example, monosaccharides such as glucose, mannose, and N-acetylglucosamine, homooligomers of two or more of these, heterooligomers composed of two or more of these, etc. Is mentioned. In addition, sugars such as α- and β-methyl glycosides, α- and β-p-nitrophenyl glycosides, O-methyl glycosides, 1-aminoglycosides, 4-methylumbelliferyl glycosides and pyridyl amination Further, it may be a saccharide, or asparagine labeled with a chromogenic substance or a fluorescent substance at the end of these saccharides, or a complex saccharide in which a polypeptide is bound via the asparagine.

Further, in the formula [I], the acceptor D
As for the saccharides whose non-reducing end is free at the C-4 position, saccharides or glycoconjugates react well, and among saccharides free at the C-4 position, the steric groups at the C-4 and C-5 positions are free. The conformation of N-acetylglucosamine is the same. Therefore, monosaccharides such as N-acetylglucosamine, glucose, glucosamine, N-acetylmannosamine, mannose, mannosamine, and allose, or sugars or complex sugars having these sugars as non-reducing terminals are good acceptors. In addition, these sugars such as α- and β-methyl glycosides, α- and β-p-nitrophenyl glycosides, O-methyl glycosides, 1-aminoglycosides, 4-methylumbelliferyl glycosides and pyridyl amination It also acts on saccharides, or complex sugars in which amino acids such as asparagine, serine, threonine, etc., or amino acids such as asparagine, serine, threonine modified with chromogenic substances, fluorescent substances, protective groups, etc. are bound to these saccharides. .

The glycosyl transfer reaction of the present invention is usually carried out by using a sugar or a glycoconjugate as a donor, a sugar or a glycoconjugate as an acceptor, and an endo-β-N-acetylglucosaminidase M as a catalyst (hereinafter, endo-M). And) and a buffer solution. The amount of the donor or acceptor used is not particularly limited, and a saturated amount can be used. The amount of End M used is not particularly limited, but may be about 10 mU to 10 U per 1 ml of the reaction solution. Buffer pH
It is sufficient to use an appropriate buffer solution of about 5 to 11
Use potassium phosphate buffer around 6.0. The transfer reaction of the present invention reacts even if an organic solvent, an inorganic salt or the like is added to the reaction solution. For example, for poorly water-soluble sugars or complex sugars, methanol can be used as the organic solvent. In addition, DMSO (dimethyl sulfoxide) and DMF
(N, N-dimethylformamide) can also be used.

The transfer reaction of the present invention is usually carried out at a temperature of room temperature to about 50 ° C., preferably about 30 to 40 ° C. The transfer reaction is completed in about several minutes to several tens hours depending on the reaction conditions. The produced remodeling saccharide or complex saccharide can be easily separated and purified from the reaction-terminated liquid by a method known in the art. For example, gel filtration column chromatography and the like can be mentioned as the method, and it can be recovered by further operations such as desalting, concentration and freeze-drying.

The carbohydrate or glycoconjugate produced by the method of the present invention serves as a substrate for various glycolytic enzymes and glycosyltransferases and can be used for searching various useful enzymes. Of the asparagine-linked sugar chains, it is particularly useful for preparing a substrate for an enzyme involved in the complex type. For example, the following formula [V]:

[0028]

[Chemical 8]

The following formula [VI] obtained by reacting a compound represented by the formula with α-p-nitrophenyl glucose (p-NP-α-Glc):

[0030]

[Chemical 9]

The compound represented by can be used for detection and measurement of endo-β-N-acetylglucosaminidase, which cleaves complex type asparagine-linked sugar chain in a sample. That is, if the target enzyme is present in the sample, p-NP-
When α-Glc is released and α-glucosidase is allowed to act on it, the free p-nitrophenol shows a yellow color, and it is possible to easily measure the enzyme activity in the sample without using a radioisotope or fluorometer. it can. Examples of the sample include organisms such as microorganisms, insects, animals and plants, and cell culture solutions thereof. Also, instead of p-NP-α-Glc, for example, X-Glc (5-bromo-4-chloro-3-indolyl-D
-Glucoside) can be used, in which case it exhibits a blue color.

Further, for example, by reducing the p-nitrophenylated saccharide of the present invention to prepare p-aminophenylated saccharide, and thereafter binding it to activated carboxyl agarose or cyanogen bromide activated agarose, A carrier having a sugar bound thereto can be prepared. This sugar-binding carrier is useful for purifying various glycolytic enzymes, glycosyltransferases, lectins and the like. In addition, the methyl glycosidated sugar synthesized in the present invention can be easily immobilized on epoxy activated agarose or the like, and the prepared sugar-bonded carrier can be used for the same purpose as described above.

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, by utilizing the catalytic action of endo-M, it is possible to transfer a wide variety of sugar chains, which was difficult with the conventional techniques, and particularly with asparagine-linked sugar chains. It is possible to provide a remodeling method for a carbohydrate and a complex carbohydrate capable of transferring any of a high-mannose type, a hybrid type and a complex type.

Remodeling of sugar chains of sugars or complex sugars is extremely useful industrially because it can be used for the development and production of drugs with few side effects.

[0035]

EXAMPLES The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples. [Example 1] of human asialotransferrin sugar chain
Transfer reaction to GlcNAc Purification of Endo M was performed by S. Kadowaki et al.
And Biological Chemistry (Agric. Bio
l. Chem.), 54, 97-106 (1990)].

Mucor hiemalis SK-314 with glucose 0.5
%, Peptone 0.5%, yeast extract 0.5% at 28 ° C. for 3 to 4 days in a liquid medium (pH 6.5) with shaking, and cells were removed by ultrafiltration to obtain a culture filtrate. Using this culture filtrate as a crude enzyme solution, CM-cellulose treatment, 70% saturated ammonium sulfate precipitation, DEAE-Sepharose, CL-6B Sephadex G-200, Hydroxyla
A purified preparation was obtained through a purification process using a total of five types of patite, TSK-gel HW-65F, and Con A-Sepharose, and was used in the following examples. This strain is designated Mucor hiemalis SK-314 and has been deposited as FERM BP-4377 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology.

2.5 mg of human transferrin (manufactured by Sigma) asialo body, 100 mM GlcNAc (manufactured by Nacalai Tesque), 66 mM potassium phosphate (manufactured by Wako Pure Chemical Industries) buffer (pH 6.0), 32 mU End M Reaction mixture containing 1.5 ml at 37 ℃
After reacting for a time, trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added so that the final concentration was 5% to stop the reaction. The reaction solution was subjected to gel filtration column chromatography, lyophilized, pyridylamination (hereinafter abbreviated as PA) as described below, and the transfer product was identified by HPLC analysis.

100 μl of 2-aminopyridine acetic acid solution [276 m
g 2-Aminopyridine (manufactured by Wako Pure Chemical Industries) /0.1 ml acetic acid (for precision analysis manufactured by Wako Pure Chemical Industries)], then sealed and kept at 90 ° C.
After heating for 60 minutes and returning to room temperature, 100 μl of 20% dimethylamine-borane acetic acid solution (2 g dimethylamine-borane (manufactured by Wako Pure Chemical Industries) / 10 ml acetic acid (for precision analysis manufactured by Wako Pure Chemical Industries)) In addition, it was further heated for 50 minutes at 80 ° C. 2 ml of 75% methanol was added to 200 μl of the obtained reaction solution, and after stirring,
Methanol was removed by vacuum distillation, and the same procedure was repeated.
Repeated 3-4 times with% methanol. Saturated sodium hydrogencarbonate (Wako Pure Chemical Industries, Ltd.) aqueous solution was added to the vacuum concentrate.
An aqueous solution of 100% acetic anhydride (manufactured by Wako Pure Chemical Industries, Ltd.) was added in an amount of 500 μl each, and the mixture was stirred and allowed to stand for 10 minutes. To this, 1.5 ml of benzene (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and after vigorous stirring, the mixture was allowed to stand for 10 minutes, the upper layer of benzene was removed, and the same benzene extraction was repeated 7 times. The obtained aqueous layer was distilled under reduced pressure and then Se
It was subjected to gel filtration column chromatography using a phadex G-10 column and used as a sample for HPLC analysis. The sugar chain structure analysis by HPLC analysis was performed by N. Tomiya et al. [Analytical Biochemistry (Anal. B.
ichm.), 171, pp. 73-90 (1988)].

The following formula [VII]:

[0040]

[Chemical 10]

As a result of comparison using a commercially available compound (Takara Shuzo Co., Ltd.) represented by
Compound represented by the following formula [VII]:

[0042]

[Chemical 11]

The compound was identified as: [Example 2] Transfer reaction of human asialotransferrin sugar chain to (GlcNAc) ₂ 0.5 mg asialo form of human transferrin (manufactured by Sigma), 50 mM (GlcNAc) ₂ , 67 mM potassium phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) ) A reaction solution (0.3 ml) containing a buffer solution (pH 6.0) and 6.4 mU End M was reacted at 37 ° C for 6 hours and then heat-treated at 100 ° C for 3 minutes to stop the reaction. The reaction solution was subjected to gel filtration column chromatography and then freeze-dried. After PA conversion in the same manner as in Example 1 above, the transfer product was analyzed by HPLC using a normal phase column, and from the change in molecular weight, the following formula [VIII]:

[0044]

[Chemical 12]

The compound was identified as: Then this
MS analysis of PA compound confirmed the sugar chain structure. [Example 3] Transfer reaction of human asialotransferrin sugar chain to (GlcNAc) ₂ -PA 0.17 mg of human transferrin (manufactured by Sigma) asialo body, 2.2 mM (GlcNAc) ₂ -PA, 75 mM potassium phosphate ( 0.1 ml of a reaction solution containing 2.0 mU End M buffer (pH 6.0) manufactured by Wako Pure Chemical Industries, Ltd. was reacted at 37 ° C. for 6 hours. The reaction solution is M
S analysis is performed, and the transfer product is represented by the following formula [VIII]:

[0046]

[Chemical 13]

The compound was identified as: Example 4 of human asialotransferrin sugar chain
Transfer reaction to GlcNAc-Asn-DNS 0.15 mg of human transferrin (manufactured by Sigma) asialo body, 1M GlcNAc-Asn-DNS, 67 mM potassium phosphate (manufactured by Wako Pure Chemical Industries) buffer (pH 6.0), 4mU end M
0.145 ml of the reaction liquid containing the mixture was reacted at 37 ° C. for 30 minutes. The reaction solution was analyzed by HPLC using a reverse phase column, and the transfer product was
The following formula [IX]:

[0048]

[Chemical 14]

It was confirmed to be a compound represented by: [Example 5] Transfer reaction of double-chain complex type sugar chain to p-NP-α-Glc 1M p-NP-α-Glc (manufactured by Sen Chemicals) aqueous solution 3
To 0 μl, add 30 μl of 1M acetate buffer (pH 6.0) and End M
Add 100 μl of enzyme solution containing 0.14 mU and 40 μl of deionized water, and add 37
Pre-incubation at 10 ° C for 10 minutes. Then, the following formula [X]:

[0050]

[Chemical 15]

100 μl (60 nmol) of the double-chain complex type sugar chain (manufactured by Biocarb Chemicals) was added at 37 ° C.
After reacting at 100 ° C. for 100 minutes, heat treatment was performed at 100 ° C. for 3 minutes to stop the reaction. The reaction solution was separated and fractionated by HPLC using a PALPAK Type N column (manufactured by Takara Shuzo Co., Ltd.) (underlined in FIG. 7,), composition analysis, NMR, and MS analysis were performed, and the sugar chain structure was represented by the following formula [VI ]:

[0052]

[Chemical 16]

It was confirmed to be a transfer product represented by:

[Brief description of drawings]

FIG. 1 is a diagram showing a high-mannose type sugar chain structure in an asparagine-linked sugar chain.

FIG. 2 is a diagram showing a hybrid sugar chain structure in an asparagine-linked sugar chain.

FIG. 3 is a diagram showing a complex type sugar chain structure (1-branched type) in an asparagine-linked sugar chain.

FIG. 4 is a diagram showing a complex type sugar chain structure (two-branched type) in an asparagine-linked sugar chain.

FIG. 5 is a diagram showing a complex type sugar chain structure (3-branched type) in an asparagine-linked sugar chain.

FIG. 6 is a diagram showing a complex sugar chain structure (four-branched type) in an asparagine-linked sugar chain.

FIG. 7 is a diagram showing a result of HPLC in Example 5.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kenji Yamamoto 1-17-14-403 Nakasho, Otsu City, Shiga Prefecture

Claims (6)

[Claims] 1. In the presence of endo-β-N-acetylglucosaminidase M, the following formula [I]: (In the formula, A is a carbohydrate, GlcNAc is N-acetylglucosamine,
C and D represent a saccharide or a complex saccharide), and a method for producing a saccharide or a complex saccharide is carried out. 2. In the presence of endo-β-N-acetylglucosaminidase M, the following formula [II]: (Wherein A and B are saccharides, GlcNAc is N-acetylglucosamine, and C and E are saccharides or complex saccharides). Production method. 3. In the presence of endo-β-N-acetylglucosaminidase M, the following formula [III]: (Wherein A and B are saccharides, GlcNAc is N-acetylglucosamine, and C and E are saccharides or complex saccharides). Production method. 4. The method for producing a sugar or a glycoconjugate according to claim 1, 2 or 3, wherein the glycoconjugate is a glycoprotein, a glycolipid or a proteoglycan. 5. The method for producing a sugar or a complex sugar according to claim 1, 2 or 3, wherein the sugar chain transferred to the sugar or the complex sugar is an asparagine-bonded sugar chain. 6. The sugar or complex according to claim 5, wherein the sugar chain transferred to the sugar or the complex sugar is an asparagine-bonded type, and the sugar chain is a mixed type or a complex type. Method for producing sugar.