JPH1033194A - Carbohydrate and complex carbohydrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound which is useful in the fields of glycotechnology and medicines by rearranging a specific saccharide in the presence of a glycosidase specifically acting only on the lacto-N-bioside of a type 1 carbohydrate structure. SOLUTION: In the presence of a lacto-N-biosidase originating from Streptomyces sp 142 (FERM BP-4569) as a glycosidase specifically acting on only the lacto-N-bioside bond of a type 1 carbohydrate structure, a saccharide of the type 1 carbohydrate structure represented by formula I (Gal is galactose; GlcNac is N-acetyl-glucosamine; X is a chemical substance which can become a saccharide, conjugated polysaccharide or aglycon), for example, p- nitrophenyl-β-lacto-N-bioside or a combination thereof with a conjugated polysaccharide is subjected to rearrangement reaction to readily give the objective saccharide or conjugated polysaccharide of formula II (Y is a saccharide or conjugated polysaccharide substance).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a remodeled carbohydrate or complex carbohydrate utilizing the carbohydrate transfer ability of an enzyme, which is useful in the field of carbohydrate engineering or medicine, and a method for producing the carbohydrate or complex carbohydrate. It relates to the obtained saccharide or complex saccharide.

[0002]

2. Description of the Related Art Carbohydrates and glycoconjugates (glycoproteins, glycolipids, proteoglycans, etc.) are present in living cells, body fluids, fruits, seeds, microbial cells or cultures thereof, and are found in living organisms. It is known that it is involved in various important physiological activities. Therefore, in recent years, attempts have been made actively to utilize the physiological activity as a pharmaceutical. Further, attempts have been made to modify or replace sugar chains of saccharides and complex saccharides to modify their physiological activities to create new types of drugs. There are chemical methods and enzymatic methods for modifying or substituting sugar chains of saccharides and glycoconjugates. Among these methods, the enzymatic method can be carried out in an aqueous solution such as a buffer solution that does not cause protein denaturation, and is used because the enzyme acts specifically. Currently widely used remodeling techniques include the European Journal of Biochemistry,
Vol. 191, pp. 75-83 (1990). Sequential conversion of sugar chains from non-reducing ends using exoglycosidase or glycosyltransferase. In addition, reports of glycosyltransfer reactions using endoglycosidases can be found in the Journal of Biological Chemistry, Vol.
Vol. 61, 12000-1200 (1986), Flavobacterium meningosepticum (Flavob)
acterium meningosepticum) and those of endo-β-N-acetylglucosaminidase F (endo-F) and Journal of Biological Chemistry, Vol. 264, pp. 1989-19897 (198).
9) Diplococcus pneumoniae (Diploc
occus pneumoniae). The former is a report that glycerol becomes an acceptor, and the latter is a report that glycerol, tris, p-nitrophenol, serine, and threonine become acceptors. On the other hand, as a report on a remodeling mechanism of a sugar chain in which a carbohydrate or a complex carbohydrate directly serves as an acceptor, see JP-A-5-64
No. 594, an endo-β-derived from Arthrobacter protophormiae.
Those relating to N-acetylglucosaminidase (Endo-A) and those disclosed in JP-A-7-59587.
There is something about. As described above, if the stability and physiological activity of glycoconjugates obtained by remodeling sugar chains are enhanced as compared with natural glycoconjugates, it is very advantageous when applied to pharmaceuticals. In the sugar chain, two types of structures, a type 1 type Galβ1-3GlcNAc structure and a type 2 type Galβ1-4GlcNAc structure, are often found at the non-reducing end. In order to analyze the sugar chain structure and function, a sugar chain remodeling technique of removing the sugar chain structure with a sugar hydrolase or adding the sugar chain structure with a glycosyltransferase is extremely useful. However, in the conventional remodeling method using exoglycosidase or glycosyltransferase, it is necessary to carry out an enzymatic reaction for each sugar residue, and the number of reaction steps increases, which is very complicated. Also, in the case of the above-described glycosyltransfer reaction using endo-F or endo-α-N-acetylgalactosaminidase derived from Diprococcus pneumoniae, carbohydrates and complex carbohydrates do not become acceptors. On the other hand, a transglycosylation reaction using Endo-A or Endo-M is a remodeling reaction of a sugar chain in which a carbohydrate or a complex carbohydrate directly serves as an acceptor. It is. However, these cannot specifically remodel the type 1 or type 2 sugar chain structure at the non-reducing end of the sugar chain.

[0003] The present inventors previously had an enzyme that specifically acts on the type 1 sugar chain structure to release lacto-N-biose,
Lacto-N-biosidase [Proceedings of the National Academy of
Sciences of the USA (Proceedings of the USA)
National Academy of Sciences of the USA), Vol. 89, pp. 8512-8516 (1992), Journal of Biological Chemistry, 268.
Vol. 18560-18566 (1993)]. Due to the specificity of this enzyme, the type 1 structure in the sugar chain and the type 2 structure
The type structure can be identified, and the structural analysis has become extremely easy. However, it is not easy to add a type 1 type sugar chain structure and a type 2 type sugar chain structure to carbohydrates and complex carbohydrates. So far, two types of glycosyltransferases exhibiting strict transfer specificity have been proposed. Action, that is, β-
It is known to use the action of β-1,3 and β-1,4 galactosyltransferase following the action of N-acetylglucosamyltransferase.

[0004]

As described above, sugar chain remodeling using an enzymatic method is highly useful due to mild reaction conditions and the like. However, conventional exoglycosidase and endoglycosidase cannot specifically and simply add a type 1 sugar chain, and use β-N-acetylglucosamyltransferase and β-1,3 galactosyltransferase. The method has been extremely difficult due to problems such as setting reaction conditions and increasing the number of reaction steps, making the method very complicated. An object of the present invention is to provide a remodeling reaction for specifically and simply adding a type 1 sugar chain, and a simple method for producing a saccharide or a complex saccharide using the remodeling reaction.

[0005]

SUMMARY OF THE INVENTION In summary, the first invention of the present invention relates to a method for producing a saccharide or a complex saccharide, and comprises a lacto-N-type having a type 1 sugar chain structure. In the presence of a glycosidase that specifically acts only on a bioside bond, the following formula (Formula 1):

[0006]

Embedded image Galβ1-3GlcNAc-X + Y →
Galβ1-3GlcNAc-Y + X

(Where Gal is galactose, GlcNA)
c is N-acetylglucosamine, X is saccharide, complex saccharide,
Or a chemical substance that can be an aglycone, and Y represents a carbohydrate or a complex carbohydrate). Further, the second invention of the present invention is the first invention of the present invention.
The present invention relates to a saccharide or a complex saccharide obtained by the production method of the invention.

[0008] The present inventors use a glycosidase which can be expected to be able to transfer type 1 sugar chains specifically and in a one-step reaction, and transfer the sugar chains to carbohydrates and complex carbohydrates. In order to produce new carbohydrates and glycoconjugates with enhanced conventional stability and activity, we conducted intensive research and found that type 1 type sugar chains are specifically recognized and transferred to carbohydrates and glycoconjugates. As a result of finding a glycosidase having activity and further studying a reaction method thereof, the present invention has been completed. The present invention provides a saccharide and a saccharide characterized by remodeling the sugar chain of a saccharide and a complex saccharide using a glycosidase specifically acting only on a lacto-N-bioside bond of a type 1 sugar chain structure. This is a method for producing a glycoconjugate.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Lacto-N- having a type 1 sugar chain structure in the present invention
Glycosidases specifically acting only on bioside bonds include Streptomyces sp 142 (Streptomyc
es sp 142)) [Proceedings of the National Academy of Sun Encies of the USA, Vol. 89, No. 8,
521-8516 (1992)]. The enzyme acts on a sugar chain represented by the following formula (Formula 2) to produce lacto-
Substrate specificity of hydrolyzing only N-bioside bonds is described.

[0010]

Embedded image Galβ1-3GlcNAc-R (R represents a sugar residue)

However, it is not known that this enzyme preserves the activity of performing a sugar chain transfer reaction. The lacto-N-biose transfer reaction to a sugar chain or glycoconjugate has been found for the first time by the present inventors. The present invention is not limited to these, and native enzymes and genetic engineering can be used as long as the glycosidase has a transfer activity and specifically acts only on the lacto-N-bioside bond of the type 1 sugar chain structure. A gene obtained by introducing an enzyme, such as a gene encoding a target enzyme, into a vector, and transforming the vector into a host (eg, Escherichia coli), culturing a transformant, and culturing the culture The recombinant enzyme prepared from the above, the gene encoding the enzyme of interest is modified using site-directed mutagenesis, etc., introduced into a vector, and the vector is transformed into a host (eg, Escherichia coli). The transformed enzyme is cultured, and the recombinant enzyme prepared from the culture is also included in the glycosidase specifically acting only on the lacto-N-bioside bond of the type 1 sugar chain structure of the present invention. .

In the present invention, X in formula (1)
Is a chemical substance that can be a carbohydrate, a complex carbohydrate or an aglycone, and when the lacto-N-biosidase of the present invention is used, glucose, mannose, N-acetylglycosamine, galactose, fucose, xylose, p-nitrophenyl glycoside derivative And monosaccharides such as methylglycoside derivatives, homooligomers of these two or more monosaccharides, and carbohydrates such as heterooligomers composed of two or more of these components, and a polypeptide bound to Asn or a terminal via Asn. Glycoconjugate, Thr or Ser at the end
Alternatively, a chemical substance which can be an aglycone such as a glycoconjugate having a polypeptide bonded thereto via Thr or Ser, a nitrophenyl derivative, a 3-indole derivative, a 4-methylumbelliferone derivative, a naphthyl derivative, a resorufin derivative, or the like may be used. Y is a saccharide or a complex saccharide, and when using the lacto-N-biosidase of the present invention, glucose, mannose, N-acetylglycosamine, galactose, fucose, xylose, a p-nitrophenyl glycoside derivative, methyl glycoside There are carbohydrates such as monosaccharides such as derivatives, homo-oligomers of these two or more mono-saccharides, and hetero-oligomers composed of these two or more components, and complex carbohydrates having Asn or a polypeptide bound via Asn at the end thereof. Examples thereof include Thr or Ser at the end thereof, and glycoconjugates to which a polypeptide is bound via Thr or Ser. In particular, for Y, it is preferable to select a saccharide or complex saccharide having a free sugar at the C-3 position at its non-reducing terminal.

The transfer reaction of the present invention is usually carried out by adding the acceptor saccharide or complex saccharide to a starting solution containing the starting saccharide or complex saccharide, lacto-N-biosidase, and a buffer. The amounts of the raw material and the acceptor are not particularly limited, and they can be used up to the saturated amount. However, it is preferable that the acceptor is present in excess, and usually, an acceptor of 200 mM or more is used. The amount of lacto-N-biosidase used is not particularly limited and can be appropriately selected from a wide range. However, it is usually 1 mU or more, more preferably about 10 mU to 10 U per 1 ml of the starting solution. As the buffer, a suitable buffer having a pH of about 4 to 10 may be used, and it is usually preferable to carry out the reaction at about pH 5 in an acetate buffer. In the transfer reaction of the present invention, an organic solvent, an inorganic salt, and the like are added to the starting solution, and the reaction may be well changed to a hydrophobic state,
If, for example, acetonitrile is used as the organic solvent, a poorly water-soluble saccharide or complex saccharide can be used. Lact-N-biosidase reacts well in the presence of 40% acetonitrile. When the lacto-N-biosidase is used, the transfer reaction of the present invention is usually carried out at room temperature to 60 ° C.
℃, preferably at a temperature of about 30-40 ℃ and pH
The reaction is carried out under a pH condition of about 4 to 10, and the transfer reaction is usually completed in about 5 to 60 minutes, preferably about 10 to 40 minutes, depending on the reaction conditions. The remodeled saccharide or complex saccharide produced by the transfer reaction of the present invention can be easily separated and purified from the reaction-terminated liquid according to known means. For example, it can be obtained by separating the remodeled saccharide or complex saccharide from the reaction-terminated liquid by gel filtration column chromatography, ion exchange resin column chromatography, or the like, and further performing concentration, desalting, freeze-drying, or the like. it can.

The carbohydrate or complex carbohydrate produced by the method of the present invention also serves as a substrate for various carbohydrate-degrading enzymes, and can be used for searching for various useful enzymes. For example, lacto-N
-Glc-X (5-bromo-4) in the presence of biosidase
-Chloro-3-indolyl-D-glucoside), and Galβ1-3GlcNAc-Glc-X obtained
Can be used for the detection and measurement of lacto-N-biosidase in a sample. That is, if the target enzyme is present in the sample, Glc-X is released. Then, when α-glycosidase is acted on, the free 5-bromo-4-chloro-3 is released.
-The indole has a blue color, and the enzyme activity in the sample can be measured without the need for a radioisotope or a fluorometer. As the sample, for example, a culture solution of a microorganism, a purified product from the culture solution, a preparation from an animal, a plant, or the like can be used.

As described above, by using the production method of the present invention, it is possible to easily add a sugar chain to a glycoprotein, which is a pharmaceutical product, or to change the structure of an existing sugar chain. It is possible to obtain a drug whose safety in vivo and biological activity are enhanced as compared with conventional products. In particular, it is known that metastasis and invasion of cancer involve a sugar chain based on a type 1 sugar chain structure (Galβ1-3GlcNAc), and the production method of the present invention suppresses or prevents these cancer metastasis. Very useful in the development of medicines for. Furthermore, the sugar chain or glycoconjugate obtained by the production method of the present invention can be used as a substrate for a carbohydrate-degrading enzyme, and is useful for searching for a useful carbohydrate-degrading enzyme.

[0016]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the scope of the following examples.

Example 1 Transfer reaction of sugar chain to lactose (1) Streptomyces sp142 is cultured according to the method described in Proceedings of the National Academy of Sciences of the USA to prepare lacto-N-biosidase. And used in the examples below. The strain was designated as Streptomyces sp 142 and was reported to the Institute of Biotechnology and Industrial Technology by the Ministry of International Trade and Industry of Japan.
Deposited as ERM BP-4569. 1.6 ml of 1 M lactose (Nacalai Tesque) aqueous solution
, 2.0 ml of 50 mM acetate buffer (pH 4.5)
And 0.2 ml of an enzyme solution containing 100 mU of lacto-N-biosidase, and then 5 mM p-nitrophenyl-β-lacto-N-bioside (Nacalai Tesque)
Was added and reacted at 37 ° C. for 30 minutes. Thereafter, the reaction solution was heated at 100 ° C. for 3 minutes to stop the reaction. After freeze-drying the reaction solution thus obtained, pyridyl- (2) -amination (hereinafter, abbreviated as PA) was carried out according to the method described in JP-A-1-1177, and purified by HPLC. The transfer product, Galβ1-3GlcNAcβ1-
3Galβ1-4Glc-PA was obtained. Next, composition analysis and NMR analysis of this PA compound were performed, and the sugar chain structure was Ga
1β1-3GlcNAcβ1-3Galβ1-4Glc
-PA was confirmed.

(2) A transfer reaction is carried out under the same conditions as in (1) above, and after the start of the reaction, 20 minutes, 40 minutes, 60 minutes, 120 minutes
After one minute each was sampled and the reaction was stopped. After freeze-drying the reaction solution thus obtained, a PA product was prepared in the same manner as in the above (1), and the yield was determined. The result is shown in FIG. That is, FIG. 1 is a diagram showing the yield when lactose was transferred to p-nitrophenyl-β-lacto-N-bioside using lacto-N-biosidase, and the horizontal axis represents the reaction time (minutes). The vertical axis shows the yield (%).
As can be seen from FIG. 1, a yield of about 3% was obtained after 40 minutes of the reaction.

[0019]

Industrial Applicability According to the present invention, a type 1 type sugar chain having a carbohydrate or a complex carbohydrate directly as an acceptor can be remodeled easily and simply by a one-step reaction. A method is provided. The method can efficiently and easily produce a target saccharide or complex saccharide, has a small amount of by-products, easily separates and purifies the target saccharide or complex saccharide, and has an important function in vivo. It is useful in the production of a substance obtained by remodeling the sugar chain of a physiologically active substance showing the following. Further, the saccharide or complex saccharide obtained by the production method of the present invention,
It can be used as a substrate for carbohydrate degrading enzymes, and is useful for searching for various useful enzymes.

[Brief description of the drawings]

FIG. 1 is a graph showing an example of the relationship between the transfer reaction time of lacto-N-biosidase and the yield.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd.

Claims (3)

[Claims] In the presence of a glycosidase which specifically acts only on a lacto-N-bioside bond of a type 1 sugar chain structure, Galβ1-3GlcNAc-X + Y →
Galβ1-3GlcNAc-Y + X (Gal is galactose, GlcNAc is N-acetylglucosamine, X is a chemical substance that can be a carbohydrate, a complex carbohydrate, or an aglycone, and Y is a carbohydrate or a carbohydrate). A method for producing a saccharide or a complex saccharide, wherein the saccharide or the complex saccharide is subjected to a rearrangement reaction. 2. The method for producing a saccharide or complex saccharide according to claim 1, wherein the glycosidase is lacto-N-biosidase. 3. A saccharide or complex saccharide obtained by the production method according to claim 1 or 2.