JP5951199B2 - Polypeptide that specifically binds to the sugar chain of gastrointestinal mucin - Google Patents

Description

  The present invention relates to a polypeptide that specifically binds to the sugar chain of gastrointestinal mucin. Specifically, the present invention relates to a gastrointestinal mucin composed of a polypeptide containing a sugar chain binding module (CBM) region of α-N-acetylglucosaminidase derived from a specific microorganism, particularly a polypeptide that specifically binds to a sugar chain of gastric mucin. This is a polypeptide necessary for isolating mucin derived from gastric gland mucus that suppresses the growth of Helicobacter pylori, which causes peptic ulcer and gastric cancer.

  Helicobacter pylori is a causative bacterium that causes peptic ulcer and chronic gastritis (Non-patent Document 1, Non-patent Document 2), and is estimated to be infected in half of the world population.

  Helicobacter pylori inhabits in the surface mucus secreted from the surface layer of the gastric mucosa, but not in the glandular mucus secreted from the mucosa or the deep mucosa. This gland mucus is composed of gastric mucin having a specific structure, and gastric mucin (mucin-type glycoprotein) is GlcNAcα1 having N-acetylglucosamine α residue (αGlcNAc residue) and galactose residue (Gal residue). → 4Galβ residue-containing O-glycan sugar chain is characteristically included. In recent years, it has also been reported that these sugar chains appear with the appearance of gallbladder cancer, pancreatic cancer, cervical cancer and other neoplasms (Non-patent Document 3).

  Nakayama et al. Found that a glycoprotein sugar chain to which a core biantennary O-glucan having an αGlcNAc residue at the non-reducing end was bound suppressed the growth of Helicobacter pylori. It has been clarified that it is an enzyme activity inhibition of glucosylcholesterol synthase (CHLαGcT) (non-patent document 4, non-patent document 5) possessed only by bacteria (non-patent document 4). Helicobacter pylori requires a glycosyl cholesterol component (CGL) for its growth, but because it cannot synthesize CGL itself, it ingests cholesterol from the outside world, adds glucose near the cell membrane of the fungus, and builds a cell wall. It is thought that there is. Therefore, it is presumed that the glycoprotein sugar chain of O-glycan having such an αGlcNAc residue has the property of inhibiting the construction of this cell wall, so that it can be expected to be applied as a growth inhibitor specific for H. pylori. It is a complex, high-molecular-weight glycoprotein sugar chain, and it must be prepared through complicated multi-steps under uncontrollable reaction conditions, and requires huge costs and large-scale production facilities, which is not practical.

  Patent Document 1 discloses a Helicobacter pylori-binding substance that is an oligosaccharide having a Galβ3GlcNAcGalβ4GlcNAc structure. However, since this substance is a polysaccharide and has a complicated structure, it must be prepared through multiple steps, and it cannot be easily prepared in large quantities.

  On the other hand, the present treatment method for Helicobacter pylori infection does not use these sugar chains, but is mainly sterilized by the combined use of one kind of proton pump inhibitor and two kinds of antibiotics. Three-drug combination therapy has a problem in that resistant bacteria appear and recur, or side effects appear.

  The development of a Helicobacter pylori growth inhibitor with a simple structure that allows people to continue to eat, drink, and take in peace is particularly desirable, as interest in health preservation and safety is particularly high for recent food and beverage products and pharmaceutical preparations. It was rare. Therefore, utilization of the above-mentioned gastric mucin inherently possessed by humans and domestic animals is considered to be extremely effective in reducing the risk from infection with H. pylori.

  Examples of the protein and polypeptide that specifically bind to gastric mucin include the monoclonal antibody (HIK1083, IgM) that has been found so far by Horita et al. (Non-patent Document 6). HIK1083 is a monoclonal antibody that recognizes αGlcNAc residues and is used for pathological examinations and the like. However, considering the preparation of gastric mucin as a functional substance, the use of expensive antibodies is not practical. On the other hand, galactose (Gal) and N-acetylgalactosamine (GalNAc) are slightly contained in the non-reducing end of the glycoprotein sugar chain of gastric mucin, and the use of lectins that recognize these may be cited as candidates. it can. However, as with antibodies, lectins are also relatively expensive to prepare and have poor binding and specificity, so their utility as a production means is not very high. Therefore, the discovery of a recombinant protein that recognizes αGlcNAc residues that are abundant in gastric mucin is thought to lead to a direct solution to the problem.

  The sugar-binding module (CBM) has been associated with a part of microbial-derived enzyme (sugar hydrolase) molecules that break down polysaccharides such as cellulose and starch. Until now, it has been identified from many types of microorganisms. The binding between these CBMs and sugar molecules can be analyzed and identified by techniques such as crystallography and NMR, and the amino acid sites necessary for binding to sugar molecules can be identified. On the other hand, the recent development of databases based on amino acid sequences suggests that CBM is also present in some hydrolyzing enzymes (derived from human intestinal resident bacteria) for human glycoprotein sugar chains. Yes. There are 59 CBM families already published on the CAZY site (Carbohydrate Active Enzymes site, http://www.cazy.org/Carbohydrate-Binding-Modules.html). It is presumed to be a protein region that binds to sugar chains. However, among these CBM families, there are reported examples of CBMs that bind to oligosaccharides and sugar chains possessed by animals including humans, ie, N-glycan and O-glycan (Gal, GalNAc, GlcNAc and fucose (Fuc)). Therefore, it is difficult to estimate what kind of glycoprotein sugar chain is bound only from the CBM family.

The present inventors have previously found an enzyme that specifically releases GlcNAc from an intestinal bacterium from an oligosaccharide chain in which GlcNAc is α1 → 4 and binds to galactose (Patent Document 3). Furthermore, since this enzyme had an activity to specifically release GlcNAc from gastric mucin, the properties of this enzyme were examined in detail. No other enzyme is known to release GlcNAc from gastric mucin. Comparative analysis data of the amino acid sequence of this enzyme based on the genome database (BLAST
SEARCH) shows that the C-terminal region has a plurality of FA58C regions that co-aggregate blood, and this region has homology to a sugar chain binding module (CBM) that is thought to bind to sugar chains. However, these estimated CBM regions have low homology with CBMs derived from other enzymes or strains that have been known to bind to sugar molecules so far (up to about 35%, most of which are 20%). In addition, according to previous reports, generally, the sugar chain to which the CBM region binds and the catalytic region of the enzyme, that is, the sugar chain that is the substrate are usually not related. It has not been clarified how to support the enzyme catalyst or what kind of sugar chain it binds.

Special Table 2003-517015 International Publication No. 2008/084561 Pamphlet JP 2007-236208 A

Marshall BJ et al., Lancet, 1984, Volume I, p. 1311-1315 Peek RM Jr et al., Nature Reviews Cancer, 2002, Volume 2, pages 28-37. Mikami Y et al., Modern Pathology, 2004, Vol. 17, p.962-972. Hirai Y et al., Journal of Bacteriology, 1995, Vol. 177, pp. 5327-5333. Kawakubo M et al., Science, 2004, Volume 305, p.1003-1006. Ishihara K et al., Biochemistry Journal, 1966, 318, pp.409-416.

  The present invention has been made to solve the above-mentioned problems, and provides a protein for efficiently separating and extracting GlcNAcα1 → 4Galβ residue-containing O-glycans that specifically inhibit the growth of H. pylori. An object is to provide a polypeptide that specifically binds to a sugar chain of digestive tract mucin, particularly gastric mucin.

  The inventors of the present invention compared the kinetic constants of the polypeptide having only the catalytic region and the full-length protein in the enzyme, and compared oligosaccharide chains and synthetic substrates (glycans having GlcNAc as α, GlcNAcα1 → 4Galβ-R). (R is paramethoxyphenyl (pMP) and the like) and paranitrophenyl N-acetylglucosamine (GlcNAc-α-pNP), although there is little difference in catalytic function, Thus, the present inventors have found that the CBM region in the C-terminal region may have a strong affinity for gastric mucin.

  Moreover, from the comparison of the primary structures of the amino acid sequences of the enzyme having the above specificity and its homologue, the following α-N-acetylglucosaminidase having a similar arrangement of the catalytic region and CBM-like region of the enzyme was found (FIG. 1). ). They commonly have one CBM and catalyst region in the N-terminal region, and multiple CBM regions in the C-terminal region. In addition, in the C-terminal region, in recent years, a FIVAR region (Found In Variant Architecture Domain) presumably present in common with extracellular proteins and presumed to play a role in retaining the proteins on the extracellular membrane is seen. . From the above information, it was inferred that these proteins have a role in promoting the interaction between the mucin-type glycoprotein sugar chain existing in or in the digestive tract and bacterial cells (intestinal bacteria).

That is, the present invention relates to the following gastric mucin-binding polypeptides.
(1) It specifically binds to a sugar chain of a gastrointestinal mucin comprising a polypeptide containing at least one sugar chain binding module (CBM) region of α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89). Polypeptide.
(2) α-N-acetylglucosaminidase belonging to glycosyl hydrolase family 89 (GH89) is a clostridium perfringens (Clostridium
perfringens), Bifidobacterium bifidum, Eubacterium
dolichum) or α-N-acetylglucosaminidase derived from Collinsella stercoris, which specifically binds to the sugar chain of the gastrointestinal mucin according to (1).
(3) The gastrointestinal mucin according to (2), comprising a polypeptide containing the N-terminal second to sixth CBM-like regions of α-N-acetylglucosaminidase derived from Clostridium perfringens strain13. A polypeptide that specifically binds to a sugar chain.
(4) a polypeptide comprising at least two CBM-like regions among the CBM-like regions from the N-terminal second to sixth N-terminal of α-N-acetylglucosaminidase derived from Clostridium perfringens strain 13 (3 ) A polypeptide that specifically binds to the sugar chain of the gastrointestinal mucin described in 1.).
(5) a polypeptide comprising an amino acid sequence from N-terminal 934 to 1625 of α-N-acetylglucosaminidase (SEQ ID NO: 1 in the sequence listing) derived from Clostridium perfringens strain 13; (3) A polypeptide that specifically binds to the sugar chain of the gastrointestinal mucin described in 1.
(6) The above-mentioned polypeptide does not contain a putative anchor region (FIVAR) on the cell membrane of α-N-acetylglucosaminidase, and the sugar chain of the gastrointestinal mucin according to (3) to (5) A polypeptide that specifically binds.

By using the CBM protein of the present invention as it is, or by fusing it and immobilizing it on a carrier, it has a strong affinity for O-glycans containing GlcNAcα1 → 4Galβ residues already inherent in humans and livestock. Therefore, the binding strength of the CBM protein of the present invention is at least μM level (Kd≈10 ⁻⁶ level), and it is expected that the binding strength to sugar chains is stronger than existing lectins, (2.3)), a safe substance that acts antibacterial against H. pylori can be obtained effectively. Since this protein can be prepared and purified in large quantities by microorganisms, it is suitable for industrial production.

  Unlike so-called lectins that have affinity for conventional sugar chains, this protein has high binding power to the sugar chains and convenience in genetic engineering. high. Furthermore, as a method of use unique to CBM, for example, it can be used as a tool for detecting a histopathological examination accompanying canceration such that GlcNAcα1 → 4Galβ residue-containing O-glycan is expressed or structural change of the glycan. . Further, in the cells having these CBM proteins and homologs thereof as described below, the cells in which they are highly expressed are transferred to in vivo sites where GlcNAcα1 → 4Galβ residue-containing O-glycans are expressed. It can be possible to reach. Furthermore, by introducing such a CBM protein gene into good bacteria such as lactic acid bacteria and bifidobacteria, it can be a highly safe and effective therapeutic agent or transport medium.

  The food and drink containing the Helicobacter pylori growth inhibitor obtained by the present invention is useful for reducing, curing or preventing gastric diseases. Since the substance exhibits a strong H. pylori growth inhibitory action, an excellent anti-H. Pylori action can be obtained just by adding a small amount of H. pylori growth inhibitor to food and drink.

  The pharmaceutical preparation containing the Helicobacter pylori growth inhibitor of the present invention is used for the treatment, symptom alleviation and prevention of gastric diseases such as chronic gastritis and gastric ulcer derived from Helicobacter pylori. In addition, since the sugar chain-containing substance exhibits a strong H. pylori-specific growth-inhibiting action, it is possible to produce an excellent anti-H. Pylori action just by taking a small amount of this pharmaceutical preparation, and no side effects occur. Useful for healing.

Primary structure of amino acid sequence of α-N-acetylglucosaminidase homolog CBM region to be prepared Binding of various CBM-GSTs to glycoproteins Binding of various CBM-GSTs to GlcNAcα1 → 4Galβ residue-containing glycoproteins Binding strength of various CBM-GST to sugar chains containing LAcNAc Binding to cells having GlcNAcα1 → 4Galβ residues on the surface of various CBM-GSTs

Mucin is a glycoprotein that exists mainly in the digestive organs of higher organisms such as humans and mice and some animals. In the present invention, gastric mucin is defined in the stomach and duodenum of humans, mice, and pigs. It is a mixture containing MUC1, MUC5AC and MUC6. Among these, MUC5AC and MUC6 are characterized by having a dense structure of O-linked sugar chains, and it has been shown that MUC5AC and MUC6 exist separately without being mixed with each other. . In particular, the following GlcNAcα1 → 4Galβ residue is highly bound to the non-reducing end of the O-linked sugar chain of MUC6, and the sialic acid content is low, so that the solution is neutral. In addition, since MUC6 is secreted from subcells in the lower gastric body mucosa, as a result, it is known that a large amount of MUC6 is present in the vicinity (lower gastric mucosa), that is, in a portion near the mucosal muscle plate under the gastric mucosa. It has been. Porcine gastric mucin (PGM) used in the present invention
Type III (Sigma) and PGM Class III (purified from PGM Type III)) are known to be mucins containing a relatively large amount of MUC6 having a GlcNAcα1 → 4Galβ residue-containing O-glycan.

The protein used in the present invention is Clostridium perfringens (Clostridium).
perfringens strain13) -derived α-N-acetylglucosaminidase is particularly preferably a region comprising the second to sixth N-terminal CBM regions associated with the C-terminus, but a glycosyl hydrolase having a plurality of CBMs on the C-terminal side A homologue of α-N-acetylglucosaminidase belonging to family 89 (GH89) is Bifidobacterium.
bifidum), Eubacterium dolichum, and Collinsella stercoris, which can be used in the same manner as this method. A plurality of CBMs of α-N-acetylglucosaminidase belonging to GH89 are estimated by amino acid sequence comparison to be all family proteins called CBM32 although their homology is low (about 35% or less). Therefore, the homologue derived from the above-mentioned microbial cells having a site in which a plurality of these CBM regions are arranged in tandem is highly likely to have an affinity for the same glycoprotein sugar chain (FIG. 1).

Clostridium perfringens
The proteins from the 2nd to the 6th N-terminal of the plurality of CBM regions associated with the C-terminal of α-N-acetylglucosaminidase derived from strain13) are prepared as in the following examples with reference to Patent Document 3. be able to. In addition, CBMs from other homologs can be similarly prepared.

  By using these CBMs immobilized on a carrier, the above substances can be adsorbed and eluted, and finally anti-pylori substances can be obtained. In addition, since these CBMs can be used as fusions with other proteins, the prepared CBMs can be immobilized on a carrier or used as fusion proteins with other functional proteins.

  The conditions for exerting the affinity of the protein prepared in the present invention for GlcNAcα1 → 4Galβ residue-containing O-glycan can be used as long as they are in a buffer solution of pH 4 to 9, but specifically, as follows: Become.

  Examples of the solution include a phosphate buffer solution, a carbonate buffer solution, and a Tris buffer solution, and phosphate buffered saline (PBS) is particularly preferable. The pH of the solution is preferably near neutral, and particularly preferably from pH 6.0 to pH 8.0. The reaction temperature is preferably 15 to 40 ° C, particularly preferably 25 ° C. The reaction time is preferably several tens of minutes to several hundreds of μM and several tens of minutes to 10 hours, particularly preferably about 30 minutes to 2 hours. However, when the protein concentration is several tens of nM or less, it can be bound relatively high in 5 hours or more. Any reaction vessel may be used as long as it can control the temperature in the system and does not particularly adsorb dissolved proteins and carbohydrate compounds. In the reaction, the reaction time can be shortened by stirring and shaking.

  Hereinafter, a polypeptide that specifically binds to the sugar chain of the gastrointestinal mucin of the present invention will be specifically described with reference to examples in which its function was investigated, but the present invention is not limited in any way. .

(1.1 Preparation of CBM)
Referring to Patent Document 3, gene amplification is performed using the genome of Clostridium perfringens strain 13 as a template (amplification region and primers used are shown in FIG. 1 and Table 1), and restriction enzyme (SacI and XhoI) treatment After that, protein expression vectors (commercially available (Takara) cold shock protein expression vectors (pcold
TF) is introduced into the multicloning site of the glutathione S transferase (GST) gene) at the restriction enzyme sites (SacI on the 5 ′ end side and XhoI on the 3 ′ end side), and E. coli (BL21 (DE3)). This was precultured in LB medium (2 ml) containing an ampicillin concentration of 50 μg / ml, and further shake-cultured at 30 ° C. for 3 hours until the OD600 reached 0.6 in 1000 ml of the same culture solution. Thereafter, IPTG was added at 0.05 mM and glucose 0.5%, followed by shaking culture at 15 ° C. for 24 hours to prepare Escherichia coli expressing this protein. To the collected cells, lysozyme was added at a concentration of 1 mg / ml (total amount 4 ml), and a PBS solution was further added to make the total amount 20 ml. This suspension is sonicated on ice and centrifuged at 20000 xg for 10 minutes, and the supernatant is purified using a commercially available His-tag affinity column (MN, Protino-Ni-TED2000) or glutathione beads. Purified by a purification method using (GE Healthcare). Elution was performed using 200 mM imidazole (purification method using His-tag) or 20 mM reduced glutathione (purification method using GST-tag). By using an ultrafiltration membrane (excluded molecule size of 100,000), this solution can be obtained at 100-200 mg per liter culture as a result of removing the imidazole or reduced glutathione used for the purification. It was.

(2.1 Investigation of affinity of prepared CBM for GlcNAcα1 → 4Galβ residue-containing O-glycan, investigation by dot blot)
Various glycoprotein sugar chains and sugar chains dissolved in PBS (bovine submandibular gland mucin (Sigma, Type IS, BSM (FIG. 3)), human gastrointestinal mucin (Wako, HGM (FIG. 3)), porcine stomach Crude product of mucin (Sigma, Type III, PGM
Type III (Fig. 3)), purified porcine stomach mucin (Type III mucin) (Kanto Chemical, PGM Class III (Fig. 3)), fetuin (Sigma, Fetuin (Fig. 3)), asialofetin (Sigma, Asialo)
Fetuin (FIG. 3)), chondroitin sulfate (Biochemical, CS (FIG. 3)), heparin sulfate (Biochemical, HS (FIG. 3)) 10 μL of a 1 μg / μL solution was added to a nitrocellulose membrane (PROTRAN). (BA-80, WHATMAN) and dried at 60 ° C. for 1 hour. The membrane was blocked by being immersed in 3% bovine serum albumin (BSA) dissolved in PBS containing 0.05% Tween 20 for 3 hours at room temperature. 1 mL (1 μM) of a solution in which each CBM-GST protein was dissolved in PBS was added to the membrane after blocking and incubated at 25 ° C. for 2 hours. The membrane was washed 5 times with 4 mL of PBS containing 0.05% Tween 20, and 1 mL of horseradish peroxidase-fused anti-GST monoclonal antibody (Nacalai Tesque, (1: 25,000)) was added. After incubation at room temperature for 1 hour, the membrane was washed 5 times with 4 ml of PBS containing 0.05% Tween 20, and the bound monoclonal antibody was detected by luminescence with a chemiluminescence reagent (ECL plus, GE Health Science). The results are shown in FIG.

From the above results, the prepared CBM has a strong binding property to mucin-type glycoprotein, and in particular, the GST fusion containing the CBM4 region is a sugar having an αGlcNAc-containing PGMCclassIII mucin, that is, a GlcNAcα1 → 4Galβ residue-containing O-glycan. It was shown to bind strongly to protein sugar chains. It was also found that it was difficult to bind to other sugar chains. Furthermore, it was found from comparison of each CBM-GST that at least the CBM4 region is necessary for binding to GlcNAcα1 → 4Galβ residue-containing O-glycan, that is, αGlcNAc at the non-reducing end. It was also found that CBM4-5-GST binds to heparin. This is probably because heparin has a GlcNAcα- or GlcN-SO ₄ residue, so that the CBM4 region is also involved in heparin binding.

(2.2 Investigation of affinity of prepared CBM for GlcNAcα1 → 4Galβ residue-containing O-glycan, investigation by sandwich equalizer)
A sandwich ELISA kit in which 100 μL of a 0.5% solution of porcine stomach mucin (Type III, Sigma) dissolved in a PBS solution is immobilized with a monoclonal antibody (HIK1083) that specifically binds to a sugar chain to which GlcNAc is bonded with α. It was injected into each well of an ELISA plate of Kanto Chemical). After allowing the plate to stand at 37 ° C. for 2 hours, each well was washed 5 times with 400 μL of PBS containing 0.05% Tween20. Next, an anti-GST monoclonal antibody (Nacalai Tesque, (1: 25,000)) fused with 100 μL of horseradish peroxidase (HRP) was added. After incubating at room temperature for 1 hour, each well was washed 5 times with 400 μL of PBS containing 0.05% Tween 20, and the bound monoclonal antibody was allowed to stand for 30 minutes after adding the HRP reaction reagent attached to the kit, and then stopped. Detection (420 nm) with a microplate reader. The results are shown in FIG.

  The results showed that CBM2-6-GST strongly bound to GlcNAcα1 → 4Galβ residue-containing glycoprotein with a significant difference. According to the result of the dot blot shown in Example 2, CBM2-4-GST, CBM2-5-GST, and CBM2-6-FIVAR-GST are also bound to GlcNAcα1 → 4Galβ residue on the plate. Although it is considered, it was not shown in the present method with a significant difference compared to CBM2-6-GST.

  From the above results, it was shown that the CBM2-6 protein provided by the present invention binds to a glycoprotein having an activity of inhibiting H. pylori growth. From these results, the GH89 family α-N-acetylglucosaminidase containing a plurality of CBM32 (or FA58C) regions in the C-terminal region includes a plurality of CBM regions present in the C-terminal direction from the catalytic region. It was estimated that the protein binds to αGlcNAc-containing mucins. That is, it was found that the preparation of a protein containing this region is important for the preparation of a glycoprotein having an inhibitory effect against H. pylori growth.

(2.3 Verification of binding force to Galβ1 → 4GlcNAc-R compound containing fluorescent group of prepared CBM)
A compound (LAcNAc-TMR, MW; 1141.4) in which the reducing end (R) of N-acetyllactosamine (Galβ1 → 4GlcNAc-R) is bound to a tetramethylrhodamine group via polyethylene glycol is dissolved in PBS. , Prepared various CBM-GST (CBM2-3-GST, CBM3-4-GST, CBM4-5-GST, CBM5-6-GST, CBM2-6-GST, CBM3-6-GST, CBM4-6-GST, GST was added to PBS at a final concentration (from 1 nM to 1 μM), and the diffusion time at that time was measured by Olympus MF-20, and as a result, CBM3-6-> CBM2- was compared by comparing the binding strength to LAcNAc-TMR. 6->CBM5-6->CBM4-5->CBM4-6->GST> CBM3-4- It was decided.

  From the above results and the results of dot blots and the like (Example 2.1 and Example 2.2), it was inferred that CBM2 and CBM5 are involved in at least binding to galactose (Gal). Furthermore, in the above method, the degree of binding of CBM2-3-GST and CBM2-6-GST to LAcNAc-TMR can be determined by measuring the degree of fluorescence polarization (mP) simultaneously with the change in protein concentration. ) As 1.422 μM and 4.007 μM, respectively (FIG. 5).

The binding property of CBM protein to AGS cells (Non-patent Document 5) stably expressing αGlcNAc residues and control cells was examined.
Cells were cultured overnight on glass slides placed in 12-well cell culture plates. The slide glass was washed once with PBS and then immersed in a 3.7% Paraformaldehyde solution for 10 minutes to immobilize the cells. Further, after washing once with PBS, it was immersed in a 0.1% Triton X-100 solution for 5 minutes. After washing 3 times with PBS, blocking was performed using a 1% BSA solution for 30 minutes. 0.4 μg / ml CBM2-3-GST solution and 100 μl of CBM2-5-GST solution were placed on a glass slide and allowed to react at room temperature for 1 hour. After washing with PBS three times, the primary antibody anti-GST-tag antibody (0.5 μg / ml) was reacted for 1 hour, and further washed with PBS three times, then the secondary antibody anti-mouse IgG-FITC antibody. (0.5 μg / ml) was reacted for 30 minutes. After thoroughly washing with PBS, a cover glass was sealed from above and observed with a fluorescence microscope. As a result, it was revealed that CBM2-5-GST protein strongly binds to α-GlcNAc stably expressing AGS cells (α4GnT cell) (FIG. 6).

  With reference to Example 1, gene amplification (region encoding 5 CBMs in the C-terminal region) was performed using the genome of Bifidobacterium bifidum JCM as a template. After restriction enzyme treatment, protein expression vector (pET23a, The protein of interest was induced and purified in the same manner as in Example 3. As a result of conducting the same examination as in Example 2 using this recombinant protein, it was shown that it strongly binds to a digestive tract mucin, particularly a glycoprotein sugar chain having a GlcNAcα1 → 4Galβ residue-containing O-glycan. Similarly, it was found that it was difficult to bind to other sugar chains.

  The sugar chain separation / extraction method using the protein provided by the present invention is a completely new method using a sugar chain binding tool of enterobacteria, and the protein used has an affinity for conventional general sugar chains. Unlike the so-called lectins having the properties, they have a high binding power to the sugar chain and convenience in genetic engineering, and thus are highly industrially applicable.

  Therefore, the prepared protein serves as an activity-enhancing factor by fusion to a hydrolase or glycosyltransferase related to mucin-type glycoprotein sugar chains, and mucin-type glycoprotein sugar chains (particularly containing GlcNAcα1 → 4Galβ residues). It is also useful as a tool for pathological examination of cancer tissues that express

CBM sugar binding module FIVAR found in various architecture domain
TM transmembrane domain

Claims (5)

A polypeptide comprising an amino acid sequence from N-terminal 934 to 1625 of α-N-acetylglucosaminidase (SEQ ID NO: 1 in the sequence listing) derived from Clostridium perfringens strain 13 , which is a sugar of gastrointestinal mucin A polypeptide that specifically binds to a chain. A polypeptide comprising an amino acid sequence from N-terminal 934 to 1348 of α-N-acetylglucosaminidase (SEQ ID NO: 1 in the sequence listing) derived from Clostridium perfringens strain 13 , which is a sugar of gastrointestinal mucin A polypeptide that specifically binds to a chain. A polypeptide comprising an amino acid sequence from the N-terminal 934th to 1495th of α-N-acetylglucosaminidase (SEQ ID NO: 1 in the sequence listing) derived from Clostridium perfringens strain13 , which is a sugar of gastrointestinal mucin A polypeptide that specifically binds to a chain. A polypeptide consisting of Clostridium perfringens (Clostridium perfringens) strain13 derived alpha-N-acetylglucosaminidase amino acid sequence from the N-terminal 934-th to 1922-th (SEQ ID NO: 1), the gastrointestinal mucin sugar A polypeptide that specifically binds to a chain.   The polypeptide according to any one of claims 1 to 4, wherein the sugar chain of the digestive tract mucin is a GlcNAcα1-4Galβ residue-containing O-glycan.

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