JP4781851B2 - Novel carbohydrate hydrolase that hydrolyzes αN-acetylglucosaminyl linkage - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new enzyme for isolating GlcNAc from an oligosaccharide obtained by binding &alpha;N-acetylglucosamine (GlcNAc) to galactose at positions of &alpha;1,4. <P>SOLUTION: A recombinant glycoside hydrolase is prepared by carrying out gene amplification from genome of Welch bacillus, integrating the amplified gene into a protein expression vector, expressing the protein in Escherichia coli, subjecting the expressed protein to affinity chromatography utilizing a histidine tag and then concentrating the refined solution with an ultrafilter. The enzyme has a characteristic acting on a mucin type sugar protein sugar chain containing the oligosaccharide chain at 37&deg;C in a buffer solution having a pH of 7.0-8.0. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a recombinant protein, and more particularly, to a novel exo-type carbohydrate hydrolase useful for carbohydrate structural analysis and medical diagnostic fields.

In recent years, GlcNAcα1-4Gal-, an oligosaccharide chain in which N-acetylglucosamine (GlcNAc) is α and galactose (Gal) is linked to the non-reducing end of mucin-type glycoprotein sugar chains in the stomach and duodenal mucus of higher organisms (For example, refer nonpatent literature 1 etc.). Recently, this sugar chain has attracted attention as a substance that sterilizes or inhibits the growth of Helicobacter pylori, which is a cause of gastric cancer and gastric ulcer (see, for example, Non-Patent Document 2). In this research and development field, in addition to visual information such as the distribution of sugar chains on the tissue, information from the analysis results of the chemical structure plays an important role. In this case, it is essential to obtain a sugar hydrolase having high specificity for GlcNAc bound at the terminal α.
On the other hand, the enzyme that hydrolyzes the α-glycoside bond of GlcNAc in a broad sense is one that hydrolyzes human heparin oligosaccharide (-GlcNAcα1-4GlcAβ1-3-) and the synthetic substrate methylumbelliferyl N-acetyl of bee-derived enzyme There are no reports other than those for -α-D-glucosamine (GlcNAc-α-MEU) (see, for example, Non-Patent Documents 3 and 4). In addition, these sugar hydrolases do not show a degrading activity for the above disaccharide (GlcNacα1-4Gal), and since these are extracted from nature, they cannot be a large-scale preparation method.

Therefore, in order to achieve the above object, a new enzyme preparation method having hydrolytic activity specific to the above disaccharide and capable of mass production has been demanded.
Here, GlcA is an abbreviation for glucuronic acid.
Ishihara, K. et al., Biochem. J., 318 (1996), 409-416. Kawakubo, M. et al., Science, 305 (2004), 1003-1006. Zhao, GH et al., Proc. Natl. Acad. Sci. USA, 93 (1996), 6101-6105. Nok, JA et al., J. Biochem Mol. Toxico., 15 (2001), 221-227.

  An object of the present invention is to provide a large amount of an enzyme that liberates GlcNAc from an oligosaccharide chain (GlcNAcα1-4Gal-) in which N-acetylglucosamine (GlcNAc) is α and bound to galactose (Gal) at the non-reducing end. There is.

  In view of the above situation, the present inventors have made several studies on the basis of some DNA sequence information considered to be an enzyme gene for the purpose of searching for an enzyme that releases GlcNAc from a sugar chain having a GlcNAcα1-4Gal-site. As a result of producing a recombinant protein, it was found that a protein having a certain amino acid sequence has an activity of releasing GlcNAc from a GlcNAcα1-4Gal-sugar chain, and the present invention has been achieved.

That is, the present invention has the amino acid sequence of up to 1133 from 23 th of SEQ ID NO: 1, oligosaccharides (GlcNAcα1-4Gal-R; R is OH, a sugar chain, a protein or lipid, GlcNAc is N- acetylglucosamine , Gal represents galactose.) Is a recombinant carbohydrate hydrolase that liberates N-acetylglucosamine (GlcNAc) from a glycoprotein sugar chain or the like containing non-reducing terminal at the non-reducing end, and has the following properties.
(1) Acts on GlcNAcα1-4Gal-R to release N-acetylglucosamine (GlcNAc).
(2) Acts on 4-nitrophenyl N-acetyl-α-D-glucosamine (GlcNAcα-pNP) to produce N-acetylglucosamine (GlcNAc) and 4-nitrophenol (pNP). It does not act on N-acetyl-α-D-galactosamine (GalNAcα-pNP) or 4-nitrophenyl N-acetyl-β-D-glucosamine (GlcNAcβ-pNP).
(3) It has activity at near neutral pH (7-8) and 37 ° C., and its molecular weight is about 165000 or 167000.

  The enzyme of the present invention is a novel enzyme that specifically releases GlcNAc from a GlcNAcα1-4Gal-oligosaccharide site present in a mucin-type carbohydrate, and has a complex sugar chain structure such as a mucin-type glycoprotein sugar chain. Useful for elucidation.

Hereinafter, the present invention will be described in detail. Proteins of the present invention are recombinant proteins made from a portion of a gene having the Werushe bacteria (Clostridium perfringens strain13) strains, those having a sequence from the N-terminus to 1133 has a water-decomposable activity (SEQ ID NO: 1) . Particularly desirably, a protein having an amino acid sequence deleted up to the 23rd N-terminal has a strong activity, and it is necessary for the activity to have this amino acid sequence.
The amino acid sequence of the recombinant protein of the present invention may have several amino acid substitutions, deletions, insertions or transfers that do not substantially affect the enzymatic properties. Those having the above are also included in the enzyme of the present invention. In the present specification, “several amino acids” refers to the number of amino acids that may be mutated to such an extent that the enzymatic properties of the enzyme do not change, and are usually 5% or less of the total number of amino acids.

  In the production of the protein of the present invention, after performing gene amplification using the above-mentioned genome as a template, digestion with restriction enzyme sites (SacI on the 5 ′ end side and XhoI on the 3 ′ end side) After being incorporated into a system vector (pcoldTF: Takara) and transformed into host E. coli, it can be expressed and purified by the following method. In addition, the protein of this invention is obtained as a fusion protein with the following protein by the said vector.

The structure of the protein of the present invention will be outlined. From the amino (NH ₂ ) terminal upstream region, six histidine sites (His-tag affinity sites), which are amino acid sequences necessary for protein purification, Trigger factor for forming a structure that becomes a solubilized protein when expressed, Protease for specifically recognizing the protease necessary to cleave the fused site after expression and acquisition of active protein ( (Thrombin) recognition site and the protein site of the present invention, leading to the carboxyl terminus (COOH). For protein expression and purification, induce expression of the target protein into a solution (LB medium containing carbenicillin concentration 50 μg / ml) with an OD600 value of 0.6 of E. coli transformed with the above vector (BL21 (DE3)) After adding isopropylthiogalactoside (IPTG), which is a substance to be treated, at 0.1 to 0.5 mM, the cells expressing the protein of the present invention can be obtained by further shaking culture at 15 ° C. for 24 hours. For protein extraction, the bacterial cells are sonicated, and the supernatant obtained after centrifugation is subjected to His-tag affinity chromatography, whereby the partially purified product of the protein of the present invention is added to 30-liter per 1 liter of culture solution. Can be obtained with 50mg. This fraction is a substantially main component (70% or more of the total protein) of the protein of the present invention by SDS polyacrylamide electrophoresis, and can be a partially purified product.

(Protein acquisition)
Using the genome of Welsh bacteria (Clostridium perfringens strain strain 13) as a template, sense primer (cggcgagctcggtagtgcaattaaggtaagggcatca) antisense primer (ccgctcgagttagaagctctaaaaactcaacatttt), DNA polymerase Prime Star (manufactured by Takara) reaction, thermal cycler Research PTC100 (M The gene amplification (reaction conditions (in order of 98 ° C. for 10 seconds, 56 ° C. for 15 seconds, 72 ° C. for 2 minutes 30 seconds for 30 cycles)) was performed to obtain DNA fragments after amplification. After subjecting the obtained product to restriction enzyme (SacI and XhoI) treatment, the cold shock protein expression vector pcold TF (manufactured by Takara) was transferred to the restriction enzyme site (SacI and 3 'end side on the 5' end side). And then transformed into E. coli (BL21 (DE3)) as an expression host, and the transformant was obtained on an LB agar medium containing a carbenicillin concentration of 50 μg / ml. This was pre-cultured in LB medium (2 ml) containing a carbenicillin concentration of 50 μg / ml, further inoculated into 1000 ml of the same culture solution, and shake-cultured at 30 ° C. for 3 hours until OD600 reached 0.6. Thereafter, IPTG was added at 0.25 mM, followed by shaking culture at 15 ° C. for 24 hours to prepare Escherichia coli expressing this protein. After centrifugation, the supernatant was removed, and then lysozyme was added to the collected cells at a concentration of 1 mg / ml (total amount 4 ml), and a PBS solution was further added to make the total volume 20 ml. This suspension solution was sonicated on ice, centrifuged at 20000 × g for 10 minutes, and the supernatant was purified by a purification method using His-tag affinity chromatography (Protino-Ni-TED2000, manufactured by Nippon Genetics). . By using an ultrafiltration membrane (excluded molecule size 100000), this solution can be used to remove the imidazole and the like used in the above purification, and as a result, the target protein can be obtained as an intermediate purified product at 30-50 mg per liter culture. did it.

Enzyme activity (relative to 4-nitrophenylglycoside)
Take 10 μl of each 4-nitrophenyl substrate (GlcNAc-α-pNP, GlcNAc-β-pNP, GalNAc-α-pNP) 30 mM solution in separate containers, and add 50 μl, 10 μl of the protein solution prepared above to 0.5 μg / μl. 10 μl of double concentrated PBS (pH 7.4) solution was added to make up a total volume of 100 μl. The mixture was incubated at 37 ° C. while stirring. After reacting for a certain period of time, the amount of released pNP was calculated by measuring with an absorption altimeter, and the reactivity to each substrate was compared. After 24 hours of incubation, GlcNAc-α-pNP was 100% hydrolyzed, but GlcNAc-β-pNP and GalNAc-α-pNP were less than 2%.

Enzyme activity (evaluation of activity against glycoprotein)
10 μl of the protein solution 0.5 μg / μl prepared above (or the above protein solution inactivated by heat at 95 ° C. for 10 minutes), 10 μl of 10-fold concentrated PBS (pH 7.4) solution, porcine gastric mucin glycoprotein (Kanto Chemical) , Partially purified product) 50 μl each was added to make a total volume of 100 μl, and incubated for 20 hours at 37 ° C. with stirring. After the reaction, it was measured by the following methods.

(1) The solution after the above reaction was filtered through a microtube-sized ultrafiltration membrane, and the mucin glycoprotein was separated and removed by HPLC (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; Shodex NH2P-50E (Showa When it was subjected to Denko)), the released GlcNAc was clearly confirmed. On the other hand, it was not confirmed at all in the case of the heat-inactivated protein solution.

(2) The amount of α-bound GlcNAc in the solution after the reaction was detected using a gastric gland mucous mucin detection kit (manufactured by Kanto Chemical Co., Ltd.) using the monoclonal antibody HIK1083 against disaccharide (GlcNAcα1-4Gal). After the reaction, dilute the solution to 1/100 (0.03%) (100 μl in total) and inject it into the well of the microtiter plate coated with the attached HIK1083 (monoclonal antibody that recognizes GlcNAcα1-4Gal). Let stand for hours. The wells were washed with 400 μl of PBS solution, and the attached HIK1083 antibody fused with horseradish-derived hydroxyperoxidase was added and allowed to stand at room temperature for 1 hour. Thereafter, the wells were washed with 400 μl of a 50 mM phosphate buffer aqueous solution, developed and stopped with the attached coloring kit, and the absorbance was quantified at 450 nm with an immunoplate reader, immuno mini NJ-2300 (SIC System Instruments). From the results obtained, the heat-inactivated protein solution showed an absorbance of 10 times or more compared to the other protein solution. This showed that the enzyme of the present invention released GlcNAc in an exo form from the GlcNAcα1-4Gal sugar chain present in porcine gastric mucin, and the binding to the HIK1083 antibody was reduced.

  The hydrolase of the present invention hydrolyzes αGlcNAc bonds specifically expressed in the stomach and duodenum, and thus plays an important role in determining the structure of the mucin-type sugar chain of the stomach that has not yet been elucidated. By this structure determination, information on the substance structure for suppressing the growth of Helicobacter pylori that affects gastric cancer and stomach cancer becomes clearer, which can be useful knowledge for the pharmaceutical industry and the food industry.

Claims (2)

The recombinant protein according to any one of the following (a) to (d).
(A) A recombinant protein consisting of the amino acid sequence from the 23rd to 1133rd positions of SEQ ID NO: 1.
(B) A recombinant protein consisting of the amino acid sequence from the 1st to 1133rd positions of SEQ ID NO: 1.
(C) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the 23rd to 1133th amino acid sequences of SEQ ID NO: 1, and acting on the compound of the following [Chemical Formula 1] , A recombinant protein having an activity of releasing N-acetylglucosamine (GlcNAc).
[Chemical formula 1] GlcNAcα1-4Gal-R (I)
(In [Chemical Formula 1] , R represents OH, sugar chain, protein or lipid, GlcNAc represents N-acetylglucosamine, and Gal represents galactose.)
(D) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the first to 1133th amino acid sequences of SEQ ID NO : 1 and acting on the compound of [Chemical Formula 1] , A recombinant protein having an activity of releasing N-acetylglucosamine (GlcNAc). The carbohydrate hydrolase comprising the recombinant protein according to claim 1, which has the following physicochemical properties.
(1) Acts on the compound of [Chemical Formula 1] to release N-acetylglucosamine (GlcNAc).
(2) Acts on 4-nitrophenyl N-acetyl-α-D-glucosamine (GlcNAcα-pNP) to produce N-acetylglucosamine (GlcNAc) and 4-nitrophenol (pNP). It does not act on N-acetyl-α-D-galactosamine (GalNAcα-pNP) or 4-nitrophenyl N-acetyl-β-D-glucosamine (GlcNAcβ-pNP).
(3) Near neutral pH (7-8) and active at 37 ° C., molecular weight is about 165000 or 167000.