JP5425481B2 - Novel carbohydrate hydrolase that hydrolyzes α-N-acetylglucosaminyl linkage - Google Patents

Description

  The present invention relates to a novel protein, and more particularly to a novel carbohydrate hydrolase that hydrolyzes α-N-acetylglucosaminyl linkages of carbohydrates.

  In higher organisms such as humans, it has been reported that heparin in which N-acetylglucosamine (GlcNAc) is bound at α and oligosaccharide chains (GlcNAcα1-4Gal-) bound to galactose (Gal) exist ( For example, refer nonpatent literature 1 grade | etc.,). These sugar chains are not only ubiquitously present on certain tissues, but are also attracting attention as important substances related to various diseases in recent years (see, for example, Non-Patent Document 2). Therefore, in the clinical and research and development fields, in addition to visual information such as the distribution of sugar chains on tissues, information from the analysis results of the chemical structure plays an important role. In this case, an antibody against GlcNAc conjugated with α is useful, but at present, only one type has been reported, and there are very few reported examples of its specificity. For this reason, it is considered that the use of a sugar hydrolase having high specificity for a sugar chain containing GlcNAc bound by α will greatly contribute to the above-mentioned field.

  So far, the enzyme that hydrolyzes the α-glycoside bond of GlcNAc (aGNase) is a recombinant enzyme derived from our bacterium (Clostridium perfringens (hereinafter aGNaseC)), and is a sugar chain mainly containing GlcNAcα1-4Gal. Patent Document 1), wild-type enzyme hydrolyzing human heparin oligosaccharide (-GlcNAcα1-4GlcAβ1-3-) and snake venom-derived enzyme (synthetic substrate methylumbelliferyl-N-acetyl-α-D) There is a report example of glucosamine (wild-type enzyme acting on GlcNAc-α-MEU) (see, for example, Non-Patent Documents 3 and 4). Since these sugar hydrolases are biosafety level 2 enzymes that act on GlcNAcα1-4Gal from bacteria, their use is limited, and those derived from humans and snake venoms are extracted from natural and cultured cells Therefore, it is difficult to be a mass preparation method.

Therefore, in order to achieve the above-described object, a new enzyme preparation method that is derived from a safe host and can be prepared in large quantities has been demanded.
Here, GlcA is an abbreviation for glucuronic acid.

JP 2007-236208 A

Ishihara, K. et al Biochem. J., 318 (1996), 409-416 Kawakubo, M. et al Science, 305 (2004), 1003-1006 Zhao, G. H. etal Proc. Natl. Acad. Sci. USA, 93 (1996), 6101-6105 Nok, J. A. etal J. Biochem Mol. Toxico., 15 (2001), 221-227

  An object of the present invention is to provide a large amount of an enzyme that releases GlcNAc from an oligosaccharide (GlcNacα1-R) or a sugar derivative in which N-acetylglucosamine (GlcNAc) is bonded to α at the non-reducing end from a safe cell. Is to make it.

  In light of the above situation, the present inventors have produced several recombinant proteins based on some DNA sequence information considered to be homologous genes of human aGNase and aGNaseC. As a result, the present inventors have found an enzyme that does not act on GlcNAcα1-4Gal-R but acts on GlcNAcα-OR and releases GlcNAc.

  That is, the present invention relates to a protein having the amino acid sequence from 22nd to 732th of SEQ ID NO: 1, the amino acid sequence from 21st to 730th of SEQ ID NO: 2, or the 34th to 744th amino acid sequence of SEQ ID NO: 3. It is.

Further, the present invention has amino acid sequences from the 22nd to the 732rd of SEQ ID NO: 1, the 21st to the 730th of SEQ ID NO: 2, or the 34th to 744th of the SEQ ID NO: 3, and the following formula (1) Is a recombinant carbohydrate hydrolase that liberates N-acetylglucosamine (GlcNAc) from water, and in particular has the following physicochemical properties:
GlcNAcα-OR (1)
(In the formula, R represents aromatic and saturated, unsaturated hydrocarbon, sugar chain, protein, sugar chain, protein or lipid containing various substituents, and GlcNAc represents N-acetylglucosamine.)

(1) 4-aminophenyl-N-acetyl-α-D-glucosamine (GlcNAcα-pNP) in the amino acid sequence from 22nd to 732th of SEQ ID NO: 1 or 21st to 730th of SEQ ID NO: 2 To produce N-acetylglucosamine (GlcNAc) and 4-nitrophenol (pNP), but 4-nitrophenyl-N-acetyl-α-D-galactosamine (GalNAcα-pNP), 4-nitrophenyl- α-L-fucose (Fucα-pNP), 4-nitrophenyl-β-L-fucose (Fucβ-pNP), 4-nitrophenyl-α-D-glucose (Glcα-pNP), 4-nitrophenyl-α- D-galactose (Galα-pNP), 4-nitrophenyl-β-D-galactose (Galβ-pNP), 4-nitrophenyl-α-D-mannose (Manα-pNP), 4-nitrophenyl-β-D -Glucuronic acid (GlcAβ-pNP) does not work.
In the amino acid sequence from the 34th to the 744th of SEQ ID NO: 3, 4-nitrophenyl-N-acetyl-α-D-glucosamine (GlcNAcα-pNP), 4-nitrophenyl-α-D-glucose (Glcα-pNP) ), And 4-nitrophenyl-β-D-glucuronic acid (GlcAβ-pNP) to produce each free sugar and 4-nitrophenol (pNP), but 4-nitrophenyl-N-acetyl -α-D-galactosamine (GalNAcα-pNP), 4-nitrophenyl-α-L-fucose (Fucα-pNP), 4-nitrophenyl-β-L-fucose (Fucβ-pNP), 4-nitrophenyl-α 4-Nitrophenyl-α-D-mannose (Manα-pNP) does not act on -D-galactose (Galα-pNP), 4-nitrophenyl-β-D-galactose (Galβ-pNP).
(2) It does not act on a disaccharide (GlcNAcα-Gal) in which GlcNAc is bonded with α at the 3-position, 4-position and 6-position of Gal, and a sugar chain or derivative thereof having this disaccharide at the non-reducing end.
(3) All the above proteins are active at near neutral pH (7-8) and 37 ° C,
(4) The molecular weight of a fusion protein having an affinity tag (6 × His) for protein purification in the molecule and a trigger factor that is a chaperone in the N-terminal region is about 130,000. The above region can be removed by protease (Factor Xa). The molecular weight after removal is about 82,000.

  The enzyme of the present invention does not act on the GlcNAcα1-4Gal-oligosaccharide site present in the mucin-type glycoprotein sugar chain present in the human body, and is a novel enzyme that acts on other GlcNAc-containing sugar chains and compounds It is useful for elucidating the complex sugar chain structure expressed on the tissue.

The enzyme activity of aGNase B1, B2, and B3 with respect to 4-nitrophenyl glycoside of Example 2 is shown. The enzyme activity of aGNaseB1 with respect to 4-methoxyphenyl glycoside of Example 3 is shown. The enzyme activity of aGNaseB2 with respect to 4-methoxyphenyl glycoside of Example 3 is shown. The enzyme activity of aGNaseB3 with respect to 4-methoxyphenyl glycoside of Example 3 is shown. The enzyme activity of aGNaseB3 with respect to the sulfated 4-methylumbelliferyl-sulfated saccharide of Example 4 is shown. The enzyme activity of aGNaseB3 with respect to the porcine stomach mucin glycoprotein of Example 5 is shown.

  Hereinafter, the present invention will be described in detail. The protein of the present invention is three recombinant proteins produced from a part of the gene of Bacteroides thetaiotaomicron VPI5482 strain, from N-terminal 22nd to 732rd (SEQ ID NO: 1, aGNaseB2), from N-terminal 21st A protein having an amino acid sequence from the 730th (SEQ ID NO: 2, aGNaseB3) and the N-terminal 34th to 744th (SEQ ID NO: 3, aGNaseB1) has strong hydrolytic activity, and having this amino acid sequence is active is necessary.

  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. Thereafter, the fused site can be removed by cleaving at the Factor Xa site using a protease.

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). In protein expression and purification, a substance that induces expression of the target protein into a solution (LB medium containing carbenicillin concentration of 50 μg / ml) having an OD600 value of 0.6 of E. coli transformed with the above vector (MC1061 strain). After adding isopropylthiogalactoside (IPTG) at 0.25 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, so that the partially purified product of the protein of the present invention is 15 to 15 per liter of culture solution. Can be obtained with 20mg. This fraction is a substantially main component (80% or more of the total protein) of the protein of the present invention by SDS polyacrylamide electrophoresis, and can be a partially purified product. Furthermore, 0.2 mg / ml of the fusion protein was treated with protease (Factor Xa) 15 unit / ml for 16 hours at 4 ° C, the fused portion was cleaved and then unreacted with an ultrafiltration filter (Amicon Ultra 15 MWCO 100K). Is removed, and the recovered filtrate is passed through His-tag affinity chromatography, whereby a protein having no His-tag and trigger factor fusion moiety can be recovered in a yield of 65 to 90%.

Protein acquisition Using the genome of Bacteroides thetaiotaomicron VPI5482 as a template, each sense primer (underlined indicates the restriction enzyme SacI site) (B1: SEQ ID NO: 4 cggc gagctc ccaatcgattcattggcagcacgt, B2: SEQ ID NO: 5 cggc gagctc gaaacaggaaa and SEQ ID NO 6cggc gagctc gatactggatcaagtaacccagta), antisense primer (underline indicates the restriction enzyme XhoI site) (B1: SEQ ID NO 7ccg ctcgag ttactttgcttcaatcgctgtccgata, B2: SEQ ID NO 8ccg ctcgag ttattgtgctttggtaaagtacgtatc, B3: and SEQ ID NO: 9ccg ctcgag ttaataaaaatattgcatatatttctc), DNA Gene amplification by polymerase cycler PTC100 (manufactured by MJ Research) using polymerase Prime Star (Takara) reaction (reaction conditions (in order, 28 cycles at 98 ° C for 10 seconds, 56 ° C for 15 seconds, 72 ° C for 2 minutes 30 seconds)) The DNA fragment was obtained 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). (XhoI) was subjected to a ligation reaction and transformed into E. coli (MC1061 strain) 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, and cultured with shaking at 15 ° C. for 24 hours, whereby E. coli expressing this protein could be prepared. 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 of 100,000), this solution can be used to remove the imidazole and the like used for the purification, and as a result, the target protein can be obtained as an intermediate purified product at 15 to 20 mg per liter culture. did it. Furthermore, 0.2mg / ml of the fusion protein was treated with protease (Factor Xa) 15unit / ml for 16 hours at 4 ° C, the fused part was cleaved, and then unreacted fusion protein using ultrafiltration membrane (exclusion molecule size 100000) And the collected filtrate is passed through His-tag affinity chromatography, whereby the protein from which the His-tag and the trigger factor fusion moiety have been removed can be recovered.

Enzyme activity (relative to 4-nitrophenyl derivative)
Monosaccharide substrates that are 4-nitrophenyl derivatives (GlcNAc-α-pNP, GlcNAc-β-pNP, GalNAc-α-pNP, Fuc-α-pNP, Fuc-β-pNP, Glc-α-pNP, Gal- (α-pNP, Gal-β-pNP, Man-α-pNP, GlcA-β-pNP) Take 20 μl of 15 mM solution in separate containers, and 2 μl of the protein solution prepared above, 2 μl, 10-fold concentrated PBS (pH 7.4) 10 μl of the solution was added to make a total volume of 100 μl. This was incubated at 37 ° C. with agitation. After reacting for a certain period of time, the amount of released pNP was calculated by measuring with an absorptiometer, and the reactivity to each substrate was compared. In aGNaseB2 and aGNaseB3, after incubation for 1 hour, only GlcNAc-α-pNP was hydrolyzed to 100%, and other substrates were not hydrolyzed at all. In aGNaseB1, after incubation for 6 hours, GlcNAc-α-pNP was hydrolyzed by about 20%, Glc-α-pNP was hydrolyzed by 75%, and only GlcA-β-pNP was hydrolyzed by 35%. It did not hydrolyze (FIG. 1).

Enzyme activity (relative to 4-methoxyphenyl derivative)
After reacting each 4-methoxyphenyl derivative disaccharide substrate (GlcNAcα1-4Galβ-pMP, GlcNAcα1-3Galβ-pMP, GlcNAcα1-6Galβ-pMP) 3 mM with a protein solution 0.6 μg / μl for a certain period of time as described above, When subjected to HPLC (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; ODS-3 (4.6X250mm), detection; UV 283nm), Galβ-pMP, which is considered to be released after GlcNAc cleavage, was confirmed in any substrate. There wasn't. (FIGS. 2, 3 and 4)

Enzyme activity (for 4-methylumbelliferyl derivatives)
Each 4-methoxyphenyl derivative monosaccharide substrate (GlcNAcα-MEU, 2-sulfamino-GlcNAcα-MEU, 6-sulfo-GlcNAcα-MEU) 1 mM as above with a protein solution (aGNaseB3) 0.6 μg / μl for a certain period of time After reaction, HPLC (HPLC apparatus; GL7400 (manufactured by Shimadzu), analytical column; ODS-3 (4.6X250mm), detection; excitation wavelength: 360 nm, fluorescence wavelength: 450 nm), unsulfated GlcNAcα Although cleaved almost completely (95% or more) in 1 h, the release of methylumbelliferone (MEU) was detected, but the sulfated monosaccharide was 2% or less without being cleaved. (Figure 5)

Enzyme activity (evaluation of activity against glycoprotein)
About 30 μg (10.0 mUnit / ml) of the protein solution prepared above (or the above protein solution heat-inactivated at 95 ° C. for 10 minutes), 10 μl of 10-fold concentrated PBS (pH 7.4) solution, porcine gastric mucin glycoprotein ( 50 μl of a 1.5% solution of Kanto Chemical Co., Ltd. (partially purified product) was added to make a total volume of 100 μl, and the mixture was incubated at 37 ° C. for 20 hours with stirring. After the reaction, it was measured by the following methods. As a positive control, a partially purified form of aGNaseC was used.

  The amount of α-bound GlcNAc in the solution after the reaction was detected using a gastric gland mucus 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 (0.0075%) (100 μl in total) and inject it into the well of a 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). The obtained results showed that all of the above-mentioned aGNaseB1, aGNaseB2, and aGNaseB3 did not react at all. As a positive control, it was shown that when about 10 μg (2.4 mUnit / ml) of partially purified aGNaseC was added, it was significantly hydrolyzed. (Fig. 6)

  The hydrolase of the present invention does not act on sugar chains containing GlcNAcα1-4Gal expressed in association with canceration of the stomach, duodenum, and each organ, and specifically hydrolyzes sugar derivatives containing GlcNAc in α. From the degradation (aGNase B2 and B3), it may be possible to easily obtain information such as the distribution of other sugar chains containing GlcNAc in α in vivo in combination with antibodies and aGNaseC. Furthermore, since the aGNase is derived from Bacteroides belonging to Biosafety Level 1, there are few restrictions on its use. In addition, this enzyme may be used as a heparanase-hydrolyzing enzyme because it is similar in sequence to an enzyme that exhibits heparanase activity from the results of in silico computer analysis.

Claims (2)

From the following formula (1), consisting of the amino acid sequence from the 22nd to the 732th sequence of SEQ ID NO: 1 or the 21st to 730th sequence of the SEQ ID NO: 2 and having the following physicochemical properties , N- A recombinant carbohydrate hydrolase that releases acetylglucosamine (GlcNAc) .
GlcNAcα-OR (1)
(In the formula, R represents aromatic and saturated, unsaturated hydrocarbon, sugar chain, protein, sugar chain, protein or lipid containing various substituents, and GlcNAc represents N-acetylglucosamine.)
(1) It acts on 4-nitrophenyl-N-acetyl-α-D-glucosamine (GlcNAcα-pNP) to produce N-acetylglucosamine (GlcNAc) and 4-nitrophenol (pNP). Phenyl-N-acetyl-α-D-galactosamine (GalNAcα-pNP), 4-nitrophenyl-α-L-fucose (Fucα-pNP), 4-nitrophenyl-β-L-fucose (Fucβ-pNP), 4 -Nitrophenyl-α-D-glucose (Glcα-pNP), 4-nitrophenyl-α-D-galactose (Galα-pNP), 4-nitrophenyl-β-D-galactose (Galβ-pNP) , 4- nitro It does not act on phenyl-α-D-mannose (Manα-pNP) or 4-nitrophenyl-β-D-glucuronic acid (GlcAβ-pNP).
(2) It does not act on a disaccharide (GlcNAcα-Gal) in which GlcNAc is bonded with α at the 3-position, 4-position and 6-position of Gal, and a sugar chain or derivative thereof having this disaccharide at the non-reducing end.
(3) Active at near neutral pH (7-8) and 37 ° C.
(4) The molecular weight of the fusion protein having His-tag is about 130,000, and the molecular weight after cleavage with a protease is about 82,000. Recombinant sugar that releases N-acetylglucosamine (GlcNAc) from the following formula (1), which comprises the amino acid sequence from 34th to 744th of SEQ ID NO: 3 and has the following physicochemical properties: Quality hydrolase.
GlcNAcα-OR (1)
(In the formula, R represents aromatic and saturated, unsaturated hydrocarbon, sugar chain, protein, sugar chain, protein or lipid containing various substituents, and GlcNAc represents N-acetylglucosamine.)
(1) 4-nitrophenyl-N-acetyl-α-D-glucosamine (GlcNAcα-pNP), 4-nitrophenyl-α-D-glucose (Glcα-pNP), and 4-nitrophenyl-β-D-glucuron Acts on acid (GlcAβ-pNP) to produce each free sugar and 4-nitrophenol (pNP), but 4-nitrophenyl-N-acetyl-α-D-galactosamine (GalNAcα-pNP), 4 -Nitrophenyl-α-L-fucose (Fucα-pNP), 4-nitrophenyl-β-L-fucose (Fucβ-pNP), 4-nitrophenyl-α-D-galactose (Galα-pNP), 4-nitro It does not act on phenyl-β-D-galactose (Galβ-pNP) or 4-nitrophenyl-α-D-mannose (Manα-pNP).
(2) It does not act on a disaccharide (GlcNAcα-Gal) in which GlcNAc is bonded with α at the 3-position, 4-position and 6-position of Gal, and a sugar chain or derivative thereof having this disaccharide at the non-reducing end.
(3) Active at near neutral pH (7-8) and 37 ° C.
(4) The molecular weight of the fusion protein having His-tag is about 130,000, and the molecular weight after cleavage with a protease is about 82,000.

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