Enzymatic Attachment of Glycosaminoglycan Chain to Peptide Using the Sugar Chain Transfer Reaction with Endo-β-xylosidase
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m112183200
ISSN1083-351X
AutoresKeinosuke Ishido, Keiichi Takagaki, Mito Iwafune, Syuichi Yoshihara, Mutsuo Sasaki, Masahiko Endo,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoEndo-β-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-β-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-β-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-β-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-β-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan. Endo-β-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-β-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-β-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-β-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-β-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan. At present, it is possible to mass produce extremely useful proteins due to remarkable developments in gene engineering. However, many reports have described that those proteins synthesized by gene recombination have few biophysical activities because of the incompletion or the lack of carbohydrate chains (1.Simonsen C.C. Shepard H.M. Gray P.W. Leung D.W. Pennica D. Yerverton E. Derynck R. Sherwood P.J. Levinson A.D. Goeddel V. Merigan T.C. Friedman R.M. Interferons. 25. Academic Press, New York1982: 1-14Crossref Google Scholar, 2.Hirose S. Ohsawa T. Inagami T. Murakami K. J. Biol. Chem. 1982; 257: 6316-6321Abstract Full Text PDF PubMed Google Scholar). This is because DNA incorporated by gene recombination has no direct information about biosynthesis of the carbohydrate chains. Furthermore, because the elongation mechanisms of carbohydrate chains were complicated and not clear, it was very difficult to regulate them by gene technology. As a result, the method of attaching the carbohydrate chains to those proteins by glycotechnology plays an important role. Recently, the transglycosylation reaction of glycosidase has received much attention as a method for attaching the carbohydrate chains to protein (3.Takegawa K. Tabuchi M. Yamaguchi S. Kondo A. Kato I. Iwahara S. J. Biol. Chem. 1995; 270: 3094-3099Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 4.Yamamoto K. Takegawa K. Trends Glycosci. Glycotechnol. 1997; 9: 339-354Crossref Scopus (18) Google Scholar). This reaction was regarded as a specific reaction using a reverse reaction of hydrolysis in which the carbohydrate moiety of the substrate was transferred to the hydroxyl groups of acceptor compounds (5.Trimble R.B. Atkinson P.H. Tarentino A.L. Plummer T.H. Maley F. Tomer K.B. J. Biol. Chem. 1986; 261: 12000-12005Abstract Full Text PDF PubMed Google Scholar, 6.Bardales R.M. Bhavanandan V.P. J. Biol. Chem. 1989; 264: 19893-19897Abstract Full Text PDF PubMed Google Scholar). Because the carbohydrate moiety could be transferred to the acceptor compound by block unit using this reaction, the transglycosylation reaction of glycosidase played important roles in glycotechnology. Yamamoto et al. (7.Yamamoto K. Fujimori K. Haneda K. Mizuno M. Inazu T. Kumagai H. Carbohydr. Res. 1997; 305: 415-422Crossref PubMed Scopus (77) Google Scholar) described that the complex type of oligosaccharide from human transferrin glycoprotein was transferred to peptidyl-N-acetylglucosamine using the transglycosylation of endo-β-N-acetylglucosaminidase from Mucor hiemalis. Ashida et al. (8.Ashida H. Yamamoto K. Murata T. Usui T. Kumagai H. Arch. Biochem. Biophys. 2000; 373: 394-400Crossref PubMed Scopus (26) Google Scholar) reported that endo-α-N-acetylgalactosaminidase from Bacillussp. had the transglycosylation activity and succeeded in synthesizing neo-oligosaccharide by using this activity. In the field of proteoglycan, Takagaki and co-workers (9.Saitoh H. Takagaki K. Majima M. Nakamura T. Matsuki A. Kasai M. Narita H. Endo M. J. Biol. Chem. 1995; 270: 3741-3747Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 10.Takagaki K. Munakata H. Majima M. Endo M. Biochem. Biophys. Res. Commun. 1999; 258: 741-744Crossref PubMed Scopus (26) Google Scholar, 11.Takagaki K. Munakata H. Kakizaki I. Majima M. Endo M. Biochem. Biophys. Res. Commun. 2000; 270: 588-593Crossref PubMed Scopus (29) Google Scholar) reported the reconstruction of GAG 1The abbreviations used are: GAGglycosaminoglycanChSchondroitin sulfateCh6Schondroitin 6-sulfateDSdermatan sulfateHSheparan sulfateHAhyaluronic acidΔGlcAd-gluco-4-enepyranosyluronic acidBoct-butyloxycarbonMCA4-methylcoumaryl-7-amideAMC7-amide-4-methylcoumarinPA2-aminopyridineHPLChigh pressure liquid chromatographyProteoproteochondroitinpeptidopeptidochondroitin. All sugars mentioned in this paper are of D configuration chains by recombining different disaccharide units from various GAGs by using the transglycosylation reaction of bovine testicular hyaluronidase. However, there have been no reports of any enzymes capable of transferring the intact GAG chain. Therefore, we focused our attention on the endo-type glycosidase that released the intact GAG chains from proteoglycan, and we investigated a method for attaching the GAG chain directly to peptide using the transglycosylation reaction of the enzyme. glycosaminoglycan chondroitin sulfate chondroitin 6-sulfate dermatan sulfate heparan sulfate hyaluronic acid d-gluco-4-enepyranosyluronic acid t-butyloxycarbon 4-methylcoumaryl-7-amide 7-amide-4-methylcoumarin 2-aminopyridine high pressure liquid chromatography proteochondroitin peptidochondroitin. All sugars mentioned in this paper are of D configuration Previously, we have purified and characterized three kinds of glycosidases that act on the linkage region of proteoglycan as follows: 1) endo-β-glucuronidase (12.Takagaki K. Nakamura T. Majima M. Endo M. J. Biol. Chem. 1988; 263: 7000-7006Abstract Full Text PDF PubMed Google Scholar) from rabbit liver, which liberates the glucuronyl galactose (GlcAβ1–4Gal) linkage; 2) endo-β-galactosidase (13.Takagaki K. Nakamura T. Takeda Y. Daidouji K. Endo M. J. Biol. Chem. 1992; 267: 18558-18563Abstract Full Text PDF PubMed Google Scholar) from the mid-gut gland of the molluscus Patinopecten, which liberates the galactosyl galactose (Galβ1–3Gal) linkage; and 3) endo-β-xylosidase (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar) from the mid-gut gland of the molluscus Patinopecten, which liberates the xylosyl serine (Xyl-β1-O-Ser) linkage. All of these enzymes were endo-type glycosidases releasing the intact GAG chains from proteoglycan. Because endo-β-xylosidase cleaved the direct linkage, Xyl-β1-O-Ser linkage, between a core protein and a GAG chain, it was highly likely that the transglycosylation reaction of this enzyme would be an important tool for attaching the GAG chain to protein. In this report, we describe the transglycosylation (GAG chain transfer) activity of endo-β-xylosidase and our success in transferring the intact GAG chain to peptide using this activity. This is the first report on attaching the GAG chain to peptide enzymatically. Chemicals—Butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-me- thylcoumaryl-7-amide) (Boc-Leu-Ser-Thr-Arg-MCA) was purchased from Peptide Institute Inc. (Osaka, Japan). Activated protein C was purchased from Sigma. Hyaluronic acid (HA, average molecular mass, 41 kDa) was obtained from Research Center, Denki Kagaku Co. (Tokyo, Japan). Dermatan sulfate (DS, from pig skin, average molecular mass, 32 kDa) and chondroitin 6-sulfate (Ch6S, from shark cartilage, average molecular mass, 64 kDa) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Chondroitin (Ch, average molecular mass, 10 kDa) was prepared from chondroitin 6-sulfate by modification (15.Nakamura T. Takagaki K. Majima M. Kimura S. Kubo K. Endo M. J. Biol. Chem. 1990; 265: 5390-5397Abstract Full Text PDF PubMed Google Scholar) of the method of Kantor and Schubert (16.Kantor T.G. Schubert M. J. Am. Chem. Soc. 1957; 79: 152-153Crossref Scopus (198) Google Scholar). 2-Aminopyridine (PA) was purchased from Wako Pure Chemical Co. (Osaka, Japan) and recrystallized from hexane. Sephadex G-50, Sephacryl S-200, and Sepharose CL-4B were purchased from Amersham Biosciences. DEAE-cellulose (DE32) was purchased from Whatman Chemical Separation (Maidstone, UK). Hyaluronidase (from Streptomyces hyalurolyticus), chondroitinase ABC (from Proteus vulgaris), chondroitinase AC-II (from Arthrobacter aurescens), and heparitinase II (from Flavobacterium heparinum) were purchased from Seikagaku Kogyo Co. Actinase E was purchased from Kaken Kagaku Co. (Tokyo, Japan). β-Galactosidase (from bovine testis) was purchased from Sigma. Endo-β-galactosidase and endo-β-xylosidase were purified from the molluscus Patinopecten as described previously (13.Takagaki K. Nakamura T. Takeda Y. Daidouji K. Endo M. J. Biol. Chem. 1992; 267: 18558-18563Abstract Full Text PDF PubMed Google Scholar, 14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). All other chemicals were obtained from commercial sources. Fluorescence (PA) labeling of the reducing terminals of GAGs (HA, Ch6S, DS, and Ch) was carried out as described previously (17.Kon A. Takagaki K. Kawasaki H. Nakamura T. Kojima K. Kato I. Majima M. Endo M. J. Biochem. (Tokyo). 1991; 110: 132-135Crossref PubMed Scopus (71) Google Scholar), based on the method of Hase et al. (18.Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Crossref PubMed Scopus (391) Google Scholar). PA-GAGs were used as the standard marker of molecular weight for gel filtration HPLC. For detection of PA-GAGs, an excitation wave length of 320 nm and an emission wave length of 400 nm were used. The donor substrates for the GAG chain transfer reactions, proteochondroitin sulfate (Proteo-ChS), proteodermatan sulfate (Proteo-DS), and proteoheparan sulfate (Proteo-HS), were purified from bovine tracheal cartilage, pig skin, and bovine lung and purified using standard procedures described by Heinegard and Hascall (19.Heinegard D. Hascall V.C. J. Biol. Chem. 1974; 249: 4250-4256Abstract Full Text PDF PubMed Google Scholar). These procedures relied on extraction of proteoglycan with 4 m guanidine HCl in 0.05 mTris-HCl buffer, pH 8.0, containing the following protease inhibitors: 10 mm EDTA, 0.1 mεamino-n-capronic acid, 10 mm N-ethylmaleimide, 5 mm benzamidine HCl, 5 mm phenylmethylsulfonyl fluoride, and 0.36 mpepstatin subsequent purification by ion-exchange chromatography on DEAE-cellulose eluted in 7 m urea with salt gradients, and gel chromatography on Sepharose CL-4B. Proteo-ChS, Proteo-DS, and Proteo-HS prepared as above were dialyzed against distilled water and lyophilized. To obtain peptidochondroitin sulfate (peptido-ChS), peptidodermatan sulfate (peptido-DS), and peptidoheparan sulfate (peptido-HS), their proteoglycans were digested with actinase E in 0.1 mTris-HCl buffer, pH 8.0, containing 10 mmCaCl2, at 50 °C for 24 h. After digestion, the peptidoglycans were purified by DEAE-cellulose column chromatography and by Sephacryl S-200 chromatography. The molecular masses of the glycosaminoglycan chains of the peptido-ChS, peptido-DS, and peptido-HS were about 30, 32, and 19 kDa, respectively, which were estimated from gel filtration chromatography. After each peptidoglycan was digested by endo-β-xylosidase, the reducing terminals of the liberated GAG chains were labeled by PA. Then each PA-GAG chain was hydrolyzed in 2 n HCl at 100 °C for 2 h. PA-xylose was detected by HPLC as described previously (20.Takagaki K. Nakamura T. Kawasaki H. Kon A. Ohishi S. Endo M. J. Biochem. Biophys. Methods. 1990; 21: 209-215Crossref PubMed Scopus (21) Google Scholar). The molar amount of the peptidoglycan was determined by the molar amount of the PA-xylose. To obtain two peptido-oligosaccharides (ΔGlcA-GalNAc(S)-GlcA-Gal-Gal-Xyl-peptide and ΔGlcA-Gal-Gal-Xyl-peptide), peptido-ChS was digested with chondroitinase ABC and chondroitinase AC-II as described by Yamagata et al. (21.Yamagata T. Saito H. Habuchi O. Suzuki S. J. Biol. Chem. 1968; 243: 1523-1535Abstract Full Text PDF PubMed Google Scholar) and purified by Sephadex G-50 column chromatography. To obtain peptido-xyloside (Xyl-peptide), peptido-ChS was digested with endo-β-galactosidase (13.Takagaki K. Nakamura T. Takeda Y. Daidouji K. Endo M. J. Biol. Chem. 1992; 267: 18558-18563Abstract Full Text PDF PubMed Google Scholar), followed by digestion by β-galactosidase (22.Distler J.J. Jourdian G.W. J. Biol. Chem. 1973; 248: 6772-6780Abstract Full Text PDF PubMed Google Scholar), as described previously, and purified by HPLC. The typical GAG chain transfer reaction of endo-β-xylosidase was carried out as follows. The peptidoglycan (250 nmol) as a donor and the Boc-Leu-Ser-Thr-Arg-MCA (1 μmol) as an acceptor were incubated with 10 milliunits of endo-β-xylosidase in 100 mm sodium acetate buffer, pH 3.0, at 37 °C for 12 h. The reaction was terminated by immersion in a boiling water bath for 3 min. Hydrolysis reaction by endo-β-xylosidase was carried out as described previously (100 mm sodium acetate buffer, pH 4.0 at 37 °C for 24 h) (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). A high performance liquid chromatograph (HPLC, Hitachi L-6200, Hitachi Co., Tokyo, Japan) connected to a fluorescence detector (model F-1150, Hitachi Co.) was used. HPLC analysis for the GAG chain transfer reaction was carried out with a TSK gel DEAE-5PW column (7.5 × 75 mm, Tosoh Co., Tokyo, Japan) under the following conditions. For solution A 0.2 m NaCl and for solution B 1 m NaCl were prepared; the column was equilibrated with solution A, and the ration of solution B to solution A was increased linearly to 100% over 60 min after sample injection; the flow rate was fixed at 1 ml/min; and column temperature was 30 °C. HPLC analysis for the enzymatic digestion products was carried out with Shodex OHpak SB-803HQ and SB-804HQ columns (both 8.0 × 300 mm, Seikagaku Kogyo Co.) in series, which was eluted with 0.2 m NaCl at a flow rate of 0.5 ml/min. HPLC analysis for the reaction product after endo-β-xylosidase treatment was carried out with TSK gel ODS 120 T (4.6 × 250 mm, Tosoh Co.) under the following conditions. Solution C was 0.1% trifluoroacetic acid, and solution D was 0.1% trifluoroacetic acid and 80% acetonitrile. The column was equilibrated with solution C, and the ratio of solution D to solution C was increased linearly from 0 to 60% over 60 min after sample injection; the flow rate was fixed at 1.0 ml/min, and the column temperature was 35 °C. The eluate was monitored on the basis of the fluorescence of Boc-Leu-Ser-Thr-Arg-MCA at excitation and emission wavelengths of 330 and 400 nm, respectively. Native polyacrylamide gel electrophoresis was done in 7.5% polyacrylamide gel at 4 °C. Protein was stained with Coomassie Brilliant Blue R-250. Cellulose acetate membrane electrophoresis was carried out using Separax (6 × 22 cm, Jookoo Co., Tokyo, Japan) in 0.47 m formic acid, 0.1 mpyridine buffer, pH 3.0, at 1 mA/cm for 60 min (23.Mathews M.B. Decker L. Biochim. Biophys. Acta. 1968; 156: 419-421Crossref PubMed Scopus (10) Google Scholar). For determination of fluorescence, membrane strips were cut into pieces 3-mm wide and then extracted with 1 ml of water. Staining of GAG on the cellulose acetate membrane was done with 0.05% Alcian blue in 70% ethanol. Samples were digested with the following enzymes: chondroitinase ABC (100 mm Tris-HCl buffer, pH 8.0) (21.Yamagata T. Saito H. Habuchi O. Suzuki S. J. Biol. Chem. 1968; 243: 1523-1535Abstract Full Text PDF PubMed Google Scholar), chondroitinase AC-II (100 mm sodium acetate buffer, pH 6.0) (21.Yamagata T. Saito H. Habuchi O. Suzuki S. J. Biol. Chem. 1968; 243: 1523-1535Abstract Full Text PDF PubMed Google Scholar), Streptomyces hyaluronidase (20 mm sodium acetate buffer, pH 5.0) (24.Ohya T. Kaneko Y. Biochim. Biophys. Acta. 1970; 198: 607-609Crossref PubMed Scopus (454) Google Scholar), and heparitinase II (100 mm sodium acetate buffer, pH 7.0) (25.Hovingh P. Linker A. Carbohydr. Res. 1974; 37: 181-192Crossref PubMed Scopus (53) Google Scholar). Mass spectrum was obtained on a Sciex API-III triple-quadruple mass spectrometer (Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization source, as described previously (26.Takagaki K. Kojima K. Majima M. Nakamura T. Kato I. Endo M. Glycoconj. J. 1992; 9: 174-179Crossref PubMed Scopus (68) Google Scholar). The samples were dissolved in 50% methanol and injected at 2 μl/min with a micro-HPLC syringe pump. In positive ion mode, scanning was done from m/z 400–1000 during the 1-min scan (10 cycles). The reaction product was blotted onto a poly(vinylidene difluoride) membrane (Millipore, Co.). N-terminal amino acid sequence of the reaction product was determined with a protein sequencer (490 Procise, PerkinElmer Life Sciences). The activity of activated protein C was measured according to the method of Ohno et al. (27.Ohno Y. Kato H. Morita T. Iwanaga S. Takada K. Sakakibara S. Stenflo J. J. Biochem. (Tokyo). 1981; 90: 1387-1395Crossref PubMed Scopus (44) Google Scholar). Two nmol of the reaction product were incubated in 0.05 m of Tris-HCl buffer, pH 8.5, containing 0.15m NaCl and 1 mm CaCl2 with 1.0 μg of activated protein C. The fluorescence of liberated 7-amide-4-methylcoumarin (AMC) was measured on a spectrofluorimeter (Hitachi F-4500, Hitachi, Tokyo, Japan) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. To investigate the GAG chain transfer activity of endo-β-xylosidase, Boc-Leu-Ser-Thr-Arg-MCA was used as an acceptor for the carbohydrate chain. This peptide contained two amino acids (serine and threonine) that had hydroxyl groups required for the transfer region of the GAG chain and also contained a fluorescence substrate. For those reasons, this peptide was useful for the analysis of reaction products on HPLC. As a donor of the carbohydrate chain, peptido-ChS from bovine tracheal cartilage was used. Endo-β-xylosidase was purified as described previously (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). In Fig. 1, native PAGE of this purified enzyme in 7.5% polyacrylamide gel shows a single band. The acceptor peptide and peptido-ChS were incubated at 37 °C with endo-β-xylosidase in 100 mm sodium acetate buffer, pH 3.0 (total volume, 100 μl). After incubation, the reaction was terminated by immersion in a boiling water bath for 3 min. The aliquot of reaction mixture was subjected to anion-exchange HPLC (TSKgel DEAE-5PW). As a result, only one acceptor peak (Fig. 2, peak 1) was obtained before the incubation (Fig. 2a). However, as the reaction proceeded, another fluorescent peak (Fig. 2, peak 2) was generated at the retention time of 41 min. This elution position corresponded to the elution position of PA-Ch6S. Peak 2 was collected and subjected to cellulose acetate membrane electrophoresis. As a result, the reaction product band corresponded to the standard Ch6S, and at this part of membrane, the same fluorescence as the acceptor peptide was detected (Fig. 3). From these results, it was shown that the reaction product had the same negative charge and the same fluorescence as the acceptor peptide.Figure 3Electrophoresis on a cellulose acetate membrane of the reaction product. Electrophoresis was carried out as described using 0.47 m formic acid, 0.1 mpyridine buffer, pH 3.0, at 1 mA/cm for 60 min. Staining was done with 0.05% Alcian blue in 70% ethanol. For determination of fluorescence, the membrane was cut into 3-mm pieces, and each piece was extracted with 1 ml of water.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the structure of the reaction product (Fig. 2, peak 2), the reaction product was digested with various GAG-degrading enzymes. After digestion, reaction mixture was subjected to gel filtration HPLC (Shodex OH-pak SB-803HQ and SB-804 HQ in series). As a result, the reaction product appeared at the retention time of 27 min (Fig. 4A). Judging from the elution position, the molecular mass of the reaction product was estimated to about 30 kDa, corresponding to the molecular mass of the chondroitin sulfate chain of the donor. The reaction product was sensitive to chondroitinase ABC and chondroitinase AC-II digestions (Fig. 4, B and C), although it was not sensitive to Streptomyces hyaluronidase and heparitinase II digestions (Fig. 4, D and E). For these results, it was confirmed that the reaction product had the chondroitin sulfate. To clarify that the chondroitin sulfate of the reaction product was attached to the acceptor peptide, the reaction product was incubated under the optimal condition for hydrolysis reaction of endo-β-xylosidase. After hydrolysis, the reaction mixture was subjected to a reversed phase HPLC (TSKgel ODS 120T). As a result, the reaction product peak was shifted to the position corresponding to the authentic acceptor peptide (Fig. 5). The fluorogenic component was collected, and ion-spray mass spectrometry analysis was performed. The spectra of the new fluorogenic component showed a peak at m/z [M + H]+ ion that was detected at m/z 733.4 [M + H]+ (Fig. 6). The combined results of HPLC analysis and ion-spray mass spectrometry indicated that this fluorogenic component was Boc-Leu-Ser-Thr-Arg-MCA. From these results, it was clarified that the acceptor peptide was liberated from the reaction product after hydrolysis by endo-β-xylosidase. Thus, it was confirmed that the chondroitin sulfate chain from the donor was attached to the acceptor peptide through the xyloside linkage.Figure 6Ion spray mass spectrum of products obtained from the reaction product after hydrolysis by endo-β-xylosidase. The fluorogenic peak (bar in Fig. 5B) obtained from the reaction product after hydrolysis by endo-β-xylosidase was recovered and analyzed by ion-spray mass spectrometry. The conditions for mass spectrometry were described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ChS attachment site of the acceptor peptide was investigated. First, the acceptor peptide without the Boc residue was prepared by trifluoroacetic acid treatment. Next, the chondroitin sulfate chain was attached to this peptide by the GAG chain transfer reaction of endo-β-xylosidase. After that, the reaction product was subjected to N-terminal amino acid sequence analysis. As a result, N-terminal amino acid sequence of the reaction product was determined as Leu-X-Thr-Arg. The second amino acid, the serine residue, was not identified. This result indicated that the hydroxyl group of the serine residue was substituted, that is the ChS chain was attached to the serine residue. From these results, it was confirmed that the ChS chain from the donor was transferred selectively to the serine residue of the acceptor peptide. Thus, it was shown that novel peptidoglycan, Boc-Leu-Ser(ChS)-Thr-Arg-MCA, was synthesized using the GAG chain transfer reaction of endo-β-xylosidase. Time course change of the GAG chain transfer reaction was investigated (Fig. 7A). It was shown that the reaction products were increased as the incubation time increased. However, after 12 h of incubation, they were hydrolyzed, and then decreased gradually. To investigate the effect of pH on the GAG chain transfer reaction, the reaction was performed at various pH values, and the amounts of the reaction product were measured after 12 h at 37 °C (Fig. 7B). It was shown that the optimal pH of the GAG chain transfer reaction was 3.0, differing from the optimal pH 4.0 of hydrolysis of this enzyme (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). The effect of the acceptor peptide concentration on the GAG chain transfer reaction was investigated by performing the reaction with various concentrations of the acceptor peptide for 12 h at pH 3.0 (Fig. 7, C). The reaction product was increased depending on the concentration of the acceptor peptide. The effect of the donor concentration on the GAG chain transfer reaction was investigated in the presence of various concentrations of peptido-ChS at pH 3.0 (Fig. 7D). The reaction product was increased with increasing amounts of peptido-ChS up to 250 nmol (final, 2.5 mm). Because of the solubility, however, we could not add or test higher concentrations (above 2.5 mm) of peptido-ChS. The effect of several metal ions on the GAG chain transfer reaction was investigated (Table I). It was found that the GAG chain transfer activity was strongly inhibited by Cu2+ and Ag+.Table IEffects of various metal ions on the GAG chain transfer reactionCompounds (10 mm)Relative activity%H2O100CuSO417MgSO465AgNO326CaCl242CoCl286ZnCl2108MnCl255CdCl2108BaCl251KCl74NaCl91EDTA103The GAG chain transfer reaction of endo-β-xylosidase was done under the normal conditions except for the presence of various metal ions. Values indicate the mean of duplicates. Open table in a new tab The GAG chain transfer reaction of endo-β-xylosidase was done under the normal conditions except for the presence of various metal ions. Values indicate the mean of duplicates. The effect of the chain length of the donor on the GAG chain transfer reaction was investigated (Table II). The intact peptido-ChS was digested exhaustively with chondroitinase ABC and chondroitinase AC-II, and ΔGlcA-GalNAc(S)-GlcA-Gal-Gal-Xyl-peptide and ΔGlcA-Gal-Gal-Xyl-peptide were prepared. These two peptido-oligosaccharides were used as donors of the GAG chain transfer reaction of endo-β-xylosidase. It was shown that the linkage hexasaccharide and the linkage tetrasaccharide of the donor could be transferred to the acceptor peptide; however, the reaction product was limited to 25 and 15%, respectively. When Xyl-peptide and chondroitin sulfate without peptide (after hydrolysis by endo-β-xylosidase) were used as donors, the GAG chain transfer reactions were not observed at all.Table IIEffects of the length of the GAG cahin on the GAG chain transfer reactionDonorsRelative activityaThe activity shows the relative rate of the reaction product when the products of the GAG chain transfer reaction using intact peptido-ChS as a donor is 100%. Values indicate the mean of duplicates.(%)(GlcA-GalNAc)n-GlcA-GalNAc-GlcA-Gal-Gal-Xyl-peptide100ΔGlcA-GalNAc-GlcA-Gal-Gal-Xyl-peptide25ΔGlcA-Gal-Gal-Xyl-peptide15Xyl-peptide0(GlcA-GalNAc)n-GlcA-GalNAc-GlcA-Gal-Gal-Xyl0Each of the donors (250 nmol) and Boc-Leu-Ser-Thr-Arg-MCA (1 μmol) were incubated with endo-β-xylosidase under the typical conditions of the GAG chain transfer reaction.a The activity shows the relative rate of the reaction product when the products of the GAG chain transfer reaction using intact peptido-ChS as a donor is 100%. Values indicate the mean of duplicates. Open table in a new tab Each of the donors (250 nmol) and Boc-Leu-Ser-Thr-Arg-MCA (1 μmol) were incubated with endo-β-xylosidase under the typical conditions of the GAG chain transfer reaction. The ability of the GAG chain transfer reaction on various donors was investigated (Table III). By using peptido-DS, peptido-HS, and HA as donors, the GAG chain transfer reaction was done as described above. As a result, it was shown that both DS chain and HS chain were transferred to the acceptor peptide; however, HA was not transferred at all.Table IIIEffects on the GAG chain transfer reaction of various GAGs used as donorsDonors (peptidoglycan)Relative activityaThe activity shows the relative rate of the reaction product when the products of the GAG chain transfer reaction using intact peptido-ChS as a donor is 100%. Values indicate the mean of duplicates.(%)ChS (bovine tracheal cartilage, 30 kDa)100DS (pig skin, 32 kDa)75HS (bovine lung, 19 kDa)43HA (bacteria, 41 kDa)0Each of the peptidoglycans (250 nmol) as donors and Boc-Leu-Ser-Thr-Arg-MCA (1 μmol) were incubated with endo-β-xylosidase under the typical conditions of the GAG chain transfer reaction.a The activity shows the relative rate of the reaction product when the products of the GAG chain transfer reaction using intact peptido-ChS as a donor is 100%. Values indicate the mean of duplicates. Open table in a new tab Each of the peptidoglycans (250 nmol) as donors and Boc-Leu-Ser-Thr-Arg-MCA (1 μmol) were incubated with endo-β-xylosidase under the typical conditions of the GAG chain transfer reaction. The activity of activated protein C, which was a kind of protease, toward the Boc-Leu-Ser(ChS)-Thr-Arg-MCA was investigated. It was shown that the activity of the activated protein C toward Boc-Leu-Ser(ChS)-Thr-Arg-MCA was decreased to 30% compared with the activity toward Boc-Leu-Ser-Thr-Arg-MCA (Fig. 8). This result showed that the Boc-Leu-Ser(ChS)-Thr-Arg-MCA was resistant against activated protein C compared with Boc-Leu-Ser-Thr-Arg-MCA. Previously, we have isolated and characterized endo-β-xylosidase from the mid-gut gland of the molluscus Patinopecten (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). This enzyme was an endo-type glycosidase that hydrolyzed the xylosyl serine linkage between a core protein and a GAG chain and, as a result, released the intact GAG chain from proteoglycan. In this study, we clarified that endo-β-xylosidase had the transglycosylation activity and succeeded in attaching the intact GAG chain to the peptide by using this activity. The reaction scheme is depicted in Fig. 9. The transglycosylation activity of this enzyme had the following significant characteristics. 1) It could transfer the intact GAG chain of relatively high molecular weight (20–30 kDa) to peptide. 2) It could transfer the different type of GAG chains (ChS, DS, and HS) similarly to peptide. For these reasons, we named this reaction “GAG chain transfer reaction.” In recent years, some reports (28.Haneda K. Inazu T. Mizuno M. Iguchi R. Yamamoto K. Kumagai H. Aimoto S. Suzuki H. Noda T. Bioorg. Med. Chem. Lett. 1998; 8: 1303-1306Crossref PubMed Scopus (63) Google Scholar, 29.Ajisaka K. Miyasato M. Ishii-Karakasa I. Biosci. Biotechnol. Biochem. 2001; 65: 1240-1243Crossref PubMed Scopus (10) Google Scholar, 30.Li Y.-T. Carter B.Z. Rao B.N.N. Schweingruber H. Li S.-C. J. Biol. Chem. 1991; 266: 10723-10726Abstract Full Text PDF PubMed Google Scholar) have described the transglycosylation reactions using endo-type glycosidases. As well as these reactions, the GAG chain transfer reaction of endo-β-xylosidase was regarded as a specific reaction using a reverse reaction of hydrolysis. Therefore, it was very important for the GAG chain transfer reaction to accelerate a reverse reaction by changing the optimal conditions of hydrolysis of this enzyme. For instance, although the optimal pH of hydrolysis of endo-β-xylosidase was 4.0, the yields of the GAG chain transfer reaction were increased by shifting the pH of the reaction mixture from 4.0 to 3.0. Takagaki et al. (8.Ashida H. Yamamoto K. Murata T. Usui T. Kumagai H. Arch. Biochem. Biophys. 2000; 373: 394-400Crossref PubMed Scopus (26) Google Scholar) has reported that the optimal pH of the transglycosylation reaction of bovine testicular hyaluronidase was 7.0, differing from the optimal pH 5.0 of hydrolysis for this enzyme. It was suggested that changing pH was strongly connected with accelerating a reverse reaction. Furthermore, increasing the concentrations of the donor and the acceptor was necessary for accelerating the reverse reaction, namely the GAG chain transfer reaction. Although the low molecular weight GAG chain (linkage tetrasaccharide and linkage hexasaccharide) could be transferred to the peptide by the GAG chain transfer reaction of endo-β-xylosidase, the high molecular weight GAG chain could be transferred more effectively. Previously, we have investigated the substrate specificity of hydrolysis of endo-β-xylosidase, and we clarified that this enzyme released the high molecular weight GAG chain more effectively (14.Takagaki K. Kon A. Kawasaki H. Nakamura T. Tamura S. Endo M. J. Biol. Chem. 1990; 265: 854-860Abstract Full Text PDF PubMed Google Scholar). Therefore, it was suggested that the higher molecular weight GAG chain could be transferred because of this substrate specificity of this enzyme. Moreover, it was shown that three kinds of GAG chains (ChS, DS, and HS) could be transferred to the peptide by using the GAG chain transfer reaction. However, HA was not transferred by this reaction at all. GAG chains of proteoglycan were attached to the serine residue of the core protein through the linkage region (GlcA-Gal-Gal-Xyl) (31.Lindahl U. Roden L. Gottschalk A. Glycoprotein. Elsevier Science Publishing Co., Inc., New York1972: 491-517Google Scholar). However, it was known that HA was a GAG without a linkage region and did not bind to the core protein (32.Weissman B. Meyer K. J. Am. Chem. Soc. 1954; 76: 1753-1757Crossref Scopus (216) Google Scholar). HA could not be the substrate of endo-β-xylosidase because it did not have the xylosyl serine linkage. Therefore, HA was not transferred to peptide by the GAG chain transfer reaction of endo-β-xylosidase. Yamamoto et al. (7.Yamamoto K. Fujimori K. Haneda K. Mizuno M. Inazu T. Kumagai H. Carbohydr. Res. 1997; 305: 415-422Crossref PubMed Scopus (77) Google Scholar) succeeded in attaching the N-linked oligosaccharide from human transferrin to peptide T by using the transglycosylation reaction of endo-β-N-acetylglucosaminidase from M. hiemalis and described that this glycosylated peptide T was highly stable against proteolysis in comparison to native peptide T. The acceptor peptide used in the current study was the substrate for activated protein C, a kind of protease. It was observed that the ChS-attached acceptor peptide was resistant against the activated protein C. It was suggested that the attached ChS chain inhibited the attack of the enzyme on the substrate as well as the N-linked oligosaccharide of the glycosylated peptide T. This result indicates that the ChS chain of proteoglycan inhibits the proteolysis of core protein. The GAG chain transfer reaction of endo-β-xylosidase released the GAG chain from the donor and transferred it selectively to the serine residue of the acceptor peptide, although the threonine residue also had the hydroxyl group required as the transfer region of the GAG chain. The mechanism of the selective transfer to the serine residue was not clarified in this study. Furthermore, the acceptor specificity, the transfer ability to the high molecular weight protein, and the regioselective transfer of the GAG chain remain problems to be solved in the future. GAG chains of proteoglycan have important roles in the expression and the regulation of its biological function (33.Silbert J.E. Sugumaran G. Biochim. Biophys. Acta. 1995; 1241: 371-384Crossref PubMed Scopus (64) Google Scholar, 34.Kanayama N. Maehara K. Suzuki M. Fujise Y. Terao T. Biochem. Biophys. Res. Commun. 1997; 238: 560-564Crossref PubMed Scopus (24) Google Scholar, 35.Nadanaka S. Clement A. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). GAG has a basic structure made of repeating disaccharides (typically a repeat of 40–100 times), which consist of uronic acid and hexosamine (36.Hascall V.C. Hascall G.T. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Publishing Corp., New York1981: 39-63Crossref Google Scholar). However, it is known that the bioactive domain consists of particular sugar chain sequences, such as pentasaccharides with activity of anticoagulation of heparin (antithrombin III binding activity) (37.Casu B. Oreste P. Torri G. Zoppetti G. Choay J. Lormeau J.-C. Petitou M. Sinay P. Biochem. J. 1981; 197: 559-609Crossref Scopus (277) Google Scholar) and hexasaccharides with the heparin cofactor II binding domain in sugar chain of dermatan sulfate (38.Maimone M.M. Tollefsen D.M. J. Biol. Chem. 1990; 265: 18263-18271Abstract Full Text PDF PubMed Google Scholar). Recently, Takagaki et al. (39.Takagaki K. Munakata H. Majima M. Kakizaki I. Endo M. J. Biochem. (Tokyo). 2000; 127: 695-702Crossref PubMed Scopus (24) Google Scholar, 40.Takagaki K. Ishido K. Trends Glycosci. Glycotechnol. 2000; 12: 295-306Crossref Scopus (16) Google Scholar) succeeded in synthesizing custom-made GAGs consisting of different combinations of disaccharide units by using the transglycosylation reaction of bovine testicular hyaluronidase. Therefore, it is possible to recombine disaccharide units of the GAG chain of the donor peptidoglycan in order to make it bioactive. Furthermore, by using the GAG chain transfer reaction, the reconstructed bioactive GAG chain could be attached to the peptide. As a result, because the peptide would have unprecedented biological function, it would be possible to synthesize a novel bioactive proteoglycan. In this report, we described the attachment of the GAG chain to peptide by using the GAG chain transfer reaction of endo-β-xylosidase as a glycotechnological tool for artificial synthesis of proteoglycan. The GAG chain transfer reaction of endo-β-xylosidase is expected to open a new avenue in proteoglycan glycotechnology.
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