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J. Biol. Chem., Vol. 277, Issue 14, 11889-11895, April 5, 2002

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Enzymatic Attachment of Glycosaminoglycan Chain to Peptide Using the Sugar Chain Transfer Reaction with Endo--xylosidase^*

Keinosuke Ishido, Keiichi Takagaki, Mito Iwafune, Syuichi Yoshihara^§, Mutsuo Sasaki^§, and Masahiko Endo^¶

From the  Department of Biochemistry and ^§ Second Department of Surgery, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan

Received for publication, December 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endo--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--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--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--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--xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, 2). 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, 4). 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, 6). 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) described that the complex type of oligosaccharide from human transferrin glycoprotein was transferred to peptidyl-N-acetylglucosamine using the transglycosylation of endo--N-acetylglucosaminidase from Mucor hiemalis. Ashida et al. (8) reported that endo--N-acetylgalactosaminidase from Bacillus sp. had the transglycosylation activity and succeeded in synthesizing neo-oligosaccharide by using this activity. In the field of proteoglycan, Takagaki and co-workers (9-11) reported the reconstruction of GAG¹ 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.

Previously, we have purified and characterized three kinds of glycosidases that act on the linkage region of proteoglycan as follows: 1) endo--glucuronidase (12) from rabbit liver, which liberates the glucuronyl galactose (GlcA1-4Gal) linkage; 2) endo--galactosidase (13) from the mid-gut gland of the molluscus Patinopecten, which liberates the galactosyl galactose (Gal1-3Gal) linkage; and 3) endo--xylosidase (14) from the mid-gut gland of the molluscus Patinopecten, which liberates the xylosyl serine (Xyl-1-O-Ser) linkage. All of these enzymes were endo-type glycosidases releasing the intact GAG chains from proteoglycan. Because endo--xylosidase cleaved the direct linkage, Xyl-1-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--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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ChemicalsButyloxycarbonyl-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) of the method of Kantor and Schubert (16). 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). -Galactosidase (from bovine testis) was purchased from Sigma. Endo--galactosidase and endo--xylosidase were purified from the molluscus Patinopecten as described previously (13, 14). All other chemicals were obtained from commercial sources.

Pyridylamination of Glycosaminoglycans-- Fluorescence (PA) labeling of the reducing terminals of GAGs (HA, Ch6S, DS, and Ch) was carried out as described previously (17), based on the method of Hase et al. (18). 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.

Preparation of Donors (Peptidoglycan)-- 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). These procedures relied on extraction of proteoglycan with 4 M guanidine HCl in 0.05 M Tris-HCl buffer, pH 8.0, containing the following protease inhibitors: 10 mM EDTA, 0.1 M amino-n-capronic acid, 10 mM N-ethylmaleimide, 5 mM benzamidine HCl, 5 mM phenylmethylsulfonyl fluoride, and 0.36 M pepstatin 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 M Tris-HCl buffer, pH 8.0, containing 10 mM CaCl₂, 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--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). The molar amount of the peptidoglycan was determined by the molar amount of the PA-xylose.

Preparation of Peptido-oligosaccharide-- To obtain two peptido-oligosaccharides (GlcA-GalNAc(S)-GlcA-Gal-Gal-Xyl-peptide and GlcA-Gal-Gal-Xyl-peptide), peptido-ChS was digested with chondroitinase ABC and chondroitinase AC-II as described by Yamagata et al. (21) and purified by Sephadex G-50 column chromatography. To obtain peptido-xyloside (Xyl-peptide), peptido-ChS was digested with endo--galactosidase (13), followed by digestion by -galactosidase (22), as described previously, and purified by HPLC.

Enzymatic Reactions of Endo--xylosidase-- The typical GAG chain transfer reaction of endo--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--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--xylosidase was carried out as described previously (100 mM sodium acetate buffer, pH 4.0 at 37 °C for 24 h) (14).

HPLC Analysis-- 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--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.

Electrophoresis-- 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 M pyridine buffer, pH 3.0, at 1 mA/cm for 60 min (23). 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.

Enzymatic Digestion-- Samples were digested with the following enzymes: chondroitinase ABC (100 mM Tris-HCl buffer, pH 8.0) (21), chondroitinase AC-II (100 mM sodium acetate buffer, pH 6.0) (21), Streptomyces hyaluronidase (20 mM sodium acetate buffer, pH 5.0) (24), and heparitinase II (100 mM sodium acetate buffer, pH 7.0) (25).

Mass Spectrum Measurement-- 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). 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).

N-terminal Amino Acid Sequence-- 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).

Measurement of Activated Protein C-- The activity of activated protein C was measured according to the method of Ohno et al. (27). Two nmol of the reaction product were incubated in 0.05 M of Tris-HCl buffer, pH 8.5, containing 0.15 M 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GAG Chain Transfer Reaction of Endo--xylosidase-- To investigate the GAG chain transfer activity of endo--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--xylosidase was purified as described previously (14). 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--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).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   PAGE of purified endo--xylosidase. PAGE was done in 7.5% polyacrylamide gel at constant 20 mA. Proteins were stained with Coomassie Brilliant Blue.

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.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Anion-exchange HPLC of the reaction products. Peptido-ChS (250 nmol) as a donor and Boc-Leu-Ser-Thr-Arg-MCA (1 mmol) as an acceptor were incubated with endo--xylosidase at 37 °C for 0 (a), 3 (b), and 6 h (c) in 100 mM sodium acetate buffer, pH 3.0. The reaction products were subjected to HPLC, using an anion-exchange column (TSKgel DEAE-5PW, 7.5 × 75 mm). The chromatographic conditions were described under "Experimental Procedures." Solid line is fluorescence intensity, and dashed line is molarity of NaCl (M). Arrow indicates the elution position of PA-Ch6S.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Electrophoresis on a cellulose acetate membrane of the reaction product. Electrophoresis was carried out as described using 0.47 M formic acid, 0.1 M pyridine 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.

Enzymatic Analysis of the Reaction Product-- 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.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Gel filtration HPLC of the reaction product digested with various enzymes. The reaction product (Fig. 2, peak 2) was recovered (A). Then aliquots were digested with chondroitinase ABC (B), chondroitinase AC-II (C), Streptomyces hyaluronidase (D), and heparitinase II (E) and then subjected to HPLC (Shodex OHpak SB-803HQ and SB-804HQ in series). The chromatographic conditions were as described under "Experimental Procedures."

Hydrolysis of the Reaction Product by Endo--xylosidase-- 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--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--xylosidase. Thus, it was confirmed that the chondroitin sulfate chain from the donor was attached to the acceptor peptide through the xyloside linkage.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Reversed phase HPLC of the reaction products after hydrolysis by endo--xylosidase. The reaction products (Fig. 2, peak 2) were recovered (A). Then aliquots were digested with endo--xylosidase under the optimal conditions (pH 4.0) for the hydrolysis (B) and then subjected to HPLC (TSKgel ODS 120 T). Solid line is fluorescence intensity, and dashed line is concentration of acetonitrile (%). The chromatographic conditions were described under "Experimental Procedures." Arrow indicates the elution position of Boc-Leu-Ser-Thr-Arg-MCA.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Ion spray mass spectrum of products obtained from the reaction product after hydrolysis by endo--xylosidase. The fluorogenic peak (bar in Fig. 5B) obtained from the reaction product after hydrolysis by endo--xylosidase was recovered and analyzed by ion-spray mass spectrometry. The conditions for mass spectrometry were described under "Experimental Procedures."

Analysis of ChS Attachment Site of the Acceptor Peptide-- 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--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--xylosidase.

Characterization of the GAG Chain Transfer Reaction-- 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.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Characterization of the GAG chain transfer reaction of endo--xylosidase. A, the effect of reaction time on the GAG chain transfer reaction; B, the effect of pH on the GAG chain transfer reaction (closed squares, 100 mM glycine HCl buffer; closed circles, 100 mM sodium acetate buffer; closed triangles, 100 mM glycine NaOH buffer); C, the effect of varying acceptor peptide concentrations on the GAG chain transfer reaction; D, the effect of varying of peptido-ChS concentrations on the GAG chain transfer reaction.

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).

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⁺.

View this table: [in this window] [in a new window]   Table I Effects of various metal ions on the GAG chain transfer reaction The GAG chain transfer reaction of endo--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 GlcA-GalNAc(S)-GlcA-Gal-Gal-Xyl-peptide and GlcA-Gal-Gal-Xyl-peptide were prepared. These two peptido-oligosaccharides were used as donors of the GAG chain transfer reaction of endo--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--xylosidase) were used as donors, the GAG chain transfer reactions were not observed at all.

View this table: [in this window] [in a new window]   Table II Effects of the length of the GAG cahin on the GAG chain transfer reaction Each of the donors (250 nmol) and Boc-Leu-Ser-Thr-Arg-MCA (1 µmol) were incubated with endo--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.

View this table: [in this window] [in a new window]   Table III Effects on the GAG chain transfer reaction of various GAGs used as donors Each of the peptidoglycans (250 nmol) as donors and Boc-Leu-Ser-Thr-Arg-MCA (1 µmol) were incubated with endo--xylosidase under the typical conditions of the GAG chain transfer reaction.

The Activity of Activated Protein C toward the Boc-Leu-Ser(ChS)-Thr-Arg-MCA-- 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.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   The activity of activated protein C toward Boc-Leu-Ser(ChS)-Thr-Arg-MCA. Boc-Leu-Ser(ChS)-Thr-Arg-MCA (2 nmol), which was produced by transferring ChS chain, was incubated in 50 mM Tris-HCl buffer, pH 8.5, containing 0.15 M NaCl and 1 mM CaCl2 with 1.0 mg of activated protein C. The fluorescence of liberated 7-amide-4-methylcoumarin was measured with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Open circles, the activity toward Boc-Leu-Ser-Thr-Arg-MCA; closed circles, the activity toward Boc-Leu-Ser(ChS)-Thr-Arg-MCA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previously, we have isolated and characterized endo--xylosidase from the mid-gut gland of the molluscus Patinopecten (14). 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--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."

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Scheme of GAG transfer reaction of endo--xylosidase.

In recent years, some reports (28-30) have described the transglycosylation reactions using endo-type glycosidases. As well as these reactions, the GAG chain transfer reaction of endo--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--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) 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--xylosidase, the high molecular weight GAG chain could be transferred more effectively. Previously, we have investigated the substrate specificity of hydrolysis of endo--xylosidase, and we clarified that this enzyme released the high molecular weight GAG chain more effectively (14). 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). However, it was known that HA was a GAG without a linkage region and did not bind to the core protein (32). HA could not be the substrate of endo--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--xylosidase.

Yamamoto et al. (7) succeeded in attaching the N-linked oligosaccharide from human transferrin to peptide T by using the transglycosylation reaction of endo--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--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 (33-35). GAG has a basic structure made of repeating disaccharides (typically a repeat of 40-100 times), which consist of uronic acid and hexosamine (36). 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) and hexasaccharides with the heparin cofactor II binding domain in sugar chain of dermatan sulfate (38). Recently, Takagaki et al. (39, 40) 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--xylosidase as a glycotechnological tool for artificial synthesis of proteoglycan. The GAG chain transfer reaction of endo--xylosidase is expected to open a new avenue in proteoglycan glycotechnology.

	FOOTNOTES

^* This work was supported by Grants-in-aid for Scientific Research 09358013, 11121203, 11470029, 12680603, and 12793010 from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Tel: 81-172-39-5015; Fax: 81-172-39-5016; E-mail: endo-m@cc.hirosaki-u.ac.jp.

Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M112183200

	ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; ChS, chondroitin sulfate; Ch6S, chondroitin 6-sulfate; DS, dermatan sulfate; HS, heparan sulfate; HA, hyaluronic acid; GlcA, D-gluco-4-enepyranosyluronic acid; Boc, t-butyloxycarbon; MCA, 4-methylcoumaryl-7-amide; AMC, 7-amide-4-methylcoumarin; PA, 2-aminopyridine; HPLC, high pressure liquid chromatography; Proteo, proteochondroitin; peptido, peptidochondroitin. All sugars mentioned in this paper are of D configuration.

	REFERENCES

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