©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enzymic Reconstruction of Glycosaminoglycan Oligosaccharide Chains Using the Transglycosylation Reaction of Bovine Testicular Hyaluronidase (*)

(Received for publication, October 11, 1994; and in revised form, December 7, 1994)

Hiromi Saitoh (1) Keiichi Takagaki (1) Mitsuo Majima (2) Toshiya Nakamura (1) Akihiko Matsuki (1) Masaharu Kasai (1) Hozumi Narita (1) Masahiko Endo (1)(§)

From the  (1)Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036, Japan and (2)Kushiro Junior College, 1-10-42 Midorigaoka, Kushiro 085, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The reconstruction of glycosaminoglycan chains using the transglycosylation reaction of testicular hyaluronidase was investigated. First, the optimal conditions for the transglycosylation reaction catalyzed by the enzyme were determined by incubation with the enzyme, using hyaluronic acid (M(r) = 800,000) as a donor and pyridylaminated hyaluronic acid hexasaccharide having glucuronic acid at the nonreducing terminal as an acceptor. The carbohydrate chains as reaction products were determined by high performance liquid chromatography and mass spectrometry. The optimal pH for hydrolysis by the enzyme was found to be about 5.0, whereas that for the transglycosylation reaction was about 7.0. Sodium chloride in the reaction medium inhibited the transglycosylation reaction. Under the optimal conditions, the carbohydrate chains were sequentially transferred along with disaccharide units to the nonreducing terminal of the acceptor and elongated up to docosasaccharide from the acceptor, pyridylaminated hexasaccharide. Using a combination of hyaluronic acid, chondroitin, and chondroitin 4- and 6-sulfate as an acceptor and a donor, it was possible to reconstruct hybrid chains, which were natural or unnatural types of glycosaminoglycan chains. Therefore, it is highly likely that application of the transglycosylation reaction using testicular hyaluronidase would facilitate artificial reconstruction of glycosaminoglycans having some physiological functions.


INTRODUCTION

Bovine testicular hyaluronidase is one of the endotype glycosidases and hydrolyzes the internal bonds of both hyaluronic acid (HA) (^1)and chondroitin sulfates, which have the N-acetylhexosamine linkage attached through the 4-position of the uronic acid(1) . The final reaction products consist mainly of tetrasaccharides and hexasaccharides. It is known that the tetra- and hexasaccharides are derived from the transglycosylation reaction carried out by hyaluronidase itself(2, 3, 4) . In other words, hyaluronidase catalyzed the transglycosylation reaction as well as hydrolysis(2, 3, 4, 5) .

When hexasaccharide derived from hyaluronic acid is digested with testicular hyaluronidase, it is considered that hexasaccharides are cleaved to disaccharides and tetrasaccharides initially, which in turn are converted to octasaccharides. Then these octasaccharides are digested to tetrasaccharides(2, 3, 4) . However, the precise mechanism of the transglycosylation reaction catalyzed by the enzyme is not yet understood, although several effective techniques for analyzing the oligosaccharide transfers that occur rapidly during hydrolysis and transglycosylation were investigated(6, 7) .

In order to elucidate the glycosyl transfer mechanism, Kon et al.(8) recently devised a method of labeling the reducing terminal of oligosaccharides using a fluorogenic reagent, 2-aminopyridine (PA). PA-labeled and unlabeled HA oligosaccharides as a substrate were incubated with testicular hyaluronidase, and the reaction products were analyzed using ion spray mass spectrometry(9) . Using these methods, it was clarified that the hyaluronidase was found to hydrolyze the N-acetylhexosaminide linkage at the nonreducing terminal site, and the disaccharide units, glucuronosyl-beta1-3-N-acetylglucosamine, were successively released from the nonreducing terminal site. Immediately, the released disaccharide units were quickly transferred to the glucuronic acid residue at the nonreducing terminal of another oligosaccharide chain as an acceptor through the beta1-4 linkage. When heptasaccharides or larger oligosaccharides having N-acetylhexosamine at the nonreducing terminal were used as a donor, a trisaccharide, N-acetylglucosaminyl-beta1-4 glucuronosyl-beta1-3-N-acetylglucosamine was released from the nonreducing terminal and then the trisaccharide was also transferred to glucuronic acid at the nonreducing terminal of an acceptor. Thus, it was observed that oligosaccharide used as an acceptor became elongated.

Thus it appears possible to elongate glycosaminoglycan (GAG) chains in vitro using the transglycosylation reaction of testicular hyaluronidase. Recently, more attention has been directed toward remodeling of carbohydrate chains using glycosidases from a glycotechnological viewpoint(10, 11, 12) . It seems feasible to reconstruct various oligosaccharides of GAGs using the transglycosylation reaction of hyaluronidase. In this report, we describe the optimal conditions for promoting selectively the transglycosylation reaction rather than the hydrolysis reaction of testicular hyaluronidase and the synthesis of reconstructed GAGs under these conditions. A system for the reconstruction of GAG chains would open a new avenue in GAG glycotechnology.


MATERIALS AND METHODS

Chemicals

Bovine testicular hyaluronidase (type 1-S) was purchased from Sigma and was purified according to the method of Borders and Raftery(13) . It was free of beta-glucuronidase or beta-N-acetylhexosaminidase activities, as measured by the method of Barrett(14) . Bovine liver beta-glucuronidase was purchased from Sigma. Bovine kidney beta-N-acetylglucosaminidase, Streptococcus hyaluronidase, Streptomyces hyaluronidase, chondroitinase AC-II, chondroitinase B, and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enopyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi4S) were obtained from Seikagaku Kogyo Co. (Tokyo, Japan).

Sephadex G-15, G-75, and G-200 were purchased from Pharmacia Biotech Inc. Bio-Gel P-4 (400 mesh) and AG 1-X2 (200-400 mesh) were obtained from Bio-Rad.

PA was purchased from Wako Pure Chemical Co. (Osaka, Japan) and recrystallized from hexane. Sodium cyanoborohydride was purchased from Aldrich. Other reagents were of analytical grade and obtained from commercial sources.

Preparation of Substrates

For glycosaminoglycans, hyaluronic acid was prepared from human umbilical cord by the method of Danishefsky and Bella(15) , followed by further purification by AG 1-X2 chromatography and Sephadex G-200 gel-filtration chromatography as described previously(16) . The prepared hyaluronic acid contained only glucosamine as hexosamines, and amino acids were not detected. The molecular weight of hyaluronic acid was estimated to be about 800,000 by gel-filtration chromatography. The hyaluronic acid was used as a donor in the transglycosylation reaction.

Chondroitin 6-sulfate (Ch6S; from shark cartilage), chondroitin 4-sulfate (Ch4S, from whale cartilage), and dermatan sulfate (DS, from pig skin) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Chondroitin was prepared from chondroitin 6-sulfate by a modification (17) of the method of Kantor and Schubert(18) .

GAG oligosaccharides were prepared from hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, and chondroitin using the procedure described in a previous report(19) . Each GAG was digested with testicular hyaluronidase. From the digested materials the oligosaccharides (from hexa- to docosasaccharide) were purified through a Bio-Gel P-4 column. Their molecular weights were determined by mass spectrometry. Dermatan sulfate hexasaccharide was prepared using the partial hydrazine degradation method of Maimone and Tollefsen(20) . Judging from its digestibility with chondroitinase AC-II and B and beta-glucuronidase, the uronic acid components of DS hexasaccharides were identified as glucuronic acid and iduronic acid for the carbohydrate residue of the nonreducing terminal and iduronic acid for the internal site of the oligosaccharide.

Preparation of PA-oligosaccharides

Fluorescence (PA) labeling of the reducing terminal sugar of oligosaccharides was carried out as described previously(19) . Each pyridylaminated hexasaccharide (PA-HA hexasaccharide, PA-Ch4S hexasaccharide, PA-Ch6S hexasaccharide, and PA-DS hexasaccharide) was used as an acceptor in the transglycosylation reaction, and PA-HA and PA-Ch4S oligosaccharides were used as standards of molecular weight markers for HPLC.

Conditions for the Hydrolysis Reaction Catalyzed by Testicular Hyaluronidase

Hydrolysis was carried out as follows. Five micrograms of hyaluronic acid as a substrate and 1.0 NFU of testicular hyaluronidase dissolved in 50 µl of 0.5 M sodium acetate buffer (pH 5.0) containing 0.75 M NaCl was incubated at 37 °C for 1 h. The reaction was terminated by immersion in a boiling water bath at 100 °C for 3 min, and aliquots were then assayed by the method of Reissig et al.(21) , which quantified N-acetylglucosamine at the reducing termini of the oligosaccharides released by the enzyme.

Conditions for the Transglycosylation Reaction Catalyzed by Testicular Hyaluronidase

A typical transglycosylation reaction was carried out as follows. Five micrograms of GAGs (hyaluronic acid, chondroitin, chondroitin 4- and 6-sulfate, and dermatan sulfate) as donors, 2 nmol of PA-hexasaccharide as an acceptor, and 1.0 NFU of testicular hyaluronidase dissolved in 50 µl of 0.15 M Tris-HCl buffer, pH 7.0, were incubated at 37 °C for 1 h. The reaction was terminated by immersion in a boiling water bath at 100 °C for 3 min. Ten micrograms of reaction products was subjected to HPLC.

HPLC for PA-oligosaccharides

HPLC for analysis of PA-oligosaccharides was carried out with a PALPAK Type S column (4.0 times 250 mm, Takara Shuzo, Kyoto, Japan) under the following conditions: solution A containing 3% acetic acid adjusted to pH 7.3 with triethylamine and acetonitrile at a ratio of 20:80 and solution B containing the same agents at a ratio of 50:50 were prepared; the column was equilibrated with solution A, and the ratio of solution B to solution A was increased linearly to 100% over 60 min after sample injection; the flow rate was fixed at 1.0 ml/min; and the column temperature was 30 °C. A Hitachi L-6200 equipped with a fluorescence detector (Model F-1150, Hitachi Co., Tokyo, Japan) was used. Fluorescence of PA was detected at excitation and emission wavelengths of 320 and 400 nm, respectively.

Mass Spectrum Measurement

All mass spectra were obtained on a Sciex API-III triple-quadrupole mass spectrometer (Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization source(19) . The mass spectrometer was operated in the negative mode; the ion spray voltage was set at -4,000 V, the interface plate voltage was -600 V, and the orifice voltage was -100 V. The samples were introduced in 0.5 mM ammonium acetate/acetonitrile (50:50 by volume). A micro-HPLC syringe pump, JASCO Familic 100N (pump = 22, Harvard Apparatus Inc., MA), was used to deliver the samples at a flow rate of 2 µl/min. Scanning was done from m/z 300 to 1,200 during the 1-min scan (six cycles). The collisionally activated dissociation spectrum was measured using argon as the collision gas at a collision energy of 40 eV. The daughter ions of the collisionally activated dissociation spectrum were recorded from m/z 200 to 1,000.

Enzyme Digestion

Samples were digested with the following enzymes: Streptococcus hyaluronidase (0.1 M sodium acetate buffer, pH 5.0)(22) , Streptomyces hyaluronidase (0.02 M acetate buffer, pH 5.0)(23) , beta-glucuronidase (0.1 M sodium acetate buffer, pH 4.4) (24) , chondroitinase AC-II (0.1 M sodium acetate buffer, pH 6.0)(25) , and chondroitinase B (0.1 M Tris-HCl buffer, pH 8.0)(26) .


RESULTS

Effect of pH on the Transglycosylation Reaction

To estimate the optimal pH for the transglycosylation reaction, hyaluronic acid (M(r) = 800,000) as a donor and PA-HA hexasaccharide as an acceptor were incubated at 37 °C for 1 h with testicular hyaluronidase in sodium acetate buffer within a pH range of 3.0-6.0 and in Tris-HCl buffer within a pH range of 6.0-9.0. As PA-HA hexasaccharide was no longer digested with hyaluronidase, as mentioned previously(9) , PA-HA hexasaccharide was used as an acceptor for the transglycosylation reaction in order to distinguish it from the donor. After incubation at various pH values, the changes in the chain length of PA-oligosaccharides as the reaction products were determined using HPLC (PALPAK Type S). At a low buffer pH, elongation of the PA-oligosaccharide chain was very slight (Fig. 1). As the pH was increased, PA-oligosaccharides with various chain lengths were observed, the elongation progressing in disaccharide units. The progression of elongation of the PA-oligosaccharides reached a maximum at pH 7.0, when the elongation was observed at least from hexasaccharide to docosasaccharide. However, at pH 9.0, elongation was no longer evident. Representative PA-oligosaccharides that were elongated are plotted in Fig. 1(inset). On the other hand, the optimal pH for hydrolysis by the enzyme was about 5.0, as reported previously(3, 6) .


Figure 1: PA-oligosaccharides produced by the transglycosylation reaction of testicular hyaluronidase at various pH values. PA-HA hexasaccharide as an acceptor and hyaluronic acid as a donor were incubated with testicular hyaluronidase at 37 °C for 1 h in sodium acetate buffer within the pH range of 3.0-6.0 and in Tris-HCl buffer, pH range of 6.0-9.0. The reaction mixtures were then subjected to HPLC (PALPAK Type S column; 4.0 times 250 mm) with fluorescence detection. The flow rate was 1 ml/min. The elution conditions for the chromatography are described under ``Materials and Methods.'' The amounts of each reaction product were calculated on the basis of fluorescence. Arrows indicate the elution positions of PA-HA oligosaccharide standards (6, PA-HA hexasaccharide; 8, PA-HA octasaccharide; 10, PA-HA decasaccharide; 12, PA-HA dodecasaccharide; 14, PA-HA tetradecasaccharide; 16, PA-HA hexadecaaccharide; 18, PA-HA octadecasaccharide; 20, PA-HA eicosasaccharide; 22, PA-HA docosasaccharide). Inset, amounts of the reaction products formed by the transglycosylation reaction in Fig. 1were plotted. PA-octa- and PA-decasaccharide as representative short chains and PA-HA hexadecasaccharide as a representative long chain were plotted. circle, octa-; up triangle, deca-; box, hexadecasaccharide in sodium acetate buffer. bullet, octa-; , deca-; , hexadecasaccharide in Tris-HCl buffer.



Effect of NaCl Concentration on the Transglycosylation Reaction

The effects of NaCl concentration on the transglycosylation reaction were investigated at various concentrations of NaCl at pH 7.0 at 37 °C for 1 h. The elongation of the PA-oligosaccharide decreased with increasing NaCl concentration, and hardly any elongation occurred at a concentration of 0.5 M NaCl (Fig. 2). Therefore, the presence of NaCl was not necessary for the transglycosylation reaction, although hydrolytic activity was dependent on the presence of NaCl, as reported previously(6) .


Figure 2: Effects of NaCl concentration on the transglycosylation reaction. PA-HA hexasaccharide as an acceptor and hyaluronic acid as a donor were incubated with testicular hyaluronidase at 37 °C for 1 h in Tris-HCl buffer, pH 7.0, containing various concentrations of NaCl. The reaction products were subjected to HPLC (PALPAK Type S), and the amounts of elongated oligosaccharides were calculated. The conditions for HPLC are described under ``Materials and Methods.'' PA-HA octa-, PA-deca-, and PA-hexadecasaccharide as the representative reaction products were plotted: bullet, octa-; , deca-; , hexadecasaccharide.



Time Course of the Transglycosylation Reaction

The time course of the transglycosylation reaction was investigated at pH 7.0 (Fig. 3). It was observed that the PA-oligosaccharides elongated as the reaction products increased with prolonged incubation time and that 80% of the PA-HA hexasaccharide added as the acceptor was used for transglycosylation during the 1-h incubation, the chain length of PA-oligosaccharides reaching docosasaccharide.


Figure 3: Time course of PA-HA oligosaccharides produced by the transglycosylation reaction of testicular hyaluronidase. PA-HA hexasaccharide as an acceptor and hyaluronic acid as a donor were incubated with the hyaluronidase at 37 °C for 0 (a), 15 (b), 60 (c), and 90 min (d) in Tris-HCl buffer, pH 7.0, and then subjected to HPLC (PALPAK Type S). The conditions for HPLC are described under ``Materials and Methods.'' Arrows indicate the elution positions of PA-HA oligosaccharide standards.



Effect of Acceptor Concentration on the Transglycosylation Reaction

To investigate the effects of the acceptor concentration on the transglycosylation reaction, PA-HA hexasaccharide at various concentrations as an acceptor was incubated for 1 h with hyaluronic acid as a donor under the optimal conditions for the transglycosylation reaction. PA-octa-, PA-deca-, and PA-hexadecasaccharide as the representative reaction products detected by HPLC are plotted in Fig. 4. The amounts of the elongated PA-oligosaccharides increased in proportion to the concentration of the acceptor.


Figure 4: The effects of acceptor concentration on the transglycosylation reaction. PA-HA hexasaccharide at various concentrations as an acceptor was incubated with hyaluronic acid as a donor under the conditions (pH 7.0) optimal for the transglycosylation reaction at 37 °C for 1 h. The reaction products were subjected to HPLC (PALPAK Type S), and the amounts of each elongated oligosaccharide were calculated on the basis of fluorescence. PA-octa-, PA-deca-, and PA-hexasaccharide as representatives are plotted: bullet, PA-octa-; , PA-deca-; , PA-hexadecasaccharide.



Effect of Donor Concentration on the Transglycosylation Reaction

To investigate the effects of the donor concentration on the transglycosylation reaction, hyaluronic acid at various concentrations as a donor was incubated for 1 h with 2 nmol of PA-HA hexasaccharide as an acceptor under the optimal conditions for the transglycosylation reaction (Fig. 5). PA-octa-, PA-deca-, and PA-hexadecasaccharide as representative reaction products detected by HPLC were plotted. The amounts of the elongated PA-oligosaccharides increased with increasing donor concentration up to 0.1% hyaluronic acid; after a further increase to over 0.1%, the amount of the reaction product became maximal.


Figure 5: The effects of donor concentration on transglycosylation reaction. PA-HA hexasaccharide was incubated with hyaluronic acid at various concentrations as a donor under the conditions (pH 7.0) optimal for transglycosylation at 37 °C for 1 h. The reaction products were subjected to HPLC, and the amounts of each elongated oligosaccharide were calculated on the basis of fluorescence. PA-octa-, PA-deca-, and PA-hexasaccharide as representative reaction products are plotted: bullet, PA-octa-; , PA-deca-; , PA-hexadecasaccharide.



Chemical Structure of PA-Oligosaccharides Produced Using Hyaluronic Acid as both Acceptor and Donor

The reconstructed PA-octasaccharide, which was produced by transglycosylation under the optimal conditions for 60 min using PA-HA hexasaccharide as an acceptor and hyaluronic acid as a donor (the bar in Fig. 3c), was recovered and purified by HPLC (PALPAK Type S). Then the chemical structure of the reconstructed PA-octasaccharide was analyzed by ion spray mass spectrometry (Fig. 6), which revealed multiply charged ions, [M-2H], [M-3H], [M-4H], and [M-5H] at m/z 994.2, 662.4, 496.4, and 397.0, respectively. The molecular weight of the PA-oligosaccharide was computed to be 1,990.1 ± 0.3 based on the presence of these ions. Its mass number was the same as that of native PA-HA octasaccharide pyridylaminated directly to the octasaccharide, which was obtained by hyaluronidase digestion of hyaluronic acid.


Figure 6: Ion spray mass spectrum of a transglycosylation reaction product. PA-HA hexasaccharide as an acceptor was incubated with hyaluronic acid as a donor under the conditions (pH 7.0) optimal for the transglycosylation reaction for 60 min. A reaction product considered to be PA-HA octasaccharide (bar in Fig. 3c) was recovered and purified by HPLC. Then an aliquot was subjected to ion spray mass spectrometry in 0.5 mM ammonium acetate/acetonitrile (50:50 by volume) at 2 µl/min. Details of the conditions used for mass spectrometry are described under ``Materials and Methods.''



Furthermore, the fluorescent substances obtained from the reconstructed PA-octa- and PA-decasaccharide were sensitive to Streptococcus hyaluronidase and Streptomyces hyaluronidase digestion, as was the case for the native PA-octa- and PA-decasaccharide, and the final products of both digestions were close to standard PA-hexasaccharide (data not shown). Therefore, it was confirmed that the carbohydrate chains of the reconstructed PA-HA oligosaccharides were very similar to the native hyaluronic acid chain.

Chemical Structure of PA-oligosaccharides Produced by the Transglycosylation Reaction from PA-Ch4S Hexasaccharide as an Acceptor and Hyaluronic Acid as a Donor

The chemical structures of the carbohydrate chains produced by the transglycosylation reaction using the various combinations of heterogenic GAGs were investigated. PA-Ch4S hexasaccharide as an acceptor and hyaluronic acid as a donor were incubated with hyaluronidase under the optimal conditions for the transglycosylation reaction at 37 °C for 1 h, and the reaction products were analyzed by HPLC (Fig. 7). Three peaks of newly synthesized products (fractions I, II, and III) were observed, which were PA-oligosaccharides elongated by the addition of disaccharide units to the acceptor. Each peak was eluted at a reaction time slightly later than standard PA-Ch4S oligosaccharides. Each fraction was recovered, and its chemical structure was analyzed.


Figure 7: HPLC chromatograms of the transglycosylation reaction products produced from PA-Ch4S hexasaccharide as an acceptor and hyaluronic acid as a donor. PA-Ch4S hexasaccharide and hyaluronic acid were incubated with hyaluronidase under the conditions (pH 7.0) optimal for the transglycosylation reaction at 37 °C for 0 (a) and 60 min (b) and then subjected to HPLC. The chromatographic conditions were as described under ``Materials and Methods.'' Arrows indicate the elution positions of PA-Ch4S oligosaccharide standards.



Fraction I, considered to be a reconstructed PA-octasaccharide, was digested with beta-glucuronidase, and then the resulting fluorescent substance was analyzed by HPLC (PALPAK Type S) (Fig. 8b). It was revealed that a glucuronic acid residue was attached at the reducing terminal. Next, the reconstructed PA-octasaccharide was digested with chondroitinase AC-II and then analyzed by HPLC. The retention time of the digested fluorescent substance was equivalent to the PA-unsaturated disaccharide (DeltaDi4S) (Fig. 8c).


Figure 8: HPLC of transglycosylation reaction products digested by beta-glucuronidase and chondroitinase AC-II. Reaction products (fraction I in Fig. 7b) were recovered and purified (a). Then aliquots were digested with beta-glucuronidase (b) followed by chondroitinase AC-II (c) and subjected to HPLC. The chromatographic conditions were as described under ``Materials and Methods.'' Arrows indicate the elution positions of PA-Ch4S oligosaccharide standards and PA-unsaturated disaccharide (Deltadi4S).



In addition, the molecular weight of fraction I was determined by ion spray mass spectrometry (Fig. 9). Triply and quadruply charged molecular ions were observed, from which the molecular weight of this substance was estimated to be 1,851.5 ± 1.8. The molecular mass of the reconstructed PA-octasaccharide was the same as that of the theoretical substance, which was a PA-octasaccharide having a disaccharide unit of hyaluronic acid at the reducing terminal of the Ch4S hexasaccharide.


Figure 9: Mass spectrum of transglycosylation reaction products. PA-octasaccharide recovered from HPLC (fraction I in Fig. 7b) was purified and then subjected to ion spray mass spectrometry. The conditions for mass spectrometry were as described under ``Materials and Methods.''



Fraction III, which was regarded as PA-dodecasaccharide, was incubated with Streptococcus hyaluronidase (Fig. 10). After incubation with the enzyme, the retention time of PA-dodecasaccharide was shifted closest to that of PA-Ch4S hexasaccharide. Therefore, fraction III was a reconstructed PA-dodecasaccharide having a hybrid structure made up of a hexasaccharide unit derived from hyaluronic acid transferred to the reducing terminal of PA-Ch4S hexasaccharide.


Figure 10: HPLC of a transglycosylation reaction product digested by Streptococcus hyaluronidase. Reaction products (fraction III in Fig. 7b) were recovered and purified (a). An aliquot of the purified sample was digested with Streptococcus hyaluronidase (b) and then subjected to HPLC. The chromatographic conditions were as described under ``Materials and Methods.'' Arrows indicate the elution positions of PA-Ch4S oligosaccharide standards.



Transglycosylation Reaction by a Combination of Different GAGs as both Acceptor and Donor

The transglycosylation reaction using a combination of different GAGs as both an acceptor and a donor was investigated using testicular hyaluronidase under optimal conditions. Two nanomoles of one of the PA-labeled hexasaccharides of hyaluronic acid, chondroitin 4- and 6-sulfate, and dermatan sulfate as acceptors and 5 µg of hyaluronic acid as a donor dissolved in 50 µl of 0.15 M Tris-HCl buffer, pH 7.0, were incubated with 1.0 NFU of testicular hyaluronidase under optimal conditions at 37 °C for 1 h. The same volume of the reaction products was analyzed by HPLC (Table 1). It was found that disaccharide units released from hyaluronic acid were transferred to the reducing termini of all acceptors, except for PA-DS hexasaccharide. For elongation of the oligosaccharide chains, PA-HA hexasaccharide showed the best acceptor ability; the acceptor ability of PA-Ch6S hexasaccharide was better than that of PA-Ch4S hexasaccharide. Despite prolongation of the incubation time to 3 h, PA-DS hexasaccharide showed no acceptor ability.



In order to examine donor ability, 5 µg of hyaluronic acid, chondroitin, chondroitin 4- and 6-sulfate, or dermatan sulfate as donor dissolved in 50 µl of 0.15 M Tris-HCl buffer, pH 7.0, and PA-HA hexasaccharide as an acceptor were incubated with 1.0 NFU of testicular hyaluronidase (Table 2). Disaccharide units from each GAG except for dermatan sulfate were transferred to the nonreducing terminal of the PA-HA hexasaccharide. When hyaluronic acid and chondroitin were used as donors, the chain length of PA-oligosaccharides as reaction products reached docosa- and hexadecasaccharide, respectively. The donor abilities of chondroitin 4- and 6-sulfate were lower than those of hyaluronic acid and chondroitin. Despite prolongation of the incubation time to 3 h, no transglycosylation reaction occurred with dermatan sulfate as a donor, even though the dermatan sulfate was depolymerized by the hyaluronidase digestion. No transglycosylation reaction occurred, irrespective of whether dermatan sulfate was used as a donor or an acceptor. However, it was possible to reconstruct the hybrid glycosaminoglycan oligosaccharides using different combinations of hyaluronic acid, chondroitin, and chondroitin 4- and 6-sulfate.




DISCUSSION

Recently it has been shown that a GAG chain has many kinds of domain structures performing specific physiological functions such as anticoagulant or antithrombotic activity(20, 27) . However, the relationship between biological function and structure is not yet fully understood. Therefore, it is important to develop a method for reconstructing GAG oligosaccharides to investigate their biological function and to obtain their active domains. Recently, various attempts have been made to chemically synthesize GAGs(28) . On the other hand, enzymatic reconstruction of oligosaccharides has been achieved using glycosidase transglycosylation(10, 11) , in particular, with endotype glycosidases(12) .

Recently, we succeeded in labeling the reducing terminal of a GAG chain using a fluorogenic reagent, 2-aminopyridine(8) . Using a combination of this method with HPLC, it became possible to analyze effectively the products of the transglycosylation reaction and to recognize the reducing and nonreducing termini of the chains. Moreover, we recently devised a new method for determination of GAG structure using ion spray mass spectrometry(9, 19) .

Using these methods, important aspects of the hydrolysis and transglycosylation reactions of hyaluronidase have been clarified(9) . First, hyaluronidase releases disaccharide units bearing glucuronic acid at the nonreducing terminal, that is N-acetylhyalobiuronic acid (glucuronosyl-beta1-3-N-acetylglucosamine). Then the disaccharide units are quickly transferred to the glucuronic acid at the nonreducing termini of other chains. Second, when heptasaccharides having N-acetylglucosamine at the nonreducing terminal are used as a donor, trisaccharides are released from the nonreducing terminal by the enzyme. The trisaccharides are then transferred to the glucuronic acid at the nonreducing termini of other chains.

In this study, some important aspects of the transglycosylation reaction catalyzed by testicular hyaluronidase were clarified. First, with respect to the pH dependence of the transglycosylation reaction and hydrolysis, the optimal pH of the former was about 7.0, whereas that of the latter was 5.0. At the pH optimal for the transglycosylation reaction, the disaccharides released by the hydrolysis were quickly transferred to the other chains without remaining in the reaction medium (Fig. 11).


Figure 11: Schema of the transglycosylation reaction by testicular hyaluronidase. PA-Ch6S hexasaccharide as an acceptor and hyaluronic acid as a donor were incubated with testicular hyaluronidase. The reaction product was new oligosaccharide having a hybrid structure made up of a disaccharide unit derived from hyaluronic acid transferred to the nonreducing terminal of PA-Ch6S hexasaccharide.



Second, with regard to the NaCl dependence of the transglycosylation reaction and hydrolysis, NaCl in the reaction medium was effective for hydrolysis, as reported previously(6) , whereas the transglycosylation reaction was prevented by the NaCl. Therefore, no addition of NaCl to the reaction medium was extremely effective for activating the transglycosylation activity of the enzyme.

Third, with regard to specificity for the substrate, it was shown that hyaluronic acid and chondroitin were hydrolyzed in preference to chondroitin 4- and 6-sulfate. In the transglycosylation reaction, the substrate specificities as acceptor and donor were the same as that for hydrolysis.

The dermatan sulfate employed was digested with hyaluronidase and was depolymerized as a result. However, the dermatan sulfate had no activity as either a donor or an acceptor in the transglycosylation reaction with the enzyme. Since the iduronide linkage in the dermatan sulfate chain is not sensitive to the hyaluronidase, the depolymerization of dermatan sulfate by the enzyme occurs through glucuronide linkages in the inner part of the dermatan sulfate chain. Therefore, in order to transfer the disaccharide, that is glucuronosyl-beta1-3-N-acetylglucosamine to the glucuronic acid residue, it is necessary for at least two units of the disaccharide to be present successively in the carbohydrate chain. Therefore, it seems that a carbohydrate chain having such a sequence was not included in the dermatan sulfate employed.

On the other hand, the DS hexasaccharide we used had no activity as an acceptor, even though a glucuronic acid residue was partially present at the nonreducing terminal. It seems that the acceptor activity of DS hexasaccharide depends not only on the glucuronic acid residue at the noneducing terminal but also the penultimate N-acetylhexosamine, to which the sulfate ester bound varies in number and linkage position. In order to define the substrate specificity as an acceptor, it will be necessary to examine closely the fine chemical structure of DS hexasaccharide as an acceptor.

In order to transfer the DS oligosaccharide containing the iduronic acid residue, it will be necessary to use the real dermatan sulfate-degrading enzyme, although unfortunately this enzyme is not yet available. It has been reported that some urinary glycosaminoglycans have iduronic acid at the reducing terminal(29) . This suggests that endotype iduronidase acting on the intrachain iduronide linkage of dermatan sulfate is present in human tissue. Therefore, the isolation of a dermatan sulfate-degrading enzyme is expected in the future.

The high molecular weight GAGs used as a donor were more effective than those of low molecular weight. Because the internal chains of high molecular weight GAGs were hydrolyzed and then depolymerized, the GAGs of low molecular weight as a donor increased rapidly in the reaction medium. Therefore, even if the donor concentration of high molecular weight GAG was increased, the yield of oligosaccharide reaction products did not increase in proportion.

These results suggested it would be possible to elongate GAG oligosaccharide chains by inhibiting the hydrolysis reaction under the conditions optimal for the transglycosylation reaction. For example, to obtain longer oligosaccharide chains, the incubation was carried out at high pH. Furthermore, it was possible to reconstruct hybrid chains by changing the combinations of GAG chains used as acceptors and donors. The GAG chains reconstructed using these various combinations contained artificial chains that do not occur naturally.

Recently, genetic engineering has made great progress. However, it is known that some recombinant glycoproteins have certain problems with their biological activity because of incompleteness of their sugar chains. Therefore, it would appear to be important to glycosylate these proteins artificially. Furthermore, it has been clarified that endotype glycosidases acting on the sugar chains of glycoproteins catalyze the transglycosylation reaction(12) . There are a few enzymes such as endoglycosidases and glycosyltransferases used in glycotechnology, which correspond to the ligases and restriction enzymes used in genetic engineering. However, there are no available enzymes capable of transferring only the oligosaccharide units of GAGs, except for testicular hyaluronidase. Recently, three endotype glycosidases acting on proteoglycan have been found(30, 31, 32) . These enzymes cleaved specifically the internal linkage between the core protein and GAG chain of a proteoglycan. As a result, intact GAG chains can be obtained using these enzymes. Therefore, testicular hyaluronidase used together with these enzymes would be an excellent tool for reconstructing various GAG chains possessing biological activities.


FOOTNOTES

*
This work was supported by Grants-in-Aid for Scientific Research 0330450, 04454153, 05274107, and 05680518 from the Ministry of Education, Science, and Culture of Japan and from the Mizutani Foundation for Glycoscience. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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, Japan. Fax: 8000-0172-32-3496.

(^1)
The abbreviations used are: HA, hyaluronic acid; GAG, glycosaminoglycan; Ch4S, chondroitin 4-sulfate; Ch6S, chondroitin 6-sulfate; DS, dermatan sulfate; DeltaDi4S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enopyranosyluronic acid)-4-O-sulfo-D-galactose; PA, 2-aminopyridine; HPLC, high performance liquid chromatography; NFU, National formulary unit.


ACKNOWLEDGEMENTS

We thank Dr. I. Kato and K. Kojima (Research Institute for Glycotechnology, Hirosaki, Japan) for help in obtaining ion spray mass spectra.


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