(Received for publication, October 11, 1994; and in revised form, December 7, 1994)
From the
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 = 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.
Bovine testicular hyaluronidase is one of the endotype
glycosidases and hydrolyzes the internal bonds of both hyaluronic acid
(HA) ()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-1-3-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
1-4
linkage. When heptasaccharides or larger oligosaccharides having N-acetylhexosamine at the nonreducing terminal were used as a
donor, a trisaccharide, N-acetylglucosaminyl-
1-4
glucuronosyl-
1-3-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.
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.
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
-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.
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;
4.0 250 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.
,
octa-;
, deca-;
, hexadecasaccharide in sodium acetate
buffer.
, octa-;
, deca-;
, hexadecasaccharide in
Tris-HCl buffer.
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: , octa-;
, deca-;
,
hexadecasaccharide.
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.
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: , PA-octa-;
, PA-deca-;
,
PA-hexadecasaccharide.
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: , PA-octa-;
, PA-deca-;
,
PA-hexadecasaccharide.
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.
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
-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 (
Di4S) (Fig. 8c).
Figure 8:
HPLC of transglycosylation reaction
products digested by -glucuronidase and chondroitinase AC-II.
Reaction products (fraction I in Fig. 7b) were
recovered and purified (a). Then aliquots were digested with
-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
(
di4S).
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.
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.
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-1-3-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-1-3-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.