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(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
Bovine testicular hyaluronidase is one of the endotype
glycosidases and hydrolyzes the internal bonds of both hyaluronic acid
(HA) ( 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- 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
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
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:
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:
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:
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
Figure 8:
HPLC of transglycosylation reaction
products digested by
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- 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- 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.
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3741-3747
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 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.
)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) .
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.
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 -glucuronidase
or -N-acetylhexosaminidase activities, as measured by the
method of Barrett(14) . Bovine liver -glucuronidase was
purchased from Sigma. Bovine kidney
-N-acetylglucosaminidase, Streptococcus hyaluronidase, Streptomyces hyaluronidase, chondroitinase
AC-II, chondroitinase B, and
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enopyranosyluronic
acid)-4-O-sulfo-D-galactose (
Di4S) were obtained
from Seikagaku Kogyo Co. (Tokyo, Japan).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.-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
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) , -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) .
Effect of pH on the Transglycosylation
Reaction
To estimate the optimal pH for the transglycosylation
reaction, hyaluronic acid (M = 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) .
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.
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) .
, 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.
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.
, 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.
, 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.
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.
-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).
-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).
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.
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.
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.
)Di4S,
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enopyranosyluronic
acid)-4-O-sulfo-D-galactose; PA, 2-aminopyridine;
HPLC, high performance liquid chromatography; NFU, National formulary
unit.
We thank Dr. I. Kato and K. Kojima (Research Institute
for Glycotechnology, Hirosaki, Japan) for help in obtaining ion spray
mass spectra.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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