(Received for publication, March 29, 1995; and in revised form, May 25, 1995 )
From the
The transglycosylation activity of
endo- Carbohydrates were found to possess important biological
functions, such as cell-cell
recognition(1, 2, 3) , lectin
binding(4, 5) , and viral
infection(6, 7) . Since the studies of the
carbohydrate functions require structurally well-defined and highly
pure compounds which are usually difficult to obtain from the natural
sources, syntheses and constructions of neoglycoconjugates have rapidly
gained attention during the past decade(8) . Chemical syntheses
of neoglycoconjugates have been aggressively developed, but they
usually involve multiple, laborious steps. Especially, synthesis of the
high mannose-type oligosaccharides proved to be quite difficult, even
with enzymatic methods. Endo- We have taken advantage of this finding, and synthesized several
functional intermediates for neoglycoconjugates, one of which was
converted into a glycopolymer with pendant Man
Figure 1:
Optimization of endo-A
transglycosylation conditions. The optimum levels of the enzyme (A) and the acetone content (B) for
transglycosylation were determined by the reactions carried out in a
mixture of 11.6 nmol of Man
Figure 2:
Synthesis of
Man
Figure 3:
Figure 4:
Gel filtration of the glycopolymer on
Sephadex G-50. The sample (1 ml) was applied onto a Sephadex G-50
column (2.5
Figure 5:
Figure Z1:
Glycopolymer havine
Man
Figure 6:
Determination of molecular weight of the
glycopolymer by HPGFC. HPGFC was performed with a size exclusion column
(7.5
Figure 7:
Inhibition of binding by serum- and
liver-MBP-CRDs by the glycopolymer. The fitted curves were obtained
using the program ALLFIT(16) . Concentrations of SBA and
glycopolymer are expressed on the bases of
Man
We have shown (38) that endo-A possesses an efficient
transglycosylation activity (>90%) in 30% acetone, much higher than
those reported for 10-30% by other
glycosidases(18, 19, 20, 21, 22, 23, 24) .
This finding is now utilized to synthesize neoglycoconjugate
intermediates which are amenable to further reactions. We found that
endo-A transfers Man Several GlcNAc
derivatives having functionalized aglycon useful for synthesis of
neoglycoconjugates were tested as acceptor for endo-A
transglycosylation. The yields based on the starting donor substrate
were found to be greater than 80% with 0.2 M acceptor and
about 50% when 0.05 M or less was used in our system. The
yield of transglycosylation can be further improved if higher acceptor
concentrations are employed, as shown in the accompanying
article(38) . We have also demonstrated a successful endo-A
transglycosylation with 5.8 µmol of donor rather than 3 nmol as
shown in Table 2. In the larger scale transglycosylation to
GlcNAc-NAP, transglycosylation yield (>90%) was even higher than
those at the analytical scale reaction. In the accompanying
article(38) , an 89% yield was obtained from the
transglycosylation to GlcNAc An endo-A transglycosylation product,
Man Clustering of monosaccharides by attaching them to simple branched
peptides enhances inhibitory potencies for some C-type
lectins(34, 35) . Formation of glycopolymers is a
convenient way to provide glycoside clustering(36) . In the
present work, a dramatic increase in the inhibition of MBP-CRDs in
comparison with that by the native glycoprotein (SBA) which contains
the same Man
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-N-acetylglucosaminidase from Arthrobacter
protophormiae (endo-A) can be enhanced dramatically by inclusion
of organic solvent in the reaction mixture (see accompanying article;
Fan, J.-Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I.,
Abeygunawardana, C., and Lee, Y. C.(1995) J. Biol. Chem. 270,
17723-17729). This finding was extended to synthesis of important
intermediates for preparation of neoglycoconjugates. When 0.2 M
GlcNAc-O-(CH
)
NH
,
GlcNAc-O-CH
CH=CH
,
GlcNAc-O-(CH
)
CH=CH
,
GlcNAc-O-(CH
)
NHCOCH=CH
,
GlcNAc-S-CH
CN,
GlcNAc-S-(CH2)
CH
, or
GlcNAc-S-CH
CONHCH
CH(OMe)
were used as acceptors in 30% acetone-containing media, the
transglycosylation was accomplished with about 80% yield. The
transglycosylation yields to benzyl
-GlcNAc (67%),
4-methylumbelliferyl
-GlcNAc (66%), p-nitrophenyl
-GlcNAc (33%), and
(GlcNAc-
-S-CH
CH
CH
)
(43%) were lower, because their poor solubilities allowed only
0.05 M or lower concentrations in the reaction mixture. A
micromole-scale synthesis of
Man
GlcNAc
-O-(CH
)
NHCOCH=CH
(Man
GlcNAc
-NAP) was accomplished with 90%
yield, and the structure of the transglycosylation product was
confirmed by
H NMR. Man
GlcNAc
-NAP
was co-polymerized with acrylamide. The ratio of sugar side chain to
acrylamide in this glycopolymer was 1:44 and the molecular weight of
glycopolymer was estimated to be between 1,500,000 and 2,000,000 by
high performance gel filtration chromatography. The glycopolymer was
shown to be a much more efficient inhibitor of binding by recombinant
rat mannose binding protein-carbohydrate recognition domains (MBP-CRD)
from serum (I
= 3.5 µM
Man
GlcNAc
-sugar chain) and liver (I
= 74.5 nM) than soybean agglutinin.
-N-acetylglucosaminidase
from Arthrobacter protophormiae (endo-A)
(
)cleaves the glycosidic bond in the core
GlcNAc
1,4GlcNAc
(
)residues of high
mannose-type and hybrid type N-linked sugar chains in
glycoprotein(9) . The enzyme was also previously reported to
have transglycosylation activity in the presence of GlcNAc and other
monosaccharides(10, 11) . In the accompanying
article(38) , we found that the transglycosylation activity of
endo-A could be enhanced by inclusion of organic solvents, such as
acetone, dimethyl sulfoxide, and N,N-dimethyl formamide in the
reaction medium. For instance, the transglycosylation could be
performed to near completion in 30% acetone-containing medium using
Man
GlcNAc
Asn as donor and GlcNAc as acceptor.
GlcNAc
chains. The glycopolymer thus prepared displays a drastically
greater inhibition of binding by mannose-binding protein from liver
over the monomer oligosaccharide.
Materials
Endo-A was purified by the reported method(9) .
ManGlcNAc
Asn was prepared from soybean
agglutinin by exhaustive Pronase digestion, followed by gel filtration
on Sephadex G-50 and further HPLC purification using a graphitized
carbon column(12) . Glycoamidase A was from Seikagaku America,
Inc. (Rockville, MD). GlcNAc was purchased from Pfanstiehl
Laboratories, Inc. (Waukegan, IL). 3-(N-Acryloylamino)-propyl
-D-GlcNAc (GlcNAc-NAP) and
GlcNAc-O-(CH
)
CH=CH
were gifts from Dr. Shin-Ichiro Nishimura of Hokkaido University,
Japan. Benzyl
-GlcNAc, 4-methylumbelliferyl
-GlcNAc, p-nitrophenyl
-GlcNAc,
GlcNAc-O-(CH
)
NH
,
GlcNAc-O-CH
CH=CH
,
GlcNAc-O-(CH
)
NHCOCH=CH
,
GlcNAc-S-CH
CN,
GlcNAc-S-(CH
)
CH
,
(GlcNAc-S-CH
CH
CH
)
and
GlcNAc-S-CH
CONHCH
CH(OMe)
were synthesized in this laboratory by previously published
methods (13) . Recombinant rat MBP-CRDs from serum and liver
were expressed and purified as described (14) using expression
plasmid-bearing bacterial strains which are gifts fromDr. Kurt
Drickamer of Columbia University.
Methods
Enzymatic Reaction
A typical enzyme reaction for
transglycosylation was performed in a mixture of 3 nmol of
ManGlcNAc
Asn, 4 µmol of acceptor, and 0.9
milliunits of endo-A in a total volume of 20 µl with 25 mM ammonium acetate buffer (pH 6.0) containing 30% acetone. After
incubation at 37 °C for 15 min, the reaction was terminated by
boiling for 3 min in a water bath. The buffer was removed with a
Speedvac using a vacuum pump. The reaction mixture was analyzed using
an HPAEC-PAD system (see below).
High Performance Anion Exchange Chromatography
(HPAEC)
An HPAEC system which consisted of a Bio-LC (Dionex
Corp., Sunnyvale, CA) equipped with a pulsed amperometric detector
(PAD-II) was used for analysis of the reaction products. The
chromatographic data were analyzed using the AI-450 chromatography
software (Dionex). The endo-A reaction products were separated using a
Dionex CarboPac PA-1 column (4 250 mm) eluted at a flow rate of
1.0 ml/min with 100 mM sodium hydroxide and a gradient of
sodium acetate from 30 to 80 mM developed in 30 min. A cycle
of 5-min washing with 100 mM sodium hydroxide, 200 mM sodium acetate followed with a 15-min equilibration period was
inserted between runs. The PAD sensitivity was set at 1 K. The
quantitative determination of Man
GlcNAc and
Man
GlcNAc
was carried out by comparison with
standard materials obtained by complete digestion of
Man
GlcNAc
Asn by endo-A and
Man
GlcNAc
AsnPhe by glycoamidase A. The
transglycosylation products using acceptors other than GlcNAc were
estimated by subtracting the remaining substrate and the hydrolysis
product from the starting substrate.
Transglycosylation by Endo-A Using GlcNAc-NAP as
Acceptor
A mixture consisting of 5.8 µmol of
ManGlcNAc
Asn, 2 mmol of GlcNAc-NAP, and 1.1
unit of enzyme in 5 ml of 10 mM NH
OAc buffer (pH
6.0) containing 35% of acetone was incubated at 37 °C for 15 min.
After stopping the reaction by a 3-min boiling period, the sample was
applied to a Sephadex G-25 column (2
140 cm) and eluted with
0.1 M acetic acid. The effluent was monitored by uv absorption
at 229 nm, and the neutral sugar was determined by the phenol sulfuric
acid method(15) . The fractions containing high molecular
weight materials were combined and lyophilized to yield 10.5 mg of
white powder.
Preparation of Glycopolymer Having Pendant Chains of High
Mannose-type Oligosaccharide
The white powder obtained from gel
filtration was used as starting material for polymerization without
further purification. A small amount of the white powder (7.2 mg, about
3.25 µmol of ManGlcNAc
-NAP) was dissolved
in 0.3 ml of H
O, followed by deaeration with a water
aspirator for 30 min. To the mixture, acrylamide (8.4 mg, 118
µmol), ammonium persulfate (0.14 µmol), and N,N,N`,N`-tetramethylethylenediamine (TEMED, 6.6 µmol)
were added, and the mixture was stirred at room temperature for 3 days,
during which time, the same amounts of ammonium persulfate and TEMED
were added to the reaction mixture daily for 2 days, and the reaction
was finally completed by incubation of the mixture at 55 °C for 3
h. The reaction mixture was applied to a column (2.5
90 cm) of
Sephadex G-50 and eluted with H
O. The fractions containing
the glycopolymer were combined and lyophilized to obtain 5.3 mg of
white powder.
Estimation of Molecular Weight of the Glycopolymer by
HPGFC
The HPGFC was performed with a Gilson HPLC system equipped
with a size exclusion column (TSK-Gel G2000SW, 7.5 600 mm,
TosoHaas, NJ) and an uv detector (Model V
, ISCO). The
eluent was 0.1 M phosphate buffer (pH 7.0) containing 0.3 M NaCl and the effluent was monitored at 220 nm. The standard
compounds for molecular weight estimation were: (i) blue dextran (M
= 2,000,000); (ii)
-amylase (M
= 200,000); (iii) alcohol dehydrogenase (M
= 150,000); (iv) bovine serum albumin (M
= 66,000), and (v) carbonic anhydrase (M
= 29,000).
MBP Binding of the Glycopolymer
The solid-phase
binding studies were carried out essentially as described
previously(14) , with some minor modifications as follows. All
steps were carried out at 4 °C. Briefly, CRD was coated onto
individual polystyrene wells (Immulon 4 Removawell Strips by Dynatech,
from Fisher Scientific) which were then blocked with 1% bovine serum
albumin in 1.25 M NaCl, 25 mM CaCl, 25
mM Tris (pH 7.8). Ligands and inhibitors were in 0.5% bovine
serum albumin in the above Tris buffer for binding and inhibition. The
reference ligand used was
I-mannose
-bovine
serum albumin (about 2000 cpm/mg). Approximately 500 cpm/well of
reference ligand were incubated for 20 h with or without inhibitors at
various concentrations. The well contents were then removed, washed,
and counted in a Packard Minaxi
-counter. Counts were corrected
for background (counts remaining in a blocked well which was not coated
with CRD), and the data were analyzed using the program ALLFIT (16) to determine I
values using a logistic
equation for curve fitting.
300 MHz NMR spectra were
recorded on a Bruker AMX 300 spectrometer and measurement of a 600 MHz
NMR was performed on a Bruker AM-600 spectrometer. The chemical shifts
were based on acetone (H Nuclear Magnetic Resonance Spectroscopy (
HNMR)
= 2.225 ppm) as an internal
standard. The samples were prepared by three cycles of dissolving in
D
O and lyophilizing followed by dissolving the residue in
0.5 ml of high purity D
O (99.96% D) immediately before
measurement. The 300 MHz data were recorded at 25 °C and the 600
MHz data, at 60 °C.
Transglycosylation of Endo-A to Water-miscible
Alcohols
The transglycosylation by endo-A using
ManGlcNAc
Asn as donor to various water-miscible
alcohols was tested. As listed in Table 1, the enzyme transferred
oligosaccharide to MeOH and EtOH with 64 and 47% yield, respectively,
although the hydrolyses were 33 and 46% in these media. The identity of
the transglycosylation products obtained from 30% MeOH or EtOH were
confirmed by mass spectrometry and the anomeric configuration of the
product with MeOH was found to be
by
H NMR (data not
shown). PrOH (8% yield) and iso-PrOH (10% yield) could also serve as
acceptors of transglycosylation, but allyl alcohol could not function
as acceptor. The enzyme seemed to be stable in 30% MeOH and EtOH, but
unstable in 30% PrOH and allyl alcohol, because the total enzyme
activities (combined hydrolysis and transglycosylation activities) in
MeOH and EtOH were shown to be similar to that in H
O, but
much lower in the higher alcohols. Glycerol was found to be as good an
acceptor as MeOH or EtOH, and the transglycosylation yield was as high
as 57%.
Transglycosylation of Endo-A to Various GlcNAc
Glycosides
The transglycosylation of endo-A to some
functionalized GlcNAc glycosides was efficient (Table 2). When
acceptor concentration was 0.2 M, endo-A transferred
ManGlcNAc to
GlcNAc-O-(CH
)
NH
(93% of
the converted substrate),
GlcNAc-O-CH
CH=CH
(99%),
GlcNAc-O-(CH
)
CH=CH
(90%), and
GlcNAc-O-(CH
)
NHCOCH=CH
(78%) with yields of 81, 81, 84, and 70% of the starting
substrate, respectively. Because of the low solubility, the
concentration of benzyl
-GlcNAc was used at 0.05 M, and
4-methylumbelliferyl
-GlcNAc and p-nitrophenyl
-GlcNAc was used under saturating conditions (below 0.05 M). Even at these concentrations, the enzyme could transfer
67, 66, and 33%, respectively, of the starting oligosaccharide chain to
them and the transglycosylation indices (the percentage of a
transglycosylation product to a digested substrate) were found to be
82, 77, and 42%, respectively. The thioglycosides of GlcNAc are good
acceptors for endo-A transglycosylation. When
GlcNAc-S-CH
CN,
GlcNAc-S-(CH
)
CH
, and
GlcNAc-S-CH
CONHCH
CH(OMe)
were used as acceptors at 0.2 M, the transglycosylation
indices were 88, 86, and 95%, with the yields of 83, 78, and 81%,
respectively. A divalent thioglycoside of GlcNAc,
(GlcNAc-S-CH
CH
CH
)
,
could be also used as acceptor for endo-A transglycosylation at low
concentration (below 0.05 M) with 50% transglycosylation index
and 43% yield.
Optimization of the Reaction Conditions for a Larger
Scale Transglycosylation by Endo-A
The optimum levels of the
enzyme and acetone were examined for the transglycosylation with the
substrate at micromole levels. As shown in Fig. 1A, the
hydrolytic product increased in proportion to the amount of the enzyme.
Yield of the transglycosylation product increased upon addition of the
enzyme up to 2.2 milliunits, then decreased as more enzyme was added.
When 2.2 milliunits of the enzyme were used, only 5.6% substrate
remained. On the other hand, the transglycosylation product increased
and the hydrolytic product decreased as the acetone content increased
up to 35% (Fig. 1B). In 35% acetone, 86%
transglycosylation, and 7% hydrolysis were observed by HPAEC analysis.
Although no hydrolytic product was found in the 40% acetone medium, the
efficiency of the reaction was lower compared with those in other
media, because a greater amount of the substrate (64% of the starting
substrate) remained.
GlcNAc
Asn (donor), 4
µmol of GlcNAc-NAP (acceptor), and various amounts of the enzyme (A) or 2.2 milliunits of enzyme (B) in 10 µl of
10 mM ammonium acetate buffer (pH 6.0) containing 30% acetone (A) or different concentrations of acetone (B). The
reaction mixtures were incubated at 37 °C for 15 min and the
products were analyzed with HPAEC-PAD.
, substrate;
,
transglycosylation product;
, hydrolytic
product.
Synthesis of Man
To prepare
ManGlcNAc
-NAP by
Transglycosylation Activity of Endo-A
GlcNAc
-NAP in a quantity useful for
polymerization, the reaction scale was raised 500-fold over that in the
optimum conditions described above. The transglycosylation product,
Man
GlcNAc
-NAP, was more than 90% by HPAEC (Fig. 2), and the hydrolysis product as well as the starting
donor substrate was barely detected. The unreacted acceptor was
recovered by gel filtration on a Sephadex G-25 column and the
Man
GlcNAc
-NAP was analyzed by
H NMR
analysis and used for polymerization without further purification.
GlcNAc
-NAP by endo-A transglycosylation. The
reaction was with 5.75 µmol of
Man
GlcNAc
Asn, 2 mmol of GlcNAc-NAP, and 1.1 unit of the enzyme in 5 ml of 10 mM ammonium acetate
buffer (pH 6.0) containing 35% acetone at 37 °C for 15 min. After
lyophilization, a sample equivalent to 0.7 nmol of
Man
GlcNAc
oligosaccharide was injected into the
HPAEC-PAD system for analysis. The elution was performed with 100
mM NaOH and a linear gradient of NaOAc: 0-10% in 20 min. A, transglycosylation product,
Man
GlcNAc
-NAP; B, hydrolytic product,
Man
GlcNAc; C, remaining substrate,
Man
GlcNAc
Asn.
H NMR was used to identify the transglycosylation
product. As shown in Fig. 3A, the signals of the
acceptor were completely assigned by the decoupling technique. The H-4
signal of GlcNAc was found at 3.436 ppm and the anomeric proton signal
was around 4.495 ppm. On the other hand, the
H NMR analysis
of the transglycosylation product showed 10 new anomeric proton
signals, suggesting that the high mannose-type sugar chain was
transferred to the acceptor. The
H NMR assignments based on
the reference values (17) are listed in Table 3. The
anomeric signals agreed with those found from
Man
GlcNAc
Asn, except two GlcNAc anomeric
protons which appeared at higher field than those from the reference
compound. This is because the linkage between GlcNAc and the aglycon in
the former is a N-amide bond, and in the latter, an O-glycosidic bond. The coupling constant of GlcNAc-2 anomeric
proton was 7.8 Hz, indicating that the linkage newly formed by endo-A
transglycosylation is in the
-configuration. The H-4 signal of
GlcNAc at the ``reducing end'' at 3.436 ppm could no longer
be seen, in agreement with the results with methyl
-GlcNAc (38) that the linkage occurs at the 4-OH of the GlcNAc. The
mass spectrometry analysis shows the expected molecular weight of the
transglycosylation product (data not shown).
H NMR (300 MHz) analysis of
GlcNAc-NAP (A) and Man
GlcNAc
-NAP (B). The labile hydrogens in sample were exchanged with
deuterium by repeating a cycle of dissolving in D
O followed
by lyophilization three times before measurement. The analyses were
done in D
O using acetone (2.225 ppm) as internal standard
at 25 °C.
Polymerization of
Man
A
glycopolymer was obtained from ManGlcNAc
-NAP with Acrylamide
GlcNAc
-NAP
and acrylamide using TEMED and ammonium persulfate as catalysts. The
fractions containing the polymer eluted at the void volume of the
Sephadex G-50 column (Fig. 4) were pooled and lyophilized.
Completion of the polymerization was indicated by
H NMR
analysis (Fig. 5) which revealed disappearance of the signals at
6.2 and 5.7 ppm, attributable to the double-bond associated protons in
the aglycon and the acrylamide monomer. The NMR also showed the
existence of 11 anomeric proton signals, and the chemical shifts were
similar to those found from the monomer (Table 3), confirming
that the polymer contains Man
GlcNAc
-sugar
chains. The sugar content of the polymer was estimated to be 37% by the
phenol-H
SO
method using mannose as standard.
Therefore, the ratio of sugar side chains to acrylamide residues is
estimated to be 1:44 as shown in Fig. Z1.
90 cm), and eluted with water. The flow rate was
approximately 30 ml/h, and 4-ml fractions were collected. The neutral
sugar was determined by the phenol-H
SO
method (dotted line, absorbance at 480 nm), and GlcNAc was monitored
by the absorbance at 220 nm (solid line). a,
indicates the fractions combined as the
glycopolymer.
H NMR (600 MHz) analysis of
the glycopolymer. The chemical shifts measured in D
O at 60
°C were based on the HDO signal at 4.441 ppm. *, denotes the
signals from the polymer back bone.
GlcNAc
sugar
chain.
Determination of the Molecular Weight of the
Glycopolymer
The molecular weight of the glycopolymer was
estimated by HPGFC using blue dextran, -amylase, alcohol
dehydrogenase, bovine serum albumin, and carbonic anhydrase as
reference compounds. The polymer appeared near the void volume, and the
retention volume was slightly greater than blue dextran (molecular
weight = 2,000,000). According to the calibration curve (Fig. 6), the molecular weight is between 1,500,000 and
2,000,000.
600 mm) and 0.1 M phosphate buffer (pH 7.0)
containing 0.3 M NaCl as an eluent at a flow rate of 1.0
ml/min. Effluent was monitored by absorbance at 220 nm.
,
glycopolymer;
, reference compounds; 1, blue dextran
(2,000,000); 2,
-amylase (200,000); 3, alcohol
dehydrogenase (150,000); 4, albumin bovine serum (66,000); and 5, carbonic anhydrase (29,000).
Inhibition Study of Mannose-binding Proteins by the
Glycopolymer
A solid-phase binding assay was carried out on
serum- and liver-MBP-CRDs, using the
ManGlcNAc
-glycopolymer and soybean agglutinin
(SBA), which contains the same Man
GlcNAc
. The
results of the assay are shown in Fig. 7. In the concentration
range of SBA tested, no significant inhibition of the serum-MBP-CRD was
observed. For the liver-MBP-CRD, however, I
values of 13.2
µM based on Man
GlcNAc
or 0.4 mg/ml
SBA were obtained. However, the glycopolymer showed an I
of 3.5 µM for the serum-MBP-CRD, and an I
of 74.5 nM for the liver-MBP-CRD. In terms of the whole
glycopolymer, the I
values would be about 2.0
10
mg/ml for the serum-MBP-CRD and 3.8
10
mg/ml for the liver-MBP-CRD, respectively. The
magnitude of inhibitory potency enhancement of the glycopolymer over
the precursor cannot be calculated with certainty for the serum form of
MBP-CRD, because Man
GlcNAc
hardly inhibits this
MBP-CRD. However, for the liver form, an enhancement was about 180-fold
based on the Man
GlcNAc
, and about 1,000-fold
based on the polymer molarity, although the sugar content of the
glycopolymer was only 5.6-fold higher than SBA.
GlcNAc
.
, SBA + serum MBP-CRD;
, SBA + liver MBP-CRD;
, glycopolymer + serum
MBP-CRD;
, glycopolymer + liver
MBP-CRD.
GlcNAc to alcohols such as MeOH, EtOH,
and PrOH also. The transglycosylation to MeOH (64% yield) and EtOH (47%
yield) compares favorably with those by
-xylosidase,
- and
-glucosidase, and
-galactosidase (20-60%) from various
sources(25, 26) . However, transglycosylation to PrOH
and iPrOH were not as effective as to MeOH and EtOH. Interestingly,
although the total enzyme activity was lower in PrOH than in iPrOH,
transglycosylation to PrOH was greater than to iPrOH. Glycerol was also
a good acceptor for endo-A transglycosylation.
Endo-
-N-acetylglucosaminidase F (27) and
endo-
-N-acetylgalactosaminidase from Diplococcus
pneumoniae(18) have been reported to transfer an
oligosaccharide to the C1(3) hydroxyl of glycerol.
-OMe at the similar scale (4
µmol).
GlcNAc
-NAP, was further polymerized with
acrylamide to form a glycopolymer. Glycopolymers having di- or
trisaccharide have been synthesized by chemical or chemo-enzymatic
methods
recently(28, 29, 30, 31, 32, 33) ,
but we are not aware of any synthesis of glycopolymers with highly
complex sugar chains. The high efficiency of endo-A transglycosylation
provides an easier way to synthesize such neoglycoconjugates.
GlcNAc
oligosaccharide was
demonstrated. In the case of the liver MBP-CRD, an approximately
180-fold enhancement of inhibitory potency over the native glycoprotein
(SBA) was attained by the glycopolymer. Similarly, although no
significant inhibition of the serum MBP-CRD was observed for SBA, the
glycopolymer derived from its oligosaccharide demonstrated surprisingly
strong inhibitory potency (I
= 3.5
µM). This is a good example of ``macro-'' versus ``micro-clustering''(37) .
-N-acetyl-D-glucosaminidase from Arthrobacter protophormiae; GlcNAc, N-acetyl-D-glucosamine; Man, mannose; NAP,
3-(N-acryloylamino)-propyl; MBP, mannose-binding protein; CRD,
carbohydrate recognition domain; SBA, soybean agglutinin; HPAEC-PAD,
high performance anion exchange chromatography with pulsed amperometric
detector; HPLC, high performance liquid chromatography; HPGFC, high
performance gel filtration chromatography;
H NMR,
H nuclear magnetic resonance spectroscopy; TEMED, N,N,N`,N`-tetramethylethylenediamine.
We thank Dr. Shin-Ichiro Nishimura of Hohhaido
University, Japan, for providing GlcNAc-NAP and
GlcNAc-O-(CH)
CH=CH
as well as valuable discussions and suggestions. We also thank
Dr. Reiko T. Lee for soybean agglutinin and Dr. Kurt Drickamer of
Columbia University for the gift of bacterial strains to produce
MBP-CRDs. We are grateful to Koji Matsuoka and Dr. C. Abeygunawardana
of The Johns Hopkins School of Medicine for
H NMR analyses.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.