(Received for publication, March 29, 1995; and in revised form, May 25, 1995)
From the
The transglycosylation activity of
endo- Although dramatic progress in molecular biology has enabled
relatively painless preparation of proteins, synthesis of glycoproteins
of defined structure is still a formidable challenge, especially with
respect to the synthesis of carbohydrate parts and condensation of the
carbohydrates to protein(1) . In recent years, synthetic
glycoconjugates (neoglycoconjugates) have become even more valuable in
basic biomedical research and medical applications(2) . To
synthesize branched oligosaccharides as found in natural
glycoconjugates by conventional chemical methods requires multiple
intricate steps of protection and deprotection. Enzymatic methods,
however, are often more advantageous, because of their high stereo- and
regio-selectivities (3) . Glycosyltransferases are most often
used for assembly of oligosaccharide chains, but construction of
complex oligosaccharide chains requires ready availability of a large
number of specific enzymes. Exo-glycosidases, although known to
perform reverse reaction (4) or
transglycosylation(5, 6, 7) , are hardly
suitable for building complex oligosaccharides because of their
relative inefficiency. Several endo-type glycosidases (8, 9, 10, 11) have been found to
perform either transglycosylation or reverse reaction. For instance,
endo- Endo- We found that the hydrolytic
activity of endo-A could be suppressed and the transglycosylation
activity can be enhanced by carrying out the reaction in media
containing organic solvent. In this paper, we report the enhancement of
transglycosylation activity of endo-A to near complete suppression of
hydrolysis in the media containing organic solvents. We also examined
the acceptor specificity of the reaction in such media.
In
order to prepare the samples for mass spectrometry, 400 nmol of
Man
Data were
recorded at 20 °C without sample spinning. All two-dimensional NMR
data sets were acquired in the phase-sensitive mode using
time-proportional phase increment (29) and 2048 data points in
acquired dimension (t All data sets were processed using FELIX 1.1 (Biosym
Inc. San Diego, CA). The data sets were apodized with a
20-60° shifted sine bell function and zero filled in both
dimensions to obtain final data matrices consisting of 2 K
Figure 1:
Time course of
endo-A action in aqueous solution. The enzymatic reactions were
performed in a mixture of 15 nmol of
Man
The
transglycosylation activity of endo-A in media containing acetone,
Me
Figure 2:
Effect of organic solvents on the
transglycosylation activity of endo-A. The reaction mixtures contained
3 nmol of Man
In
Me In DMF-containing media, the
remaining substrate increased quickly upon increasing the DMF content,
indicating the relative instability of this enzyme in DMF. This is also
reflected in the lower hydrolytic activity in 30% DMF (Table 1).
Although 30% DMF could effectively suppress hydrolytic activity, it
also suppressed transglycosylation activity to 12%. The maximum
transglycosylation activity was at 10% DMF solution, where the ratio of
transglycosylation to hydrolysis was only 3.4. The maximum ratio of
transglycosylation to hydrolysis was similar to those found in
Me
Figure 3:
Stability of endo-A in media containing
organic solvents. A mixture containing 1 milliunit of the enzyme and an
appropriate amount of acetone (A), Me
Figure 4:
Effect of GlcNAc concentration on
transglycosylation activity of endo-A. The reactions were carried out
with 3 nmol of Man
The
molecular weight of the transglycosylation product was determined by
electrospray mass spectrometry analysis as 1249 (M + H)
Figure 5:
Ion-spray mass spectrum of
transglycosylation product of endo-A. The sample used was about 50
pmol, and the infusion rate was 3 µl/min in 1:1 CH
Figure 6:
In
order to study linkage mode between the two GlcNAc residues, the
Among the
monosaccharides and their derivatives tested, endo-A could transfer
oligosaccharide to GlcNAc (97%), Glc (89%), Man (87%), 2-deoxy-Glc
(91%), and methyl The products of transglycosylation to
xylose and 3-O-Me-Glc were confirmed by mass spectrometry. The
parent mass ions of the transglycosylation product to xylose were
1812.6 (M + H)
Figure 7:
Ion-spray mass spectrum of
Man
Glycosidases can be used synthetically in two modes, reverse
hydrolysis (glycoside formation) and transglycosylation.
Transglycosylation is generally the method of choice, because the
reverse hydrolases heavily favor hydrolysis and require a high
concentration of starting substrate(5) . Even with
transglycosylation, the yield of the desired product is usually low and
laborious separation of products from transglycosylation and hydrolysis
must be performed. With endo-A, the greater transglycosylation occurred
at the earlier stage of the reaction as expected (Fig. 1), but
the maximum ratio between transglycosylation and hydrolysis, determined
with HPAEC-PAD, was still 1.4:1, in agreement with the previous results
obtained with post-column derivatization of products followed by HPLC
analysis(24) . A putative mechanism of endo-A
transglycosylation is shown in Fig. S1. After forming the
enzyme-substrate complex, the enzyme could transfer the donor
oligosaccharide to either water (leading to hydrolysis) or acceptor
(leading to transglycosylation). Since the transglycosylation product
can be the substrate for the same enzyme, the process will repeat until
all substrates are transferred to water (complete hydrolysis).
Therefore, to favor transglycosylation is to enhance the relative
binding of acceptor to the enzyme-substrate complex to compete with
water, which can be attained by employing higher concentrations of the
acceptor. Recent studies (35, 36, 37) also
showed that modification of the active center of glycosidases by
site-directed mutagenesis could change transglycosylation activity of
the enzymes. Alternatively, as in our case, hydrophilic organic
solvents can be included in the enzyme reaction mixture to decrease
water content. This approach has been used for exoglycosidase
transglycosylation(38, 39, 40, 41) ,
and especially for peptide synthesis by transpeptidation of
proteases(42, 43) . In fact, we found that by
inclusion of 30% acetone in the reaction mixture, transglycosylation of
endo-A was remarkably enhanced and the hydrolysis was almost completely
suppressed (Table 2). Me
Figure S1:
Scheme 1A putative mechanism of endo-A
transglycosylation activity.
The
effectiveness of acetone in suppression of hydrolysis is not common. In
most cases, alcohols, acetonitrile, DMF, or Me The type of newly formed linkage has
been shown to be influenced by inclusion of organic solvents and
anomeric configuration of the acceptor
glycoside(44, 45, 46, 47) .
Therefore, we have carefully characterized one of the
transglycosylation products by mass spectrometry and The transglycosylation products of endo-A to
GlcNAc, Glc, Man, and 6-deoxy-Glc were analyzed chromatographically,
and the products to GlcNAc, xylose, 2-deoxy-Glc, and
3-O-Me-Glc were confirmed by mass spectrometry. Transfer of
oligosaccharide chains by endo-A to GlcNAc, Glc, and Man was also shown
by HPLC after post-column derivatization with 2-aminopyridine
previously(25) . From the data in Table 4, it is clear
that the enzyme requires the 3- and 4-OH of GlcNAc to be equatorial and
3-O-substituted GlcNAc can also serve as an acceptor. The 1-OH
and 6-OH of GlcNAc are not important for transglycosylation. Although
endo-A was found to transfer oligosaccharide to some primary alcohols
in 30% alcohol media (see (48) ), the nonspecific
transglycosylation onto primary alcohols may be explained by the
extremely high concentrations (about 7.4 M in case of MeOH)
used for the reaction. Thus we have drastically improved the
transglycosylation efficiency of endo-A, and defined the acceptor
specificity. These results have proved to be extremely valuable in the
use of endo-A to fabricate compounds useful for glycobiology (e.g. the accompanying article (48) ).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-N-acetylglucosaminidase from Arthrobacter
protophormiae (endo-A) was enhanced by inclusion of organic
solvents in the reaction mixture. In aqueous solution, the
transglycosylation yield relative to starting substrate was 32% using
Man
GlcNAc
Asn as donor and 0.5 M GlcNAc
as acceptor. However, in the media containing 30% (v/v) acetone,
dioxane, N,N-dimethylformamide, or dimethyl sulfoxide with 0.5 M GlcNAc as acceptor, the transglycosylation attained yields
of 89, 13, 28, and 75%, respectively, as analyzed by high performance
anion exchange chromatography. The enzyme was stable in media
containing up to 30% acetone, 30% dimethyl sulfoxide, or 20% N,N-dimethylformamide at 37 °C for at least 30 min. The
acceptor (GlcNAc) concentration must be greater than 0.2 M for
efficient transglycosylation. Electrospray mass spectrometry analysis
of the transglycosylation product obtained in 30% acetone with
Man
GlcNAc
Asn as donor and methyl
-2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed
a mass ion of m/z 1249.4, consistent with the
expected molecular weight. Analysis by
H NMR of the product
revealed that transglycosylation occurred at the C-4 of GlcNAc and the
linkage was of the
-configuration. In the acetone-containing
medium, Glc, Man, 2-deoxy-Glc, and methyl
-D-GlcNAc can
serve as a good acceptor as GlcNAc. Less favorable acceptors are
xylose, fructose, 6-deoxy-Glc, and
3-O-methyl-D-glucose. On the other hand, GalNAc, Gal,
allose, and 3-deoxy-Glc could not serve as acceptors of the enzyme
transglycosylation.
-N-acetylgalactosaminidase from Diplococcus
pneumoniae was shown to have both activities, and can transfer a
disaccharide, Gal
1
3GalNAc,
(
)to
various compounds including glycerol, p-nitrophenol,
threonine, and serine(9) . Ceramide glycanase from leech has
been used for synthesis of neoglycoconjugates(10) . However, in
aqueous solution, transglycosylation and glycoside formation by these
enzymes are normally of low yields also, necessitating laborious
separation of the reaction mixture.
-N-acetylglucosaminidase hydrolyzes the
glycosidic bond in the N,N`-diacetylchitobiose moiety
of N-linked sugar chains in glycoprotein. The enzymes of this
type are found in
microorganisms(12, 13, 14, 15, 16, 17) ,
plants(18, 19) , animal (20, 21, 22) , and human(23) . Among
them, the enzyme from Flavobacterium meningosepticum (endo-F)
(
)has been shown to transfer
oligosaccharide to glycerol(11) . Recently, Takegawa et al.(24, 25) reported that an
endo-
-N-acetylglucosaminidase from Arthrobacter
protophormiae (endo-A) has transglycosylation activity, although
the hydrolytic activity still predominates. The enzyme was also used
for synthesis of neoglycoprotein by the
transglycosylation(26) .
Materials
Endo-A was isolated by the published report(27) .
ManGlcNAc
Asn and
Man
GlcNAc
AsnPhe were prepared from soybean
agglutinin by exhaustive Pronase digestion, followed by gel filtration
on Sephadex G-50, and further purification with HPLC on a graphitized
carbon column(28) . Man
GlcNAc
Asn was a
gift from Dr. Ming-Chuan Shao, The University of Texas Medical School
at Houston. Glycoamidase A (glycopeptidase A) was from Seikagaku
America, Inc. (Rockville, MD). GlcNAc, GalNAc, Glc, Gal, and Man were
purchased from Pfanstiehl Laboratories, Inc. (Waukegan, IL) and allose,
xylose, fructose, 2-deoxy-Glc, 3-deoxy-Glc, 6-deoxy-Glc, and
3-O-methyl-D-glucose were obtained from Sigma. Methyl
-2-acetamido-2-deoxy-D-glucopyranoside was synthesized by
the conventional method, and the structure was confirmed by
H NMR (300 mHz). Other reagents were commercially available
products.
Methods
Enzymatic Reactions with Endo-A
A typical
enzymatic hydrolysis was performed with a mixture of 3 nmol of
ManGlcNAc
Asn and 0.1 milliunit of enzyme, in a
total volume of 20 µl of 25 mM ammonium acetate buffer (pH
6.0). After incubation at 37 °C for 10 min, the enzyme reaction was
terminated by boiling for 3 min in a water bath, and the mixture was
dried with a Speedvac using a vacuum pump. One-fourth of the sample in
50 µl was injected into an HPAEC-PAD system (see below) for
analysis. A typical transglycosylation reaction was performed with 1
milliunit of enzyme, and by inclusion of 4 µmol of GlcNAc as
acceptor and 30% acetone (v/v) in the above reaction mixture.
GlcNAc
Asn was incubated with 70 µmol of
acceptor and 76 milliunits of endo-A in 350 µl of 0.1 M ammonium acetate buffer (pH 6.0) containing 30% acetone at 37
°C for 15 min. After terminating the reaction by boiling, the
transfer products were purified by HPLC using a graphitized carbon
column (4.6
100 mm, Shandon Scientific, United Kingdom).
High Performance Anion Exchange Chromatography
An
HPAEC system consisted of a Bio-LC (Dionex Corp., Sunnyvale, CA)
equipped with a pulsed amperometric detector (PAD-II). The
chromatographic data were managed with AI-450 chromatography software
(Dionex). The endo-A reaction products were separated using a Dionex
CarboPac PA-1 column (4 250 mm) at an elution rate of 1 ml/min,
using the following conditions: A, 100 mM sodium
hydroxide with a linear gradient of sodium acetate (30-80
mM) developed in 30 min; B, 100 mM sodium
hydroxide with a linear gradient of sodium acetate (0-150
mM) in 30 min; C, isocratic elution with 100
mM sodium hydroxide and 50 mM sodium acetate. A cycle
of a 5-min washing with 100 mM sodium hydroxide and 200 mM sodium acetate and a 15-min equilibration with the starting buffer
was inserted between runs. The PAD sensitivity was set as 1 K. Under
condition A, the Man
GlcNAc,
Man
GlcNAc
, and
Man
GlcNAc
Asn were eluted at around 20-25
min and were separated from each other. The quantitative determination
of Man
GlcNAc and Man
GlcNAc
was
carried out by comparison of the peak areas with those of standards
obtained by complete digestion of known quantities of
Man
GlcNAc
Asn by endo-A and
Man
GlcNAc
AsnPhe by glycoamidase A.
Purification of Transglycosylation Product by HPLC Using
a Graphitized Carbon Column
The purification was performed with
a Gilson HPLC system equipped with an uv detector (ISCO, Lincoln, NE),
a Rheodyne 7125 injector, and a Fiatron CH-30 column heater. The column
used was Shandon Hypercarb with a Direct-Connect guard cartridge column
(Alltech Associates Inc., Deerfield, IL). The elution was with 10
mM NHOH and a linear gradient of CH
CN
(6-11%) developed in 40 min. The column was kept at 70 °C and
eluted at 1.0 ml/min, and the effluent was monitored at 210 nm. Under
these conditions, the excess acceptor was successfully removed and the
reaction products were separated from each other.
Preparation of Man
To obtain sufficient
transglycosylation product for GlcNAc
-OMe
by Transglycosylation of Endo-A
H NMR analysis, a mixture
containing 4 µmol of Man
GlcNAc
Asn, 1.4 mmol
of methyl
-D-GlcNAc, and 1.1 unit of the enzyme in 3.5 ml
of 14 mM ammonium acetate buffer (pH 6.0) containing 30%
acetone was incubated at 37 °C for 15 min. After the reaction, the
acceptor was removed by gel filtration on a Sephadex G-25 column (2
140 cm) eluted with 0.1 M acetic acid, and the
transglycosylation product was further purified by HPLC with a
graphitized carbon column eluted with 13.5% of CH
CN at 50
°C. The final yield of the transglycosylation product was 74.9%.
Spectra were recorded on a Bruker AM-600
spectrometer. The observed H Nuclear Magnetic Resonance
Spectroscopy
H chemical shifts are reported
with reference to sodium 4,4-dimethyl-4-silapentane-1-sulfonate using
acetone as an internal standard (2.225 ppm downfield from
4,4-dimethyl-4-silapentane-1-sulfonate). The oligosaccharide sample was
prepared by three cycles of dissolving in D
O and
lyophilizing followed by finally dissolving in 0.5 ml of high purity
D
O (99.96 atom %D) before measurement.
). Double-quantum filter-COSY
(DQF-COSY)(30) , Homonuclear Hartmann-Hahn
(HOHAHA)(31) , and NOESY (32) were recorded with
standard pulse sequences. Other acquisition parameters are: for
DQF-COSY, sweep width 1154.7 Hz, 8 scans per t
value with
acquisition times of 443 and 222 ms in t
and t
,
respectively; for HOHAHA, sweep width 2049.2 Hz, 72.5 ms spin lock
time, 4 scans per t
value with acquisition times of 250 and
159 ms in t
and t
; for NOESY, sweep width 2994
Hz, 300 ms mixing time, 16 scans per t
value with
acquisition times of 342 and 171 ms in t
and t
,
respectively.
2 K
real points with digital resolution in the F
dimension better than 0.0024 ppm/point.
Electrospray Mass Spectrometry
Electrospray mass
spectra were recorded on an API-III triple-quadrupole mass spectrometer
(Perkin-Elmer Sciex Instruments, Thornhill, Canada) fitted with an
atmospheric pressure ionization source. The mass spectrometer was
operated in the positive mode; the ion-spray voltage and interface
plate voltage were set to 5300 and 650 V, respectively. The orifice
voltage was 80-105 V. The pressure of the nebulizing gas was 30
psi and the flow rate was 0.8 liter/min.
Time Course of Endo-A Action in Aqueous Solution
Containing GlcNAc
The reaction course of endo-A activity in
aqueous solution was followed by measuring product formation and
substrate disappearance using HPAEC (Fig. 1). Under the
conditions used, the substrate, ManGlcNAc
Asn,
decreased quickly, and totally disappeared after 15 min. The transfer
product, Man
GlcNAc
, was formed predominantly in
the initial stage of the reaction, but decreased after 10 min, and
dropped to 11% at 60 min. On the other hand, the hydrolysis product,
Man
GlcNAc, slowly increased during the reaction, finally
reaching 89% after 60 min.
GlcNAc
Asn, 10 µmol of GlcNAc, and 2.4
milliunits of endo-A in a total volume of 20 µl with 50 mM NH
OAc buffer (pH 6.0) at 37 °C. The enzyme
reaction was terminated by a 3-min boiling in a water bath. The buffer
was removed by evaporation in vacuum. The sample containing 1.5 nmol of
oligosaccharide in 50 µl was injected onto an HPAEC-PAD system
using condition C for elution.
, transglycosylation product;
, hydrolysis product;
; remaining
substrate.
Hydrolytic Activity of Endo-A in Media Containing Organic
Solvents
The endo-A activity was tested in several media
containing 30% of one of the organic solvents. As shown in Table I, the
enzyme remained fully active when acetone or MeSO is
included in the medium, and 79 and 64% activity remained in the
dioxane- or DMF-containing medium, respectively. The hydrolytic
activities of endo-A in the medium containing MeOH or EtOH were only 33
or 55%, partly because of the formation of transglycosylation products,
methyl or ethyl glycosides (for details, see the accompanying
article(48) ). On the other hand, the enzyme has retained only
6.9 and 9.2% activity in CH
CN- or
tetrahydorfuran-containing medium, respectively.
Transglycosylation Activity in Media Containing Organic
Solvents
In the aqueous medium, the hydrolytic product was
predominant (67%), and transglycosylation product reached only 32%
under the conditions described in Table 2. By inclusion of 30%
organic solvent, the hydrolytic activity was effectively suppressed.
The yields of transglycosylation product were 89, 75, 28, and 13% of
the starting substrate in acetone-, MeSO-, DMF- and
dioxane-containing media, respectively. The hydrolytic products were
only less than 2% or could not be detected at all. The hydrolytic
activity was also suppressed in MeOH- or EtOH-containing media,
however, because of transglycosylation to the alcohols, formation of
the desired product was lower than in other media. The
transglycosylation activity of endo-A was also lower in
CH
CN- and tetrahydrofuran-containing media.
SO, or DMF was further investigated by varying their
concentrations (Fig. 2). In the acetone-containing media,
suppression of the hydrolytic activity increased as acetone
concentration was increased to 15-20%, where transglycosylation
(about 80%) was maximum. In 30% acetone, transglycosylation was 49%,
while the hydrolytic activity was suppressed to 2.4%. However, the
remaining substrate increased sharply when the acetone concentration
was raised above 20%, suggesting that the total enzyme activity
(combined hydrolytic and transglycosylation activities) decreased at
higher acetone concentrations. The ratio of transglycosylation to
hydrolysis activity rose more sharply between 20 and 30% acetone.
GlcNAc
Asn as substrate, 4 µmol
of GlcNAc as acceptor, and 1 milliunit of enzyme in 20 µl of 25
mM ammonium acetate buffer (pH 6.0) containing different
amounts of acetone (A), Me
SO (B), or DMF (C). The reaction was carried out at 37 °C for 15 min.
After stopping the reaction by a 3-min boiling, the buffer and the
solvent were removed by freeze-drying. The analysis of the reaction
mixture was done with HPAEC-PAD using condition B for elution.
,
the remaining substrate;
, the transglycosylation product;
, the hydrolysis product;
, the ratio of transglycosylation
to hydrolysis product.
SO-containing media, transglycosylation was elevated over
a wider range of concentration (5-35%) than acetone. However,
lower ratios of transglycosylation to hydrolysis indicate that the
suppression of hydrolytic activity was not as effective in
Me
SO as in acetone. In fact, 40% Me
SO was
required to suppress the hydrolytic activity to 2.6%, a comparable
level attained with 30% acetone.
SO-containing media.
Stability of Endo-A in Acetone, Me
The stability of endo-A in acetone-,
MeSO, and DMF
Containing Media
SO-, and DMF-containing media was studied (Fig. 3). The enzyme was stable in a medium containing up to 30%
acetone medium for 30 min. However, after a 30-min preincubation in the
medium containing 40% acetone, the enzyme activity was reduced to 46%
of the original and a 5-min preincubation in 50% acetone caused a loss
of 95% of the original activity. The enzyme was stable in 30%
Me
SO, but was unstable at Me
SO concentrations
greater than 40%. The enzyme was slightly more stable in
Me
SO-containing media than in acetone-containing media. In
DMF-containing media, endo-A was stable up to 20%. However, the enzyme
lost 72% of its activity in 30% DMF after 30 min, and was completely
inactivated by exposing it to 40% or higher DMF.
SO (B), or DMF (C) with 50 mM ammonium acetate
buffer (pH 6.0) in a total volume of 10 µl was preincubated at 37
°C for 30 min at predetermined lengths of time, 1 µl of the
mixture (0.1 milliunit of enzyme) was taken into another vial
containing 9 µl of 3 nmol of Man
GlcNAc
Asn
and 25 mM NH
OAc buffer (pH 6.0), and incubated at
37 °C for 10 min to determine the remaining enzyme activity. The
enzyme reaction was terminated by boiling for 3 min in a water bath.
The buffer was removed by evaporation in vacuum, and the products were
analyzed with an HPAEC-PAD system using condition B for elution.
,
0% organic solvent;
, 20% organic solvent;
, 30% organic
solvent; ⊠, 40% organic solvent;
, 50% organic
solvent.
Effect of GlcNAc Concentration on Endo-A
The
proportion of the hydrolytic products in 30% acetone diminished rapidly
when GlcNAc concentration was increased (Fig. 4). The
transglycosylation and hydrolytic products were found as 86 and 5%,
respectively, with 0.2 M GlcNAc. No hydrolytic product could
be detected when 0.5 M GlcNAc was used under this condition.
GlcNAc
Asn, 3 milliunits of
enzyme, and GlcNAc in 20 µl of 25 mM ammonium acetate
buffer containing 30% acetone at 37 °C for 10 min. The reactions
were stopped by a 3-min boiling, and the products analyzed by HPAEC-PAD
using condition C for elution.
, hydrolysis product;
,
transglycosylation product;
, remaining
substrate.
Structural Determination of a Transglycosylation Product
of Endo-A
A larger scale transglycosylation reaction in 30%
acetone was performed with 4 µmol of
ManGlcNAc
Asn as donor substrate and 1.4 mmol of
methyl
-D-GlcNAc as acceptor to obtain a sufficient
quantity of transglycosylation product for structural determination.
The transglycosylation yield was 89% from this reaction.
and 1266 (M + NH
)
which
corresponds to the calculated values (Fig. 5). The mass ion at m/z 1087 was due to loss of Man, and m/z 1014 due to loss of GlcNAc-OMe from the parent ion,
Man
GlcNAc
-OMe.
CN, 2
mM NH
OAc. The orifice voltage was 80 V. M, Man; GN, GlcNAc.
H NMR spectrum of
the transglycosylation product (Fig. 6A) shows seven
resonance peaks characteristic of anomeric protons (Table 3) and
three upfield methyl signals (3.362 ppm for OMe and 2.064,
2.026 ppm for two NAc resonances) indicating a heptasaccharide
with OMe substitution at the reducing end and the presence of two
GlcNAc residues. Chemical shifts of the five anomeric resonances most
downfield are consistent with those of five Man signals of the starting
material (33) while the two GlcNAc anomeric signals, especially
that of the reducing terminal residue, differ substantially from the
starting product. The spectrum also shows
GlcNAc (J
= 3.6 Hz) as well as
GlcNAc (J
= 7.8 Hz), suggesting the structure to
be consistent with Man
GlcNAc-
-GlcNAc-
-OMe.
H NMR spectra of the
transglycosylation product of endo-A. A, an expanded region of
one-dimensional spectrum at 20 °C with anomeric proton of each
residue labeled as in the previous report(34) . B, a
cross-section taken through H1 of
-GlcNAc in the two-dimensional
NOESY spectrum showing the intraresidue NOE to H5 and H3 as well as to
H4 of
GlcNAc. *, distorted line shape due to strong coupling. C, an expanded region of two-dimensional-HOHAHA
spectrum.
H spin system of each residue was assigned by homonuclear
two-dimensional spectra. Tracing cross-peak connectivities between
vicinally coupled protons in the DQF-COSY spectrum (data not shown)
yielded most of the assignments. In the instances where DQF-COSY
connectivity is disrupted due to strong coupling or due to partial
cancellation of cross-peaks caused by overlap(34) , the
two-dimensional HOHAHA spectrum (Fig. 6C) was used to
extend the assignments in each residue. DQF-COSY and HOHAHA spectra
yielded complete
H assignments of the heptasaccharide (Table 3). To the best of our knowledge, this is the first
reported complete assignment of a Man
structure.
Furthermore, the two-dimensional NOESY spectrum (data not shown)
clearly shows NOE from anomeric resonances of all the Man residues to
proton(s) across the glycosidic linkage, thus confirming the earlier
linkage assignments. The anomeric proton of
GlcNAc (Fig. 6B) shows NOE to its own H3 and H5 and to a
resonance at 3.620 ppm which was assigned to H4 of
GlcNAc, clearly
indicating the linkage as GlcNAc
1,4GlcNAc which exists in the
natural Man
GlcNAc
Asn structure.
Transglycosylation to Monosaccharides and Their
Derivatives
The conditions used for the reaction were the same
as described under ``Experimental Procedures'' except: (i)
ManGlcNAc
AsnPhe was used as substrate for the
transglycosylation to Man, because of its ease of separation from the
products compared with Man
GlcNAc
Asn as
substrate. (ii) The determination of transglycosylation to 2-deoxy-Glc
and 3-O-Me-Glc were performed on HPLC with a graphitized
carbon column, since the transglycosylation products overlapped with
hydrolytic product or other peaks by HPAEC. The transglycosylation
products were quantified by subtracting the remaining substrate and
hydrolysis product from the starting substrate. The transglycosylation
was also expressed as the percentage of transglycosylation product to
the digested substrate (the sum of hydrolysis and transglycosylation
products). The results are listed in Table 4.
-D-GlcNAc (94%). These results indicate
that C-1 and C-2 substituents of GlcNAc are not important for the
transglycosylation activity. Since 6-deoxy-Glc was a fair acceptor, it
suggests that 6-OH was relatively unimportant for the
transglycosylation. However, the activity was reduced from 77%
(6-deoxy-Glc) to 24% by eliminating C-6 (xylose), suggesting that the
-CH
- group contributes to the transglycosylation in some
ways. The equatorial 4-OH is essential because GalNAc and Gal could not
serve as acceptors for transglycosylation. Since 3-deoxy-Glc could not
be an acceptor at all, and allose, the 3-epimer of Glc, failed to serve
as acceptor, the 3-OH is required to be in equatorial orientation.
Substitution of 3-OH in Glc affects the transglycosylation somewhat,
since 3-O-Me-Glc could only provide 36% transglycosylation.
Fructose also could serve as acceptor, although the transglycosylation
yield was relatively low.
and 1829.8 (M +
NH
)
(Fig. 7A) which are
expected from Man
GlcNAcXyl. The intense peaks of m/z 1680.8 and 1488.8 were due to loss of xylose (i.e. Man
GlcNAc) or two Man's (i.e. Man
GlcNAcXyl) from the parent compound. In addition, a
series of sequence ions showed the loss of Man residues from the
Man
GlcNAc or Man
GlcNAcXyl. The mass ions of the
transglycosylation product to 3-O-Me-Glc were found as 1856.8
(M + H)
and 1873.8 (M +
NH
)
(Fig. 7B), agreeing
with the calculated values. The sequence mass ions revealed the loss of
Man residues from the parent ion. These results indicated that the
compounds formed by endo-A transglycosylation to xylose and
3-O-Me-Glc are Man
GlcNAcXyl and
Man
GlcNAc(3-O-Me-Glc) as expected.
GlcNAcXyl (A) and
Man
GlcNAc(3-O-Me-Glc) (B). The samples
used were about 50 pmol, and infusion rate was 3 µl/min in 1:1
CH
CN, 2 mM NH
OAc. Orifice voltages
were 105 V (A) and 85 V (B). M, Man; GN, GlcNAc; X, xylose; MG,
3-O-Me-Glc.
SO and DMF also can be used
for suppression of the hydrolytic activity, although they are not as
efficient as acetone. The fact that the extents of transglycosylation
varied between different solvents even with the same water content
suggests that transglycosylation is not only affected by the apparent
water content but also by the nature of organic solvent added.
SO were used
for the purpose of the suppression of hydrolysis. Acetone is generally
avoided, because it is known as a precipitant for protein. However, in
our case, acetone was found to be the best among the solvents tested.
Although the efficiencies of suppression of hydrolytic activity by
Me
SO and DMF are not as high as that by acetone, they are
still attractive solvents to be considered for transglycosylation,
because many proteins dissolve in Me
SO- and DMF-containing
solvents better than acetone.
H NMR.
In order to facilitate signal assignment, the methyl
-pyranoside
of D-GlcNAc was chosen as acceptor. The
H NMR
analyses revealed that the new glycosidic bond formed by
transglycosylation is indeed the GlcNAc
1,4GlcNAc linkage, the same
as that existed in the donor substrate. We have also confirmed by
H NMR that the same
-configuration was maintained when
a
-glycoside of GlcNAc (3-(N-acryloylamino)-propyl
-GlcNAc) was used as acceptor (see the accompanying article (48) for details). Thus, we can conclude that the
stereoselectivity and regioselectivity of the endo-A transglycosylation
are not influenced by the anomeric configuration of the acceptor
glycoside and inclusion of organic solvent, when transglycosylation is
to a GlcNAc residue.
-N-acetylglucosaminidase from A. protophormiae; Me
SO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; Glc, glucose; Man, mannose;
Gal, galactose; Xyl, xylose; Fru, fructose; HPAEC-PAD, high performance
anion exchange chromatography with pulsed amperometric detector; HPLC,
high performance liquid chromatography; NOSEY, nuclear Overhauser
effect spectroscopy.
We thank Dr. Reiko T. Lee for the gift of soybean
agglutinin, and Dr. Ming-Chuan Shao of The University of Texas Medical
School at Houston for a gift of ManGlcNAc
Asn.
We are also grateful to Dr. Laixi Wang for 300 MHz
H NMR
analysis of methyl
-D-GlcNAc.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.