(Received for publication, August 17, 1994; and in revised form, October 11, 1994)
From the
We describe a novel method for the enzymatic synthesis of
neoglycoproteins. Endo--N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) had high levels of
transglycosylation activity. The enzyme activity of Endo-A was markedly
increased by adding 4-L-aspartylglycosylamine (GlcNAc-Asn) to
the reaction mixture. Digesting (Man)
(GlcNAc)
with the enzyme in the presence of GlcNAc-Asn gave a mixture of
hydrolytic ((Man)
GlcNAc) and transglycosylic
((Man)
(GlcNAc)
Asn) products. By means of
transglycosylation, (Man)
GlcNAc was transferred en bloc to the partially deglycosylated ovalbumin glycopeptide
(EEKYN(GlcNAc) LTSVL) concomitant with the hydrolysis of
(Man)
(GlcNAc)
Asn. The structure of the
transglycosylation product was designated as
(Man)
(GlcNAc)
-peptide by amino acid composition
and sequence analysis as well as ion mass spectrometry. The enzyme also
transferred oligosaccharide to partially deglycosylated ribonuclease B
(GlcNAc-protein) during the hydrolysis of
(Man)
(GlcNAc)
Asn. Native ribonuclease B had
(Man)
(GlcNAc)
as its heterogeneous N-linked sugar chains. High performance liquid chromatography
showed that all of the N-linked sugar chains of the synthetic
neoribonuclease of the pyridylamino derivatives were modified to
(Man)
(GlcNAc)
.
Asparagine-linked glycosylation begins with the transfer of the
precursor oligosaccharide
(Glc)(Man)
(GlcNAc)
to a nascent
polypeptide, and the precursor oligosaccharide is modified by Golgi
enzymes, which generate a heterogeneous assortment of oligosaccharide
structures(1) . Therefore, naturally occurring glycoproteins
have a high degree of heterogeneity in their oligosaccharide moiety.
For example, human serum immunoglobulin G contains over 30 varieties of
biantennary N-linked sugar chains(2) . It is difficult
to study the biological roles of individual oligosaccharides because
they are highly heterogeneous in glycoproteins.
There are several approaches to the analysis of the functions of the individual sugar chains in glycoproteins. Besides conventional analysis with specific glycosidases(3) , site-directed mutagenesis can now identify the role of individual sugar chains in glycoproteins(4, 5) . The properties of naturally occurring and recombinant glycoproteins produced in different heterologous cells can also be prepared(6, 7, 8, 9) . However, these DNA-mediated studies call for the isolation and preparation of the genes that code for glycoproteins, and the structures of the sugar chains vary according to the host cell. Therefore, a method for remodeling oligosaccharides that are heterogeneous in terms of size and branching into homogeneous sugar chains would be useful.
Endo--N-acetylglucosaminidase (EC 3.2.1.96) releases N-linked oligosaccharide chains from glycoproteins by cleaving
the di-N-acetylchitobiose unit. This enzyme is important in
glycoprotein research, because it can be used to recover both the N-linked oligosaccharides and partially deglycosylated
proteins without damaging them. Arthrobacter protophormiae produces endo-
-N-acetylglucosaminidase (called
Endo-A) (
)when cultured in medium containing ovalbumin; the
purification and properties of the enzyme have been
reported(10) . We showed that Endo-A has strong
transglycosylation activity and that GlcNAc is transferred to the
reducing end of (Man)
GlcNAc during chitobiose cleavage by
this enzyme(11) . Endo-A can transfer (Man)
GlcNAc
to various acceptors, such as glucose, mannose, and
gentiobiose(12) . We also found that Endo-A can transfer
(Man)
GlcNAc to 4-L-aspartylglycosylamine
(GlcNAc-Asn). Therefore, we predicted that Endo-A would be useful for
synthesizing not only novel oligosaccharides but also neoglycopeptides
or neoglycoproteins. Transglycosylation is a type of hydrolysis in
which the glycosyl moiety of the substrate, instead of water, is
transferred to other hydroxy compounds. In addition, many
exoglycosidases and endoglycosidases have transglycosylation or
transfer reaction activities that have been used in the synthesis of
various glycosides (reviewed in (13) and (14) ).
However, there is little information available on the enzymatic
synthesis of neoglycoproteins by endoglycosidases. Here we examined the
transglycosylation activity catalyzed by Endo-A toward GlcNAc-peptide
derived from ovalbumin. We also describe the synthesis of
neoglycoprotein by Endo-A.
Figure 1:
A, effect of the concentrations of
GlcNAc-Asn on the hydrolysis of
(Man)(GlcNAc)
Asn-dansyl by Endo-A. Enzyme
assays were performed under standard conditions except for the prior
incubation with varying amounts of GlcNAc-Asn (
) or GlcNAc
(
) in the reaction mixture. Enzyme activities are expressed
relative to those without sugars. B, incorporation of
asparagine residue into oligosaccharides by the transglycosylation of
Endo-A. (Man)
(GlcNAc)
(21 nmol) was incubated
for 10 min at 37 °C with 0.004 unit in 25 mM ammonium
acetate buffer (pH 6.0) in the presence of varying concentrations of
GlcNAc-Asn (total volume, 20 µl). The reaction mixtures were then
eluted through Sephadex G-15 to remove remaining GlcNAc-Asn and
lyophilized prior to amino acid analysis. The samples were hydrolyzed
with 6 M HCl for 12 h, and then the incorporation of
asparagine residues into oligosaccharides was measured using an amino
acid analyzer.
To confirm the transglycosylation products, 30 µg of
(Man)(GlcNAc)
(21 nmol) prepared from
(Man)
(GlcNAc)
Asn by hydrazinolysis was
incubated with 0.004 unit of Endo-A in 25 mM ammonium acetate
buffer (pH 6.0) in the presence of various concentrations of GlcNAc-Asn
for 10 min at 37 °C (total volume, 20 µl). After the incubation
was stopped by boiling for 3 min, the reaction mixtures were applied to
Sephadex G-15 to remove any remaining GlcNAc-Asn and then lyophilized
prior to amino acid analysis. Peaks containing neutral sugars were
collected and hydrolyzed with 6 M HCl for 12 h, and then the
incorporation of the asparagine residues into oligosaccharides was
measured using an amino acid analyzer. The asparagine incorporation
increased with increasing concentrations of GlcNAc-Asn (Fig. 1B). This result showed that GlcNAc-Asn was
attached to (Man)
GlcNAc during cleavage of
(Man)
(GlcNAc)
. The attachment of GlcNAc-Asn to
(Man)
GlcNAc was also confirmed as an increase in the
molecular weight by means of ion mass spectrometry (data not shown).
Figure 2:
A, reversed-phase HPLC elution profiles of
neoglycopeptide synthesized from ovalbumin GlcNAc-peptide by Endo-A.
Reaction mixtures containing 0.004 unit of Endo-A and 100 nmol of
GlcNAc-peptide were incubated in the absence (I) or presence (II) of (Man)(GlcNAc)
Asn (33 nmol) for
10 min at 37 °C in a total volume of 20 µl. The reaction
mixtures were then analyzed by HPLC with a Wakosil 5C-18 column
equilibrated with 0.1% trifluoroacetic acid and eluted with a linear
acetonitrile gradient at a flow rate of 1.0 ml/min. Peptides were
monitored at 213 nm. B, time course of neoglycopeptide
production from GlcNAc-peptide by Endo-A.
(Man)
(GlcNAc)
Asn (33 nmol) was incubated with
0.004 unit of Endo-A in the presence of GlcNAc-peptide (100 nmol) in 25
mM ammonium acetate buffer (pH 6.0) in a total volume of 20
µl. After incubation for the indicated times, the amounts of
neoglycopeptide were analyzed by reversed-phase HPLC with a Wakosil
5C-18 column. Yield was defined as the percentage of
(Man)
(GlcNAc)
-peptide/GlcNAc-peptide (P-2/P-1 in A).
Figure 3: The optimal reaction conditions for neoglycopeptide synthesis by Endo-A. The standard reaction conditions were followed as described in Fig. 2B. A, the effect of varying GlcNAc-peptide concentrations on neoglycopeptide production. B, the effect of varying substrate concentrations on neoglycopeptide production. C, the effect of varying Endo-A concentrations on neoglycopeptide production.
Figure 4:
ConA-Sepharose 4B column chromatography of
the reaction products liberated by Endo-A in the presence of RNase B.
(Man)(GlcNAc)
Asn (1 mg) was incubated with 0.01
unit of Endo-A in the presence of 3 mg of denatured RNase B. After
incubation for 10 min at 37 °C, the reaction mixture was eluted
through ConA-Sepharose equilibrated with 10 mM acetate buffer
containing 1 M NaCl, 1 mM MgSO
, 1 mM CaCl
, and 1 mM MnCl
(pH 6.0).
Bound RNase was then eluted with the same buffer containing 1 M
-D-methyl mannoside. The arrow indicates
the addition of
-D-methyl mannoside. Fractions of 1 ml
were collected.
Figure 5:
SDS-polyacrylamide gel electrophoresis of
neo-RNase. A and G, molecular weight markers; B, native RNase B; C, GlcNAc-RNase by
endo--N-acetylglucosaminidase digestion; D,
unbound RNase B in Fig. 6; E, bound RNase B; F, bound RNase was digested by
endo-
-N-acetylglucosaminidase.
Figure 6:
Comparison of the N-linked
sugar chains in native and neo-RNases. A portion of each PA
oligosaccharide was analyzed by HPLC with a Takara Palpak Type N
column. The HPLC conditions were as described before(12) . A, native RNase B; B, neo-RNase after
(Man)GlcNAc was transferred by Endo-A. The arrows indicate the elution positions of the standard PA oligosaccharides
M5-M9, which were
(Man)
GlcNAc-PA.
The N-linked sugar chains were compared between the native and the
neo-RNase. One of the two forms of RNase (500 µg) was incubated
with 0.1 unit of the Flavobacterium endo--N-acetylglucosaminidase at 37 °C for 12 h.
After the N-linked sugar chains were partially released from
the protein moiety, the reaction mixtures were pyridylaminated and
analyzed by HPLC. PA oligosaccharides from the native RNase B separated
into five peaks (peaks (Man)
GlcNAc-PA to
(Man)
GlcNAc-PA) (Fig. 6A). Liang et al.(28) have reported that the N-linked sugar chains
of RNase B are (Man)
(GlcNAc)
. However,
we found a small amount of (Man)
(GlcNAc)
. The N-linked sugar chain of neo-RNase consisted entirely of
(Man)
(GlcNAc)
(Fig. 6B). These
results showed that the heterogeneous N-linked sugar chains
(Man)
(GlcNAc)
of the RNase B were
converted into homogeneous (Man)
(GlcNAc)
by the
oligosaccharide-transferring mechanism of Endo-A.
Some reports have described the attachment of naturally
occurring N-linked sugar chains to proteins. Mencke and Wold (29) have reported that the glycosyl-asparagine derivatives
obtained by Pronase digestion of ovalbumin are coupled to bovine serum
albumin by reductive amination with NaCNBH. Yan (30) has reported that glycosyl-Asn derivatives can be coupled
to bovine
-casein and bovine pancreatic RNase A by a
transglutaminase. These methods are useful for attaching
oligosaccharides to proteins, but there are several problems. It is
often difficult to prepare glycosyl-asparagine derivatives by Pronase
digestion, because many glycopeptides are resistant to proteases. This
procedure also yields unexpected changes in the protein structure and
activity. Most importantly these methods cannot be used to attach the
oligosaccharides to all of the original glycosylation sites of the
native glycoprotein molecules. Therefore, these strategies may change
the conformation of the protein moiety.
Endo-A has powerful
transglycosylation activity, and, during chitobiose cleavage of
(Man)(GlcNAc)
Asn, (Man)
GlcNAc is
transferred to the C-4 hydroxy group through a
-linkage of
GlcNAc(11) . Trimble et al.(31) have reported
that N-linked oligosaccharides, when hydrolyzed by
endo-
-N-acetylglucosaminidase prepared from Flavobacterium meningosepticum(32) (Endo-F), have
glycerol attached to their reducing ends. We examined the
oligosaccharide-transferring activity of commercial preparations of
Endo-F and Endo-H (endo-
-N-acetylglucosaminidase from Streptomyces plicatus)(33) . These enzymes did not
transfer oligosaccharides to GlcNAc-peptides or GlcNAc-proteins (data
not shown). Bardales and Bhavanadan (34) have reported that
endo-
-N-acetylglucosaminidase from Diplococcus
pneumoniae has not only transglycosylation activity but also
transfer activity (reversed hydrolysis). When Gal
1
3GalNAc
was incubated with D. pneumoniae endo-
-N-acetylgalactosaminidase in the presence of
glycerol, the trisaccharide Gal-GalNAc-glycerol was synthesized by the
transfer activity of the enzyme. To examine the reverse hydrolysis
activity of Endo-A, (Man)
GlcNAc and GlcNAc-peptide from
ovalbumin were mixed and incubated with Endo-A under various
conditions. However, we could not detect any reverse hydrolysis product
(data not shown).
The substrate specificity of the Endo-A is very
similar to that of Endo-C from Clostridium
perfringens(35, 36) , and it hydrolyzes primarily
high mannose type oligosaccharides(10) . However, the molecular
weight of Endo-A (approximately 80,000) is quite different from other
endo-
-N-acetylglucosaminidases, such as Endo-H (37) (271 amino acids), Endo-Fsp (17) (267 amino
acids), and Endo-F
(38) (289 amino acids), all of
which show similar substrate specificity. There are several amino acid
residues that are well conserved in all
endo-
-N-acetylglucosaminidases(17, 39) .
We cloned and sequenced the Endo-A gene and found that the N-terminal half of the amino acid sequence of the enzyme had
low identity with other endo-
-N-acetylglucosaminidases (36, 37, 38, 39) . (
)Studies are in progress to determine which amino acid
residues or domains of Endo-A protein contribute to the high
transglycosylation activity.
Here we reported a new method for the conversion of N-linked sugar chains that uses the transglycosylation activity of Endo-A (Fig. S1). This method is useful for attaching the same N-linked sugar chains to all the original glycosylation sites of glycoprotein molecules. No effective method is currently available for the conversion of heterogeneous N-linked sugar chains to homogeneous N-linked sugar chains in glycoproteins. We described for the first time a one-step procedure for the preparation of neoglycoproteins using the transglycosylation activity of endoglycosidase A. The use of glycosyltransferases in the synthesis of neoglycoproteins has not been extensively investigated. In general, glycosyltransferases are present at low concentrations and are bound to intracellular membranes. Furthermore, the substrate specificity of the enzymes is very limited. Therefore, these transferases are not stable, are difficult to purify, and require nucleotide sugars as donors. On the other hand, Endo-A can transfer the oligosaccharides en bloc, has broad pH stability(10) , and does not require nucleotide sugars for the reaction.
Scheme 1: Scheme 1Enzymatic synthesis of neoglycoproteins by Endo-A.
Because of the substrate specificity of the enzyme,
however, the conversion of the N-linked oligosaccharides by
Endo-A is limited. Endo-A specifically acts upon high mannose type
oligosaccharides, and it did not have transglycosylation activities
toward complex oligosaccharides (data not shown). Our data showed that
the GlcNAc-peptide (10 amino acids) derived from ovalbumin is a better
acceptor than bovine RNase B (GlcNAc-protein) for the
transglycosylation reaction of Endo-A. Endo-A can readily deglycosylate
native bovine RNase B but shows very little
oligosaccharide-transferring activity toward the native form of
partially deglycosylated RNase (data not shown). Considering the steric
effects of the protein moiety on the accessibility of glycosylation
sites to Endo-A, it seems that steric factors have a more direct
influence upon the transglycosylation rather than upon hydrolytic
activity. Therefore, the reaction conditions should be further examined
to obtain the optimal transglycosylation activity of Endo-A. Fan and
Lee ()have found that Endo-A has high transglycosylation
activity in the presence of organic solvents, such as acetone,
Me
SO, and N,N,dimethylformamide.
Moreover, Endo-A exerted exclusive transglycosylation activity in a
reaction mixture containing acetone, and a hydrolysis product was
undetectable under these conditions. These organic solvents may
increase the yield of neoglycoproteins by Endo-A. The method presented
here is suitable and practical for the improved synthesis of
neoglycoproteins.