©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis of Neoglycoproteins Using Oligosaccharide-transfer Activity with Endo--N-Acetylglucosaminidase (*)

(Received for publication, August 17, 1994; and in revised form, October 11, 1994)

Kaoru Takegawa (§) Mitsuaki Tabuchi Shinya Yamaguchi Akihiro Kondo (1) Ikunoshin Kato (1) Shojiro Iwahara

From the Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07 and the Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Ohtsu, Shiga 520-21, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-beta-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)(6)(GlcNAc)(2) with the enzyme in the presence of GlcNAc-Asn gave a mixture of hydrolytic ((Man)(6)GlcNAc) and transglycosylic ((Man)(6)(GlcNAc)(2)Asn) products. By means of transglycosylation, (Man)(6)GlcNAc was transferred en bloc to the partially deglycosylated ovalbumin glycopeptide (EEKYN(GlcNAc) LTSVL) concomitant with the hydrolysis of (Man)(6)(GlcNAc)(2)Asn. The structure of the transglycosylation product was designated as (Man)(6)(GlcNAc)(2)-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)(6)(GlcNAc)(2)Asn. Native ribonuclease B had (Man) (GlcNAc)(2) 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)(6)(GlcNAc)(2).


INTRODUCTION

Asparagine-linked glycosylation begins with the transfer of the precursor oligosaccharide (Glc)(3)(Man)(9)(GlcNAc)(2) 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-beta-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-beta-N-acetylglucosaminidase (called Endo-A) (^1)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)(6)GlcNAc during chitobiose cleavage by this enzyme(11) . Endo-A can transfer (Man)(6)GlcNAc to various acceptors, such as glucose, mannose, and gentiobiose(12) . We also found that Endo-A can transfer (Man)(6)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.


EXPERIMENTAL PROCEDURES

Materials

Endo-A was obtained from culture fluid of A. protophormiae as described(10) . (Man)(6)(GlcNAc)(2)Asn was prepared from ovalbumin by the procedure of Huang et al.(15) using Dowex 50W-X2 column chromatography. Dansyl derivatives of asparaginyl glycopeptides were prepared by the method of Gray(16) , followed by paper chromatography to remove dansyl sulfuric acid. Bovine pancreatic ribonuclease B (RNase B) was purchased from Sigma (Type III-B) and further purified by ConA-Sepharose 4B column chromatography. The RNase B was partially deglycosylated by endo-beta-N-acetylglucosaminidase from Flavobacterium sp. (17, 18) as described(19) . Pepsin was purchased from Sigma. The standard oligosaccharides (Man)GlcNAc were prepared from ovalbumin or porcine thyroglobulin by endo-beta-N-acetylglucosaminidase digestion. The oligosaccharide (Man)(5)(GlcNAc)(2) was prepared from (Man)(5)(GlcNAc)(2)Asn by hydrazinolysis according to Hase et al.(20) , and GlcNAc-Asn was prepared from (Man)(6)(GlcNAc)(2)Asn by endo-beta-N-acetylglucosaminidase digestion.

Isolation of Partially Deglycosylated Glycopeptide from Ovalbumin

Partially deglycosylated glycopeptide was isolated from ovalbumin by means of pepsin digestion according to Ishihara et al.(21) with the following modifications. Crystalline ovalbumin (2 g) was heat-denatured and digested with 20 mg of pepsin in 40 ml of 0.15 M HCl-citrate buffer (pH 1.4) in the presence of toluene to avoid bacterial contamination. The reaction mixture was lyophilized and dissolved in 20 mM NH(4)HCO(3). The clear supernatant was applied to a Sephadex G-25 column (1.0 times 111 cm) equilibrated with 20 mM NH(4)HCO(3). Fractions containing the carbohydrates were pooled, dried, and applied to a Sephadex G-100 column (1.0 times 110 cm) equilibrated with 20 mM NH(4)HCO(3). Peptic glycopeptides obtained by these procedures were further separated by means of HPLC with a Wakosil 5C-18 column (Wako Pure Chemicals, Osaka, Japan), equilibrated with 0.1% trifluoroacetic acid, and eluted with a linear acetonitrile gradient at a flow rate of 1.0 ml/min. The carbohydrate peak was collected and partially deglycosylated by the Flavobacterium endo-beta-N-acetylglucosaminidase. The reaction mixture was applied once more to HPLC, and a peak of a single peptide containing a GlcNAc residue was collected. Analyses of the amino acid composition, molecular weight, and amino acid sequence revealed that the structure of the GlcNAc-containing peptide was Glu-Glu-Lys-Tyr-Asn(GlcNAc)-Leu-Thr-Ser-Val. The amino acid sequence of the GlcNAc-peptide was in agreement with the data from the mRNA sequence of ovalbumin reported by McReynolds et al.(22) .

Analytical Methods

Pyridylamination of oligosaccharides and HPLC analysis of pyridylamino (PA) oligosaccharides proceeded as described(23) . Standard PA oligosaccharides were obtained from Takara Shuzo Co., Ltd. The molecular weight of oligosaccharides or glycopeptides was estimated by ion spray mass spectrometry (Perkin-Elmer API-III) according to Bruins et al.(24) . Amino acids were hydrolyzed with 6 N HCl for 24 h at 100 °C in evacuated, sealed glass tubes and then evaluated using an amino acid analyzer (Hitachi L8500). The amino acid sequence of the GlcNAc-peptide from ovalbumin was determined using a protein sequencer (Applied Biosystems model 477A). Phenylthiohydantoin amino acid derivatives were separated and identified with an on-line phenylthiohydantoin analyzer (model 120A). The elution position of GlcNAc-Asn was directly identified by means of conventional sequencing as described(25) . Endo-beta-N-acetylglucosaminidase activity was assayed using (Man)(6)(GlcNAc)(2)Asn-dansyl as the substrate(10) . One unit was defined as the amount yielding 1 µmol of GlcNAc-Asn-dansyl/min at 37 °C under our assay conditions. SDS-polyacrylamide gel electrophoresis was performed in a 17% acrylamide gel containing 0.1% SDS by the method of Laemmli(26) . Protein was stained with Coomassie Brilliant Blue R-250.


RESULTS

Effect of GlcNAc-Asn on Enzyme Activity

We found that exposing Endo-A to a mixture of monosaccharides or oligosaccharides increased the apparent enzyme activity(11, 27) . We determined that Endo-A has powerful transglycosylation activity and that it transfers GlcNAc to the reducing end of (Man)(6)GlcNAc during chitobiose cleavage. We initially investigated the effects of GlcNAc-Asn upon Endo-A activity. About 50 µmol of (Man)(6)(GlcNAc)(2)Asn-dansyl was incubated at 37 °C with 0.005 unit of Endo-A in 50 mM acetate buffer, pH 6.0. The enzyme activity was measured after an incubation with various concentrations of GlcNAc or GlcNAc-Asn for 10 min. The enzyme activity increased with increasing amounts of GlcNAc or GlcNAc-Asn. GlcNAc-Asn enhanced activity more than GlcNAc, and the relative activity with 80 mM GlcNAc-Asn to that without GlcNAc-Asn was about 1300% (Fig. 1A). These results suggested that (Man)(6)GlcNAc was transferred to GlcNAc-Asn and that the transglycosylation caused an increase in GlcNAc-Asn-dansyl liberation.


Figure 1: A, effect of the concentrations of GlcNAc-Asn on the hydrolysis of (Man)(6)(GlcNAc)(2)Asn-dansyl by Endo-A. Enzyme assays were performed under standard conditions except for the prior incubation with varying amounts of GlcNAc-Asn (circle) or GlcNAc (bullet) 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)(6)(GlcNAc)(2) (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)(6)(GlcNAc)(2) (21 nmol) prepared from (Man)(6)(GlcNAc)(2)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)(6)GlcNAc during cleavage of (Man)(6)(GlcNAc)(2). The attachment of GlcNAc-Asn to (Man)(6)GlcNAc was also confirmed as an increase in the molecular weight by means of ion mass spectrometry (data not shown).

Synthesis of a Neoglycopeptide from Ovalbumin Glycopeptides with Endo-A

Reaction mixtures containing 0.004 unit of Endo-A, 100 nmol of GlcNAc-peptide prepared from ovalbumin as described under ``Experimental Procedures,'' and 25 mM ammonium acetate buffer (pH 6.0) in a total volume of 20 µl were incubated for 10 min at 37 °C. The reaction was started by adding 50 µg (33 nmol) of (Man)(6)(GlcNAc)(2)Asn and stopped by boiling for 3 min at 100 °C. The reaction mixture was then directly analyzed by HPLC. Only one major peak (P-1) was obtained without (Man)(6)(GlcNAc)(2)Asn in the reaction mixture (Fig. 2A-I). However, another peptide peak (P-2) was generated in the presence of (Man)(6)(GlcNAc)(2)Asn (Fig. 2A, II). This peak was collected and hydrolyzed with 6 M HCl or 2 M trifluoroacetic acid, and the amino acid or amino sugar contents were investigated using an amino acid analyzer. The amino acid composition of P-2 was identical to that of P-1, except that the GlcNAc content was doubled. This result suggested that the transfer of (Man)(6)GlcNAc to the GlcNAc-peptide occurred through the transglycosylation of Endo-A. To further confirm the structure of P-2, we analyzed P-1 and P-2 by means of ion mass spectrometry, and they yielded molecular ion peaks of 1398 and 2573, respectively (data not shown). These results indicated that a neoglycopeptide had been synthesized by the transglycosylation of Endo-A and that the product (Man)(6)(GlcNAc)(2)-peptide was easily separated from GlcNAc-peptide and detected by HPLC.


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)(6)(GlcNAc)(2)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)(6)(GlcNAc)(2)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)(6)(GlcNAc)(2)-peptide/GlcNAc-peptide (P-2/P-1 in A).



The Optimal Reaction Conditions for Neoglycopeptide Synthesis by the Enzyme

We determined the optimal conditions for neoglycopeptide formation by Endo-A as follows.

Changes during the Transglycosylation Reaction

Changes during the transfer of the GlcNAc-peptide to the oligosaccharide by Endo-A are shown in Fig. 2B. In the initial stage of the reaction, much (Man)(6)(GlcNAc)(2)-peptide was synthesized by the transglycosylation activity of the enzyme. However, as the reaction proceeded, the transglycosylation product (Man)(6)(GlcNAc)(2)-peptide was gradually hydrolyzed by the enzyme once again.

The Effect of pH on Neoglycopeptide Synthesis

To investigate the effect of pH on the yield of the neoglycopeptide, the reaction was performed at various pH values, and the amounts of the enzyme products were measured after 10 min at 37 °C. The enzyme was active and stable above pH 4.0. Transglycosylation did not occur at pH 3.0, and the optimum pH level for neoglycopeptide synthesis was 6.0 in ammonium acetate buffer (data not shown).

The Effect of the GlcNAc-Peptide Concentrations on Neoglycopeptide Synthesis

The effect of the GlcNAc-peptide concentrations on the production of the neoglycopeptide was investigated by performing the reaction with various concentrations of GlcNAc-peptide for 10 min at pH 6.0. Increasing GlcNAc-peptide concentrations increased the production of (Man)(6)(GlcNAc)(2)-peptide (Fig. 3A). Because of the solubility, however, we could not add or test higher concentrations of GlcNAc-peptide.


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.



The Effect of the Substrate Concentrations on Neoglycopeptide Production

The effect of the substrate concentration on the production of the neoglycopeptide was investigated in the presence of various concentrations of (Man)(6)(GlcNAc)(2)Asn at pH 6.0. The level of neoglycopeptide increased with increasing amounts of the substrate up to 80 nmol (final concentration of 4 mM), and concentrations above 4 mM were not as effective (Fig. 3B).

The Effect of Endo-A Concentration on Neoglycopeptide Production

The production of neoglycopeptide in the presence of various concentrations of Endo-A was investigated. The amount of neoglycopeptide increased with the enzyme concentration, of which around 8 milliunits (0.4 unit/ml) was optimal (Fig. 3C).

The Enzymatic Synthesis of Neo-RNase

The results described above show that Endo-A can synthesize a neoglycopeptide. To test whether a neoglycoprotein could be made, we synthesized neoribonuclease by means of the transglycosylation activity of Endo-A under the optimal reaction conditions described above. To raise the efficiency of the transglycosylation activity of Endo-A, RNase B, which was partially deglycosylated by Flavobacterium endo-beta-N-acetylglucosaminidase to give GlcNAc-protein, was denatured with 6 M guanidine HCl. After dialysis with distilled water for 2 days, denatured GlcNAc-RNase was lyophilized and used in this experiment. The reaction conditions for transglycosylation by Endo-A were as follows. 1 mg of (Man)(6)(GlcNAc)(2)Asn was incubated with 0.01 unit of Endo-A in 50 mM ammonium acetate buffer (pH 6.0) in the presence of lyophilized GlcNAc-RNase (3.0 mg) (total volume, 50 µl). After incubation for 10 min at 37 °C, the reaction mixture was dialyzed against 10 mM acetate buffer (pH 6.0) and placed on a column (1.5 times 6 cm) containing ConA-Sepharose 4B at 4 °C. Most of the GlcNAc-RNase passed through the ConA column. However, some bound to the column, but it was eluted with buffer containing 1 M alpha-D-methyl mannoside (Fig. 4). To characterize the ConA-bound RNase further, both forms were pooled and analyzed by SDS-polyacrylamide gel electrophoresis. RNase that did not bind to the ConA column had the same molecular weight as GlcNAc-RNase (Fig. 5D). The molecular weight of the bound RNase was increased by about 2,000, and it migrated to the position of native RNase B (Fig. 5E). When bound RNase was incubated with 0.01 unit of the Flavobacterium endo-beta-N-acetylglucosaminidase, it was deglycosylated again and migrated to the position of GlcNAc-RNase (Fig. 5F). These results showed that Endo-A transferred the oligosaccharide (Man)(6)GlcNAc from (Man)(6)(GlcNAc)(2)Asn to GlcNAc-RNase. Therefore, we called the bound RNase ``neo-RNase,'' and set out to find whether (Man)(6)GlcNAc was actually linked to it.


Figure 4: ConA-Sepharose 4B column chromatography of the reaction products liberated by Endo-A in the presence of RNase B. (Man)(6)(GlcNAc)(2)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(4), 1 mM CaCl(2), and 1 mM MnCl(2) (pH 6.0). Bound RNase was then eluted with the same buffer containing 1 M alpha-D-methyl mannoside. The arrow indicates the addition of alpha-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-beta-N-acetylglucosaminidase digestion; D, unbound RNase B in Fig. 6; E, bound RNase B; F, bound RNase was digested by endo-beta-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)(6)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-beta-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)(5)GlcNAc-PA to (Man)(9)GlcNAc-PA) (Fig. 6A). Liang et al.(28) have reported that the N-linked sugar chains of RNase B are (Man)(GlcNAc)(2). However, we found a small amount of (Man)(9)(GlcNAc)(2). The N-linked sugar chain of neo-RNase consisted entirely of (Man)(6)(GlcNAc)(2) (Fig. 6B). These results showed that the heterogeneous N-linked sugar chains (Man)(GlcNAc)(2) of the RNase B were converted into homogeneous (Man)(6)(GlcNAc)(2) by the oligosaccharide-transferring mechanism of Endo-A.


DISCUSSION

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(3). Yan (30) has reported that glycosyl-Asn derivatives can be coupled to bovine beta-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)(6)(GlcNAc)(2)Asn, (Man)(6)GlcNAc is transferred to the C-4 hydroxy group through a beta-linkage of GlcNAc(11) . Trimble et al.(31) have reported that N-linked oligosaccharides, when hydrolyzed by endo-beta-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-beta-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-alpha-N-acetylglucosaminidase from Diplococcus pneumoniae has not only transglycosylation activity but also transfer activity (reversed hydrolysis). When Galbeta13GalNAc was incubated with D. pneumoniae endo-alpha-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)(6)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-beta-N-acetylglucosaminidases, such as Endo-H (37) (271 amino acids), Endo-Fsp (17) (267 amino acids), and Endo-F(1)(38) (289 amino acids), all of which show similar substrate specificity. There are several amino acid residues that are well conserved in all endo-beta-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-beta-N-acetylglucosaminidases (36, 37, 38, 39) . (^2)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 (^3)have found that Endo-A has high transglycosylation activity in the presence of organic solvents, such as acetone, Me(2)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.


FOOTNOTES

*
This work was supported by Grant-in-aid for Scientific Research 0480616 from the Ministry of Education, Science, and Culture, Japan (to S. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07, Japan. Tel.: 81-878-98-9675; Fax: 81-878-98-7295.

(^1)
The abbreviations used are: Endo-A, endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae; HPLC, high performance liquid chromatography; PA, pyridylamino; ConA, concanavalin A; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

(^2)
K. Takegawa, M. Tabuchi, K. Yamabe, M. Mita, A. Kondo, I. Kato, and S. Iwahara, manuscript in preparation.

(^3)
J.-Q. Fan and Y. C. Lee, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Hiroyuki Iwamoto (Fukuyama University) and Drs. Kenji Yamamoto and Tatsurokuro Tochikura (Kyoto University) for helpful advice. We thank Dr. Jian-Qiang Fan and Dr. Yuan Chuan Lee (Johns Hopkins University) for communicating their results prior to publication.


REFERENCES

  1. Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631-664 [CrossRef][Medline] [Order article via Infotrieve]
  2. Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandes, D. L., Leung, A., Stanworth, D., Rademacher, T. W., Mizuochi, T., Taniguchi, T., Matsuta, K., Takeuchi, F., Nagano, Y., Miyamoto, T., and Kobata, A. (1985) Nature 316, 452-457 [Medline] [Order article via Infotrieve]
  3. Kobata, A. (1979) Anal. Biochem. 100, 1-14 [Medline] [Order article via Infotrieve]
  4. Matzuk, M. M., and Biome, I. (1988) J. Cell Biol. 106, 1049-1059 [Abstract]
  5. Matzuk, M. M., Keene, J. F., and Biome, I. (1989) J. Biol. Chem. 264, 2409-2414 [Abstract/Free Full Text]
  6. Hsieh, P., Rosner, M. R., and Robbins, P. W. (1983) J. Biol. Chem. 258, 2548-2554 [Abstract/Free Full Text]
  7. Shares, B. T., and Robbins, P. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1993-1997 [Abstract]
  8. Goto, M., Akai, K., Murakami, A., Hashimoto, C., Tsuda, E., Ueda, M., Kawanishi, G., Takahashi, N., Ishimoto, A., Chiba, H., and Sasaki, R. (1988) BioTechnology 6, 67-71
  9. Tsuda, E., Goto, M., Murakami, A., Akai, K., Ueda, M., Kawanishi, N., Sasaki, R., Chiba, H., Ishihara, H., Mori, M., Tejima, S., Endo, S., and Arata Y. (1988) Biochemistry 27, 5646-5654 [Medline] [Order article via Infotrieve]
  10. Takegawa, K., Nakoshi, M., Iwahara, S., Yamamoto, K., and Tochikura, T. (1989) Appl. Environ. Microbiol. 55, 3107-3112
  11. Takegawa, K., Yamaguchi, S., Kondo, A., Iwamoto, H., Nakoshi, M., Kato, I., and Iwahara, S. (1991) Biochem. Int. 24, 849-855 [Medline] [Order article via Infotrieve]
  12. Takegawa, K., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1991) Biochem. Int. 25, 829-835 [Medline] [Order article via Infotrieve]
  13. Nilsson, K. G. I. (1988) Trends Biotechnol. 6, 256-264
  14. Cote, G. L., and Tao, B. Y. (1990) Glycoconjugate J. 7, 145-162
  15. Huang, C.-C., Mayer, H. E., Jr., and Montgomery, R. (1970) Carbohydr. Res. 13, 127-137 [CrossRef]
  16. Gray, W. R. (1967) Methods Enzymol. 11, 139-152
  17. Takegawa, K., Mikami, B., Iwahara, S., Morita, Y., Yamamoto, K., and Tochikura, T. (1991) Eur. J. Biochem. 202, 175-180 [Abstract]
  18. Yamamoto, K., Kadowaki, S., Takegawa, K., Kumagai, H., and Tochikura, T. (1986) Agric. Biol. Chem. 50, 421-429
  19. Yamamoto, K., Takegawa, K., Fan, J., Kumagai, H., and Tochikura, T. (1986) J. Ferment. Technol. 64, 397-403
  20. Hase, S., Koyama, H., Daiyasu, H., Takemoto, H., Hara, S., Kobayashi, Y., Kyougoku, Y., and Ikeda, T. (1986) J. Biochem. (Tokyo) 100, 1-10 [Abstract]
  21. Ishihara, H., Takahashi, N., Ito, J., Takeuchi, E., and Tejima, S. (1981) Biochim. Biophys. Acta 669, 261
  22. McReynolds, L., O'Malley, B. W., Nisbet, A. D., Fothergill, J. E., Givol, D., Fields, S., Robertson, M., and Brownlee, G. G. (1978) Nature 273, 723-728 [Medline] [Order article via Infotrieve]
  23. Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Agric. Biol. Chem. 54, 2169-2170 [Medline] [Order article via Infotrieve]
  24. Bruins, A. P., Covey, T. R., and Henion, J. D. (1987) Anal. Chem. 59, 2642-2646
  25. Takegawa, K., Yoshikawa, M., Mishima, T., Nakoshi, M., and Iwahara, S. (1991) Biochem. Int. 25, 585-592 [Medline] [Order article via Infotrieve]
  26. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  27. Takegawa, K., Nakoshi, M., Yamamoto, K., Tochikura, T., and Iwahara, S. (1991) J. Ferment. Bioeng. 71, 278-288
  28. Liang, C.-J., Yamashita, K., and Kobata, A. (1980) J. Biochem. (Tokyo) 88, 51-58 [Abstract]
  29. Mencke, A. J., and Wold, F. (1982) J. Biol. Chem. 257, 14799-14803 [Free Full Text]
  30. Yan, S.-C. B. (1987) Methods Enzymol. 138, 413-418 [Medline] [Order article via Infotrieve]
  31. Trimble, R. B., Atkinson, P. H., Tarentino, A. L., Plummer, T. H., Jr., Maley, F., and Tomer, K. B. (1986) J. Biol. Chem. 261, 12000-12005 [Abstract/Free Full Text]
  32. Elder, J. H., and Alexander, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4540-4544 [Abstract]
  33. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817 [Abstract/Free Full Text]
  34. Bardales, R. M., and Bhavanadan, V. P. (1989) J. Biol. Chem. 264, 19893-19897 [Abstract/Free Full Text]
  35. Ito, S., Muramatsu, T., and Kobata, A. (1975) Arch. Biochem. Biophys. 171, 78-86 [Medline] [Order article via Infotrieve]
  36. Tai, T., Yamashita, K., Ito, S., and Kobata, A. (1977) J. Biol. Chem. 252, 6687-6694 [Medline] [Order article via Infotrieve]
  37. Robbins, P. W., Trimble, R. B., Wirth, D. F., Hering, C., Maley, F., Maley, G. F., Das, R., Gibson, B. W., Royal, N., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583 [Abstract/Free Full Text]
  38. Tarentino, A. L., Quinones, G., Schrader, W. P., Changchien, L.-M., and Plummer, T. H., Jr. (1992) J. Biol. Chem. 267, 3868-3872 [Abstract/Free Full Text]
  39. Tarentino, A. L., Quinones, G., Changchien, L.-M., and Plummer, T. H., Jr. (1993) J. Biol. Chem. 268, 9702-9708 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.