©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enhanced Transglycosylation Activity of Arthrobacter protophormiae Endo--N-acetylglucosaminidase in Media Containing Organic Solvents (*)

(Received for publication, March 29, 1995; and in revised form, May 25, 1995)

Jian-Qiang Fan Kaoru Takegawa (1) Shojiro Iwahara (1) Akihiro Kondo (2) Ikunoshin Kato (2) Chitrananda Abeygunawardana (3) Yuan C. Lee (§)

From the  (1)Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, the Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Japan, the (2)Biotechnology Research Laboratory, Takara Shuzo Co., Ltd., Ohtsu, Shiga 520-21, Japan, and the (3)Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transglycosylation activity of endo--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 ManGlcNAcAsn 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 ManGlcNAcAsn 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.


INTRODUCTION

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--N-acetylgalactosaminidase from Diplococcus pneumoniae was shown to have both activities, and can transfer a disaccharide, Gal13GalNAc,()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.

Endo--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) .

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.


EXPERIMENTAL PROCEDURES

Materials

Endo-A was isolated by the published report(27) . ManGlcNAcAsn and ManGlcNAcAsnPhe 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) . ManGlcNAcAsn 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 ManGlcNAcAsn 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.

In order to prepare the samples for mass spectrometry, 400 nmol of ManGlcNAcAsn 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 ManGlcNAc, ManGlcNAc, and ManGlcNAcAsn were eluted at around 20-25 min and were separated from each other. The quantitative determination of ManGlcNAc and ManGlcNAc was carried out by comparison of the peak areas with those of standards obtained by complete digestion of known quantities of ManGlcNAcAsn by endo-A and ManGlcNAcAsnPhe 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 CHCN (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 ManGlcNAc-OMe by Transglycosylation of Endo-A

To obtain sufficient transglycosylation product for H NMR analysis, a mixture containing 4 µmol of ManGlcNAcAsn, 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 CHCN at 50 °C. The final yield of the transglycosylation product was 74.9%.

H Nuclear Magnetic Resonance Spectroscopy

Spectra were recorded on a Bruker AM-600 spectrometer. The observed 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 DO and lyophilizing followed by finally dissolving in 0.5 ml of high purity DO (99.96 atom %D) before measurement.

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). 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.

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 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.


RESULTS

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, ManGlcNAcAsn, decreased quickly, and totally disappeared after 15 min. The transfer product, ManGlcNAc, 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, ManGlcNAc, slowly increased during the reaction, finally reaching 89% after 60 min.


Figure 1: Time course of endo-A action in aqueous solution. The enzymatic reactions were performed in a mixture of 15 nmol of ManGlcNAcAsn, 10 µmol of GlcNAc, and 2.4 milliunits of endo-A in a total volume of 20 µl with 50 mM NHOAc 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 CHCN- 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 CHCN- and tetrahydrofuran-containing media.



The transglycosylation activity of endo-A in media containing acetone, MeSO, 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.


Figure 2: Effect of organic solvents on the transglycosylation activity of endo-A. The reaction mixtures contained 3 nmol of ManGlcNAcAsn 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), MeSO (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.



In MeSO-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 MeSO as in acetone. In fact, 40% MeSO was required to suppress the hydrolytic activity to 2.6%, a comparable level attained with 30% acetone.

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 MeSO-containing media.



Stability of Endo-A in Acetone, MeSO, and DMF Containing Media

The stability of endo-A in acetone-, MeSO-, 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% MeSO, but was unstable at MeSO concentrations greater than 40%. The enzyme was slightly more stable in MeSO-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.


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), MeSO (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 ManGlcNAcAsn and 25 mM NHOAc 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.


Figure 4: Effect of GlcNAc concentration on transglycosylation activity of endo-A. The reactions were carried out with 3 nmol of ManGlcNAcAsn, 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 ManGlcNAcAsn 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.

The molecular weight of the transglycosylation product was determined by electrospray mass spectrometry analysis as 1249 (M + H) 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, ManGlcNAc-OMe.


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 CHCN, 2 mM NHOAc. 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 ManGlcNAc--GlcNAc--OMe.


Figure 6: 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.





In order to study linkage mode between the two GlcNAc residues, the 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 GlcNAc1,4GlcNAc which exists in the natural ManGlcNAcAsn structure.

Transglycosylation to Monosaccharides and Their Derivatives

The conditions used for the reaction were the same as described under ``Experimental Procedures'' except: (i) ManGlcNAcAsnPhe was used as substrate for the transglycosylation to Man, because of its ease of separation from the products compared with ManGlcNAcAsn 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.



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 -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.

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) and 1829.8 (M + NH) (Fig. 7A) which are expected from ManGlcNAcXyl. The intense peaks of m/z 1680.8 and 1488.8 were due to loss of xylose (i.e. ManGlcNAc) or two Man's (i.e. ManGlcNAcXyl) from the parent compound. In addition, a series of sequence ions showed the loss of Man residues from the ManGlcNAc or ManGlcNAcXyl. 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 ManGlcNAcXyl and ManGlcNAc(3-O-Me-Glc) as expected.


Figure 7: Ion-spray mass spectrum of ManGlcNAcXyl (A) and ManGlcNAc(3-O-Me-Glc) (B). The samples used were about 50 pmol, and infusion rate was 3 µl/min in 1:1 CHCN, 2 mM NHOAc. Orifice voltages were 105 V (A) and 85 V (B). M, Man; GN, GlcNAc; X, xylose; MG, 3-O-Me-Glc.




DISCUSSION

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). MeSO 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.


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 MeSO 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 MeSO 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 MeSO- and DMF-containing solvents better than acetone.

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 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 GlcNAc1,4GlcNAc 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.

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) ).


FOOTNOTES

*
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§
To whom correspondence should be addressed. Tel.: 410-516-7322; Fax: 410-516-8716.

All monosaccharides used in this paper are of the D-configuration.

The abbreviations used are: endo-A, endo--N-acetylglucosaminidase from A. protophormiae; MeSO, 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.


ACKNOWLEDGEMENTS

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 ManGlcNAcAsn. We are also grateful to Dr. Laixi Wang for 300 MHz H NMR analysis of methyl -D-GlcNAc.


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