©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis of Neoglycoconjugates by Transglycosylation with Arthrobacter protophormiae Endo--N-acetylglucosaminidase
DEMONSTRATION OF A MACRO-CLUSTER EFFECT FOR MANNOSE-BINDING PROTEINS (*)

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

Jian-Qiang Fan Michael S. Quesenberry Kaoru Takegawa (1) Shojiro Iwahara (1) Akihiro Kondo (2) Ikunoshin Kato (2) 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 and the (2)Biotechnology Research Laboratory, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transglycosylation activity of endo--N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced dramatically by inclusion of organic solvent in the reaction mixture (see accompanying article; Fan, J.-Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C.(1995) J. Biol. Chem. 270, 17723-17729). This finding was extended to synthesis of important intermediates for preparation of neoglycoconjugates. When 0.2 M GlcNAc-O-(CH)NH, GlcNAc-O-CHCH=CH, GlcNAc-O-(CH)CH=CH, GlcNAc-O-(CH)NHCOCH=CH, GlcNAc-S-CHCN, GlcNAc-S-(CH2)CH, or GlcNAc-S-CHCONHCHCH(OMe) were used as acceptors in 30% acetone-containing media, the transglycosylation was accomplished with about 80% yield. The transglycosylation yields to benzyl -GlcNAc (67%), 4-methylumbelliferyl -GlcNAc (66%), p-nitrophenyl -GlcNAc (33%), and (GlcNAc--S-CHCHCH) (43%) were lower, because their poor solubilities allowed only 0.05 M or lower concentrations in the reaction mixture. A micromole-scale synthesis of ManGlcNAc-O-(CH)NHCOCH=CH (ManGlcNAc-NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by H NMR. ManGlcNAc-NAP was co-polymerized with acrylamide. The ratio of sugar side chain to acrylamide in this glycopolymer was 1:44 and the molecular weight of glycopolymer was estimated to be between 1,500,000 and 2,000,000 by high performance gel filtration chromatography. The glycopolymer was shown to be a much more efficient inhibitor of binding by recombinant rat mannose binding protein-carbohydrate recognition domains (MBP-CRD) from serum (I = 3.5 µM ManGlcNAc-sugar chain) and liver (I = 74.5 nM) than soybean agglutinin.


INTRODUCTION

Carbohydrates were found to possess important biological functions, such as cell-cell recognition(1, 2, 3) , lectin binding(4, 5) , and viral infection(6, 7) . Since the studies of the carbohydrate functions require structurally well-defined and highly pure compounds which are usually difficult to obtain from the natural sources, syntheses and constructions of neoglycoconjugates have rapidly gained attention during the past decade(8) . Chemical syntheses of neoglycoconjugates have been aggressively developed, but they usually involve multiple, laborious steps. Especially, synthesis of the high mannose-type oligosaccharides proved to be quite difficult, even with enzymatic methods.

Endo--N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A)()cleaves the glycosidic bond in the core GlcNAc1,4GlcNAc()residues of high mannose-type and hybrid type N-linked sugar chains in glycoprotein(9) . The enzyme was also previously reported to have transglycosylation activity in the presence of GlcNAc and other monosaccharides(10, 11) . In the accompanying article(38) , we found that the transglycosylation activity of endo-A could be enhanced by inclusion of organic solvents, such as acetone, dimethyl sulfoxide, and N,N-dimethyl formamide in the reaction medium. For instance, the transglycosylation could be performed to near completion in 30% acetone-containing medium using ManGlcNAcAsn as donor and GlcNAc as acceptor.

We have taken advantage of this finding, and synthesized several functional intermediates for neoglycoconjugates, one of which was converted into a glycopolymer with pendant ManGlcNAc chains. The glycopolymer thus prepared displays a drastically greater inhibition of binding by mannose-binding protein from liver over the monomer oligosaccharide.


EXPERIMENTAL PROCEDURES

Materials

Endo-A was purified by the reported method(9) . ManGlcNAcAsn was prepared from soybean agglutinin by exhaustive Pronase digestion, followed by gel filtration on Sephadex G-50 and further HPLC purification using a graphitized carbon column(12) . Glycoamidase A was from Seikagaku America, Inc. (Rockville, MD). GlcNAc was purchased from Pfanstiehl Laboratories, Inc. (Waukegan, IL). 3-(N-Acryloylamino)-propyl -D-GlcNAc (GlcNAc-NAP) and GlcNAc-O-(CH)CH=CH were gifts from Dr. Shin-Ichiro Nishimura of Hokkaido University, Japan. Benzyl -GlcNAc, 4-methylumbelliferyl -GlcNAc, p-nitrophenyl -GlcNAc, GlcNAc-O-(CH)NH, GlcNAc-O-CHCH=CH, GlcNAc-O-(CH)NHCOCH=CH, GlcNAc-S-CHCN, GlcNAc-S-(CH)CH, (GlcNAc-S-CHCHCH) and GlcNAc-S-CHCONHCHCH(OMe) were synthesized in this laboratory by previously published methods (13) . Recombinant rat MBP-CRDs from serum and liver were expressed and purified as described (14) using expression plasmid-bearing bacterial strains which are gifts fromDr. Kurt Drickamer of Columbia University.

Methods

Enzymatic Reaction

A typical enzyme reaction for transglycosylation was performed in a mixture of 3 nmol of ManGlcNAcAsn, 4 µmol of acceptor, and 0.9 milliunits of endo-A in a total volume of 20 µl with 25 mM ammonium acetate buffer (pH 6.0) containing 30% acetone. After incubation at 37 °C for 15 min, the reaction was terminated by boiling for 3 min in a water bath. The buffer was removed with a Speedvac using a vacuum pump. The reaction mixture was analyzed using an HPAEC-PAD system (see below).

High Performance Anion Exchange Chromatography (HPAEC)

An HPAEC system which consisted of a Bio-LC (Dionex Corp., Sunnyvale, CA) equipped with a pulsed amperometric detector (PAD-II) was used for analysis of the reaction products. The chromatographic data were analyzed using the AI-450 chromatography software (Dionex). The endo-A reaction products were separated using a Dionex CarboPac PA-1 column (4 250 mm) eluted at a flow rate of 1.0 ml/min with 100 mM sodium hydroxide and a gradient of sodium acetate from 30 to 80 mM developed in 30 min. A cycle of 5-min washing with 100 mM sodium hydroxide, 200 mM sodium acetate followed with a 15-min equilibration period was inserted between runs. The PAD sensitivity was set at 1 K. The quantitative determination of ManGlcNAc and ManGlcNAc was carried out by comparison with standard materials obtained by complete digestion of ManGlcNAcAsn by endo-A and ManGlcNAcAsnPhe by glycoamidase A. The transglycosylation products using acceptors other than GlcNAc were estimated by subtracting the remaining substrate and the hydrolysis product from the starting substrate.

Transglycosylation by Endo-A Using GlcNAc-NAP as Acceptor

A mixture consisting of 5.8 µmol of ManGlcNAcAsn, 2 mmol of GlcNAc-NAP, and 1.1 unit of enzyme in 5 ml of 10 mM NHOAc buffer (pH 6.0) containing 35% of acetone was incubated at 37 °C for 15 min. After stopping the reaction by a 3-min boiling period, the sample was applied to a Sephadex G-25 column (2 140 cm) and eluted with 0.1 M acetic acid. The effluent was monitored by uv absorption at 229 nm, and the neutral sugar was determined by the phenol sulfuric acid method(15) . The fractions containing high molecular weight materials were combined and lyophilized to yield 10.5 mg of white powder.

Preparation of Glycopolymer Having Pendant Chains of High Mannose-type Oligosaccharide

The white powder obtained from gel filtration was used as starting material for polymerization without further purification. A small amount of the white powder (7.2 mg, about 3.25 µmol of ManGlcNAc-NAP) was dissolved in 0.3 ml of HO, followed by deaeration with a water aspirator for 30 min. To the mixture, acrylamide (8.4 mg, 118 µmol), ammonium persulfate (0.14 µmol), and N,N,N`,N`-tetramethylethylenediamine (TEMED, 6.6 µmol) were added, and the mixture was stirred at room temperature for 3 days, during which time, the same amounts of ammonium persulfate and TEMED were added to the reaction mixture daily for 2 days, and the reaction was finally completed by incubation of the mixture at 55 °C for 3 h. The reaction mixture was applied to a column (2.5 90 cm) of Sephadex G-50 and eluted with HO. The fractions containing the glycopolymer were combined and lyophilized to obtain 5.3 mg of white powder.

Estimation of Molecular Weight of the Glycopolymer by HPGFC

The HPGFC was performed with a Gilson HPLC system equipped with a size exclusion column (TSK-Gel G2000SW, 7.5 600 mm, TosoHaas, NJ) and an uv detector (Model V, ISCO). The eluent was 0.1 M phosphate buffer (pH 7.0) containing 0.3 M NaCl and the effluent was monitored at 220 nm. The standard compounds for molecular weight estimation were: (i) blue dextran (M = 2,000,000); (ii) -amylase (M = 200,000); (iii) alcohol dehydrogenase (M = 150,000); (iv) bovine serum albumin (M = 66,000), and (v) carbonic anhydrase (M = 29,000).

MBP Binding of the Glycopolymer

The solid-phase binding studies were carried out essentially as described previously(14) , with some minor modifications as follows. All steps were carried out at 4 °C. Briefly, CRD was coated onto individual polystyrene wells (Immulon 4 Removawell Strips by Dynatech, from Fisher Scientific) which were then blocked with 1% bovine serum albumin in 1.25 M NaCl, 25 mM CaCl, 25 mM Tris (pH 7.8). Ligands and inhibitors were in 0.5% bovine serum albumin in the above Tris buffer for binding and inhibition. The reference ligand used was I-mannose-bovine serum albumin (about 2000 cpm/mg). Approximately 500 cpm/well of reference ligand were incubated for 20 h with or without inhibitors at various concentrations. The well contents were then removed, washed, and counted in a Packard Minaxi -counter. Counts were corrected for background (counts remaining in a blocked well which was not coated with CRD), and the data were analyzed using the program ALLFIT (16) to determine I values using a logistic equation for curve fitting.

H Nuclear Magnetic Resonance Spectroscopy (HNMR)

300 MHz NMR spectra were recorded on a Bruker AMX 300 spectrometer and measurement of a 600 MHz NMR was performed on a Bruker AM-600 spectrometer. The chemical shifts were based on acetone ( = 2.225 ppm) as an internal standard. The samples were prepared by three cycles of dissolving in DO and lyophilizing followed by dissolving the residue in 0.5 ml of high purity DO (99.96% D) immediately before measurement. The 300 MHz data were recorded at 25 °C and the 600 MHz data, at 60 °C.


RESULTS

Transglycosylation of Endo-A to Water-miscible Alcohols

The transglycosylation by endo-A using ManGlcNAcAsn as donor to various water-miscible alcohols was tested. As listed in Table 1, the enzyme transferred oligosaccharide to MeOH and EtOH with 64 and 47% yield, respectively, although the hydrolyses were 33 and 46% in these media. The identity of the transglycosylation products obtained from 30% MeOH or EtOH were confirmed by mass spectrometry and the anomeric configuration of the product with MeOH was found to be by H NMR (data not shown). PrOH (8% yield) and iso-PrOH (10% yield) could also serve as acceptors of transglycosylation, but allyl alcohol could not function as acceptor. The enzyme seemed to be stable in 30% MeOH and EtOH, but unstable in 30% PrOH and allyl alcohol, because the total enzyme activities (combined hydrolysis and transglycosylation activities) in MeOH and EtOH were shown to be similar to that in HO, but much lower in the higher alcohols. Glycerol was found to be as good an acceptor as MeOH or EtOH, and the transglycosylation yield was as high as 57%.



Transglycosylation of Endo-A to Various GlcNAc Glycosides

The transglycosylation of endo-A to some functionalized GlcNAc glycosides was efficient (Table 2). When acceptor concentration was 0.2 M, endo-A transferred ManGlcNAc to GlcNAc-O-(CH)NH (93% of the converted substrate), GlcNAc-O-CHCH=CH (99%), GlcNAc-O-(CH)CH=CH (90%), and GlcNAc-O-(CH)NHCOCH=CH (78%) with yields of 81, 81, 84, and 70% of the starting substrate, respectively. Because of the low solubility, the concentration of benzyl -GlcNAc was used at 0.05 M, and 4-methylumbelliferyl -GlcNAc and p-nitrophenyl -GlcNAc was used under saturating conditions (below 0.05 M). Even at these concentrations, the enzyme could transfer 67, 66, and 33%, respectively, of the starting oligosaccharide chain to them and the transglycosylation indices (the percentage of a transglycosylation product to a digested substrate) were found to be 82, 77, and 42%, respectively. The thioglycosides of GlcNAc are good acceptors for endo-A transglycosylation. When GlcNAc-S-CHCN, GlcNAc-S-(CH)CH, and GlcNAc-S-CHCONHCHCH(OMe) were used as acceptors at 0.2 M, the transglycosylation indices were 88, 86, and 95%, with the yields of 83, 78, and 81%, respectively. A divalent thioglycoside of GlcNAc, (GlcNAc-S-CHCHCH), could be also used as acceptor for endo-A transglycosylation at low concentration (below 0.05 M) with 50% transglycosylation index and 43% yield.



Optimization of the Reaction Conditions for a Larger Scale Transglycosylation by Endo-A

The optimum levels of the enzyme and acetone were examined for the transglycosylation with the substrate at micromole levels. As shown in Fig. 1A, the hydrolytic product increased in proportion to the amount of the enzyme. Yield of the transglycosylation product increased upon addition of the enzyme up to 2.2 milliunits, then decreased as more enzyme was added. When 2.2 milliunits of the enzyme were used, only 5.6% substrate remained. On the other hand, the transglycosylation product increased and the hydrolytic product decreased as the acetone content increased up to 35% (Fig. 1B). In 35% acetone, 86% transglycosylation, and 7% hydrolysis were observed by HPAEC analysis. Although no hydrolytic product was found in the 40% acetone medium, the efficiency of the reaction was lower compared with those in other media, because a greater amount of the substrate (64% of the starting substrate) remained.


Figure 1: Optimization of endo-A transglycosylation conditions. The optimum levels of the enzyme (A) and the acetone content (B) for transglycosylation were determined by the reactions carried out in a mixture of 11.6 nmol of ManGlcNAcAsn (donor), 4 µmol of GlcNAc-NAP (acceptor), and various amounts of the enzyme (A) or 2.2 milliunits of enzyme (B) in 10 µl of 10 mM ammonium acetate buffer (pH 6.0) containing 30% acetone (A) or different concentrations of acetone (B). The reaction mixtures were incubated at 37 °C for 15 min and the products were analyzed with HPAEC-PAD. , substrate; , transglycosylation product; , hydrolytic product.



Synthesis of ManGlcNAc-NAP by Transglycosylation Activity of Endo-A

To prepare ManGlcNAc-NAP in a quantity useful for polymerization, the reaction scale was raised 500-fold over that in the optimum conditions described above. The transglycosylation product, ManGlcNAc-NAP, was more than 90% by HPAEC (Fig. 2), and the hydrolysis product as well as the starting donor substrate was barely detected. The unreacted acceptor was recovered by gel filtration on a Sephadex G-25 column and the ManGlcNAc-NAP was analyzed by H NMR analysis and used for polymerization without further purification.


Figure 2: Synthesis of ManGlcNAc-NAP by endo-A transglycosylation. The reaction was with 5.75 µmol of ManGlcNAcAsn, 2 mmol of GlcNAc-NAP, and 1.1 unit of the enzyme in 5 ml of 10 mM ammonium acetate buffer (pH 6.0) containing 35% acetone at 37 °C for 15 min. After lyophilization, a sample equivalent to 0.7 nmol of ManGlcNAc oligosaccharide was injected into the HPAEC-PAD system for analysis. The elution was performed with 100 mM NaOH and a linear gradient of NaOAc: 0-10% in 20 min. A, transglycosylation product, ManGlcNAc-NAP; B, hydrolytic product, ManGlcNAc; C, remaining substrate, ManGlcNAcAsn.



H NMR was used to identify the transglycosylation product. As shown in Fig. 3A, the signals of the acceptor were completely assigned by the decoupling technique. The H-4 signal of GlcNAc was found at 3.436 ppm and the anomeric proton signal was around 4.495 ppm. On the other hand, the H NMR analysis of the transglycosylation product showed 10 new anomeric proton signals, suggesting that the high mannose-type sugar chain was transferred to the acceptor. The H NMR assignments based on the reference values (17) are listed in Table 3. The anomeric signals agreed with those found from ManGlcNAcAsn, except two GlcNAc anomeric protons which appeared at higher field than those from the reference compound. This is because the linkage between GlcNAc and the aglycon in the former is a N-amide bond, and in the latter, an O-glycosidic bond. The coupling constant of GlcNAc-2 anomeric proton was 7.8 Hz, indicating that the linkage newly formed by endo-A transglycosylation is in the -configuration. The H-4 signal of GlcNAc at the ``reducing end'' at 3.436 ppm could no longer be seen, in agreement with the results with methyl -GlcNAc (38) that the linkage occurs at the 4-OH of the GlcNAc. The mass spectrometry analysis shows the expected molecular weight of the transglycosylation product (data not shown).


Figure 3: H NMR (300 MHz) analysis of GlcNAc-NAP (A) and ManGlcNAc-NAP (B). The labile hydrogens in sample were exchanged with deuterium by repeating a cycle of dissolving in DO followed by lyophilization three times before measurement. The analyses were done in DO using acetone (2.225 ppm) as internal standard at 25 °C.





Polymerization of ManGlcNAc-NAP with Acrylamide

A glycopolymer was obtained from ManGlcNAc-NAP and acrylamide using TEMED and ammonium persulfate as catalysts. The fractions containing the polymer eluted at the void volume of the Sephadex G-50 column (Fig. 4) were pooled and lyophilized. Completion of the polymerization was indicated by H NMR analysis (Fig. 5) which revealed disappearance of the signals at 6.2 and 5.7 ppm, attributable to the double-bond associated protons in the aglycon and the acrylamide monomer. The NMR also showed the existence of 11 anomeric proton signals, and the chemical shifts were similar to those found from the monomer (Table 3), confirming that the polymer contains ManGlcNAc-sugar chains. The sugar content of the polymer was estimated to be 37% by the phenol-HSO method using mannose as standard. Therefore, the ratio of sugar side chains to acrylamide residues is estimated to be 1:44 as shown in Fig. Z1.


Figure 4: Gel filtration of the glycopolymer on Sephadex G-50. The sample (1 ml) was applied onto a Sephadex G-50 column (2.5 90 cm), and eluted with water. The flow rate was approximately 30 ml/h, and 4-ml fractions were collected. The neutral sugar was determined by the phenol-HSO method (dotted line, absorbance at 480 nm), and GlcNAc was monitored by the absorbance at 220 nm (solid line). a, indicates the fractions combined as the glycopolymer.




Figure 5: H NMR (600 MHz) analysis of the glycopolymer. The chemical shifts measured in DO at 60 °C were based on the HDO signal at 4.441 ppm. *, denotes the signals from the polymer back bone.




Figure Z1: Glycopolymer havine ManGlcNAcsugar chain.



Determination of the Molecular Weight of the Glycopolymer

The molecular weight of the glycopolymer was estimated by HPGFC using blue dextran, -amylase, alcohol dehydrogenase, bovine serum albumin, and carbonic anhydrase as reference compounds. The polymer appeared near the void volume, and the retention volume was slightly greater than blue dextran (molecular weight = 2,000,000). According to the calibration curve (Fig. 6), the molecular weight is between 1,500,000 and 2,000,000.


Figure 6: Determination of molecular weight of the glycopolymer by HPGFC. HPGFC was performed with a size exclusion column (7.5 600 mm) and 0.1 M phosphate buffer (pH 7.0) containing 0.3 M NaCl as an eluent at a flow rate of 1.0 ml/min. Effluent was monitored by absorbance at 220 nm. , glycopolymer; , reference compounds; 1, blue dextran (2,000,000); 2, -amylase (200,000); 3, alcohol dehydrogenase (150,000); 4, albumin bovine serum (66,000); and 5, carbonic anhydrase (29,000).



Inhibition Study of Mannose-binding Proteins by the Glycopolymer

A solid-phase binding assay was carried out on serum- and liver-MBP-CRDs, using the ManGlcNAc-glycopolymer and soybean agglutinin (SBA), which contains the same ManGlcNAc. The results of the assay are shown in Fig. 7. In the concentration range of SBA tested, no significant inhibition of the serum-MBP-CRD was observed. For the liver-MBP-CRD, however, I values of 13.2 µM based on ManGlcNAc or 0.4 mg/ml SBA were obtained. However, the glycopolymer showed an I of 3.5 µM for the serum-MBP-CRD, and an I of 74.5 nM for the liver-MBP-CRD. In terms of the whole glycopolymer, the I values would be about 2.0 10 mg/ml for the serum-MBP-CRD and 3.8 10 mg/ml for the liver-MBP-CRD, respectively. The magnitude of inhibitory potency enhancement of the glycopolymer over the precursor cannot be calculated with certainty for the serum form of MBP-CRD, because ManGlcNAc hardly inhibits this MBP-CRD. However, for the liver form, an enhancement was about 180-fold based on the ManGlcNAc, and about 1,000-fold based on the polymer molarity, although the sugar content of the glycopolymer was only 5.6-fold higher than SBA.


Figure 7: Inhibition of binding by serum- and liver-MBP-CRDs by the glycopolymer. The fitted curves were obtained using the program ALLFIT(16) . Concentrations of SBA and glycopolymer are expressed on the bases of ManGlcNAc. , SBA + serum MBP-CRD; , SBA + liver MBP-CRD; , glycopolymer + serum MBP-CRD; , glycopolymer + liver MBP-CRD.




DISCUSSION

We have shown (38) that endo-A possesses an efficient transglycosylation activity (>90%) in 30% acetone, much higher than those reported for 10-30% by other glycosidases(18, 19, 20, 21, 22, 23, 24) . This finding is now utilized to synthesize neoglycoconjugate intermediates which are amenable to further reactions.

We found that endo-A transfers ManGlcNAc to alcohols such as MeOH, EtOH, and PrOH also. The transglycosylation to MeOH (64% yield) and EtOH (47% yield) compares favorably with those by -xylosidase, - and -glucosidase, and -galactosidase (20-60%) from various sources(25, 26) . However, transglycosylation to PrOH and iPrOH were not as effective as to MeOH and EtOH. Interestingly, although the total enzyme activity was lower in PrOH than in iPrOH, transglycosylation to PrOH was greater than to iPrOH. Glycerol was also a good acceptor for endo-A transglycosylation. Endo--N-acetylglucosaminidase F (27) and endo--N-acetylgalactosaminidase from Diplococcus pneumoniae(18) have been reported to transfer an oligosaccharide to the C1(3) hydroxyl of glycerol.

Several GlcNAc derivatives having functionalized aglycon useful for synthesis of neoglycoconjugates were tested as acceptor for endo-A transglycosylation. The yields based on the starting donor substrate were found to be greater than 80% with 0.2 M acceptor and about 50% when 0.05 M or less was used in our system. The yield of transglycosylation can be further improved if higher acceptor concentrations are employed, as shown in the accompanying article(38) .

We have also demonstrated a successful endo-A transglycosylation with 5.8 µmol of donor rather than 3 nmol as shown in Table 2. In the larger scale transglycosylation to GlcNAc-NAP, transglycosylation yield (>90%) was even higher than those at the analytical scale reaction. In the accompanying article(38) , an 89% yield was obtained from the transglycosylation to GlcNAc-OMe at the similar scale (4 µmol).

An endo-A transglycosylation product, ManGlcNAc-NAP, was further polymerized with acrylamide to form a glycopolymer. Glycopolymers having di- or trisaccharide have been synthesized by chemical or chemo-enzymatic methods recently(28, 29, 30, 31, 32, 33) , but we are not aware of any synthesis of glycopolymers with highly complex sugar chains. The high efficiency of endo-A transglycosylation provides an easier way to synthesize such neoglycoconjugates.

Clustering of monosaccharides by attaching them to simple branched peptides enhances inhibitory potencies for some C-type lectins(34, 35) . Formation of glycopolymers is a convenient way to provide glycoside clustering(36) . In the present work, a dramatic increase in the inhibition of MBP-CRDs in comparison with that by the native glycoprotein (SBA) which contains the same ManGlcNAc oligosaccharide was demonstrated. In the case of the liver MBP-CRD, an approximately 180-fold enhancement of inhibitory potency over the native glycoprotein (SBA) was attained by the glycopolymer. Similarly, although no significant inhibition of the serum MBP-CRD was observed for SBA, the glycopolymer derived from its oligosaccharide demonstrated surprisingly strong inhibitory potency (I = 3.5 µM). This is a good example of ``macro-'' versus ``micro-clustering''(37) .


FOOTNOTES

*
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. Tel.: 410-516-7322; Fax: 410-516-8716.

The abbreviations used are: endo-A, endo--N-acetyl-D-glucosaminidase from Arthrobacter protophormiae; GlcNAc, N-acetyl-D-glucosamine; Man, mannose; NAP, 3-(N-acryloylamino)-propyl; MBP, mannose-binding protein; CRD, carbohydrate recognition domain; SBA, soybean agglutinin; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detector; HPLC, high performance liquid chromatography; HPGFC, high performance gel filtration chromatography; H NMR, H nuclear magnetic resonance spectroscopy; TEMED, N,N,N`,N`-tetramethylethylenediamine.

All monosaccharides used are of the D-configuration.


ACKNOWLEDGEMENTS

We thank Dr. Shin-Ichiro Nishimura of Hohhaido University, Japan, for providing GlcNAc-NAP and GlcNAc-O-(CH)CH=CH as well as valuable discussions and suggestions. We also thank Dr. Reiko T. Lee for soybean agglutinin and Dr. Kurt Drickamer of Columbia University for the gift of bacterial strains to produce MBP-CRDs. We are grateful to Koji Matsuoka and Dr. C. Abeygunawardana of The Johns Hopkins School of Medicine for H NMR analyses.


REFERENCES

  1. Wassarman, P. M. (1991)Development 108, 1-17 [Abstract]
  2. Patankar, M. S., Oehninger, S., Barnett, T., Williams, R. L., and Clark, G. F. (1993)J. Biol. Chem. 268, 21770-21776 [Abstract/Free Full Text]
  3. Lasky, L. A.(1992) Science258,964-969 [Medline] [Order article via Infotrieve]
  4. Lee, Y. C. (1988) in The Molecular Immunology of Complex Carbohydrates (Wu, A. M., ed) pp. 105-121, Series Plenum Publishing Corp., New York
  5. Lee, Y. C., Lee, R. T., Rice, K., Ichikawa, Y., and Wong, T.-C.(1991)Pure & Appl. Chem. 63, 499-506
  6. Glick, G. D., Toogood, P. L., Wiley, D. C., Skehel, J. J., and Knowles, J. R.(1991) J. Biol. Chem. 266, 23660-23669 [Abstract/Free Full Text]
  7. Toogood, P. L., Galliker, P. K., Glick, G. D., and Knowles, J. R.(1991)J. Med. Chem.34,3140-3143 [Medline] [Order article via Infotrieve]
  8. Lee, Y. C. (1994) in Neoglycoconjugates: Preparation and Applications (Lee, Y. C., and Lee, R. T., eds) pp. 3-21, Academic Press, San Diego
  9. Takegawa, K., Nakoshi, M., Iwahara, S., Yamamoto, K., and Tochikura, T.(1989) Appl. Environ. Microbiol. 55, 3107-3112
  10. 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]
  11. Takegawa, K., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S.(1991) Biochem. Int. 25, 829-835 [Medline] [Order article via Infotrieve]
  12. Fan, J.-Q., Kondo, A., Kato, I., and Lee, Y. C.(1994)Anal. Biochem. 219,224-229 [CrossRef][Medline] [Order article via Infotrieve]
  13. Lee, Y. C., and Lee, R. T. (1992) in Glycoconjugates: Composition, Structure, and Function (Allen, H. J., and Kisalius, E. C., eds) pp. 121-165, Marcel Dekker, Inc., New York
  14. Quesenberry, M. S., and Drickamer, K.(1992)J. Biol. Chem. 267, 10831-10841 [Abstract/Free Full Text]
  15. McKelvy, J. F., and Lee, Y. C.(1969)Arch. Biochem. Biophys. 132, 99-110 [Medline] [Order article via Infotrieve]
  16. De Lean, A., Munson, P. J., and Rodbard, D.(1978)Am. J. Physiol. 235,E97-E102
  17. Vliegenthart, J. F. G., Dorland, L., and van Halbeek, H.(1983)Adv. Carbohydr. Chem. Biochem. 41, 209-373
  18. Bardales, R. M., and Bhavanandan, V. P.(1989)J. Biol. Chem. 264, 19893-19897 [Abstract/Free Full Text]
  19. Sakai, K., Katsumi, R., Ohi, H., Usui, T., and Ishido, Y.(1992)J. Carbohydr. Chem. 11, 553-565
  20. Cantacuzene, D., Attal, S., and Bay, S.(1991)Biomed. Biochim. Acta 50,S231-S236
  21. Nilsson, K. G. I. (1987)Carbohydr. Res. 167, 95-103 [CrossRef][Medline] [Order article via Infotrieve]
  22. Nilsson, K. G. I. (1989)Carbohydr. Res.188,9-17 [CrossRef][Medline] [Order article via Infotrieve]
  23. Usui, T., and Murata, T. (1988)J. Biochem.103,969-972 [Abstract]
  24. Usui, T., Suzuki, M., Sato, T., Kawagishi, H., Adachi, K., and Sano, H.(1994) Glycoconjugate J. 11, 105-110 [Medline] [Order article via Infotrieve]
  25. Shinoyama, H., Kamiyama, Y., and Yasui, T.(1988)Agric. Biol. Chem. 52, 2197-2202
  26. Shinoyama, H., and Yasui, T.(1988)Agric. Biol. Chem. 52, 2375-2377
  27. 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]
  28. Kochetkov, N. K. (1984)Pure & Appl. Chem.56,923-938
  29. Nishimura, S., Matsuoka, K., Furuike, T., Ishii, S., Kurita, K., and Nishimura, K. M. (1991)Macromolecules 24, 4236-4241
  30. Nishimura, S., Matsuoka, K., Furuike, T., Nishi, N., Tokura, S., Nagami, K., Murayama, S., and Kurita, K.(1994)Macromolecules 27, 157-163
  31. Nishimura, S., Furuike, T., Matsuoka, K., Murayama, S., Nagata, K., Kurita, K., Nishi, N., and Tokura, S.(1994)Macromolecules 27, 4876-4880
  32. Kobayashi, K., Kakishita, N., Okada, M., Akaike, T., and Usui, T.(1994) J. Carbohydr. Chem. 13, 753-766
  33. Fukase, K., Nakayama, H., Kurosawa, M., Ikegaki, T., Kanoh, T., Hase, S., and Kusumoto, S. (1994)J. Carbohydr. Chem. 13, 715-736
  34. Lee, R. T., and Lee, Y. C.(1987)Methods Enzymol. 138, 424-429 [Medline] [Order article via Infotrieve]
  35. Lee, R. T., Ichikawa, Y., Kawasaki, T., Drickamer, K., and Lee, Y. C.(1992) Arch. Biochem. Biophys.299,129-136 [Medline] [Order article via Infotrieve]
  36. Lee, R. T., and Lee, Y. C. (1994) in Neoglycoconjugates: Preparation and Applications (Lee, Y. C., and Lee, R. T., eds) pp. 23-50, Academic Press, San Diego
  37. Lee, Y. C. (1993)Biochem. Soc. Trans. 21, 460-463 [Medline] [Order article via Infotrieve]
  38. Fan, J.-Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C.(1995)J. Biol. Chem.270,17723-17729 [Abstract/Free Full Text]

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