Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shao, J. Articles by Wang, P. G. Search for Related Content PubMed PubMed Citation Articles by Shao, J. Articles by Wang, P. G. Agricola Articles by Shao, J. Articles by Wang, P. G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2002, p. 5634-5640, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5634-5640.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Jun Shao, Jianbo Zhang, Przemyslaw Kowal, and Peng George Wang^*

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

Received 11 April 2002/ Accepted 17 July 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcß13Gal14Galß14Glc) and isoglobotetraose (GalNAcß13Gal13Galß14Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant ß-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The P blood group antigens are glycan structures displayed by membrane-associated glycosphingolipids on red blood cells and other tissues, such as the urothelium. Three distinct serologic specificities, P, P₁, and P^K, have been described, which differ from each other solely in oligosaccharide content. The P antigen, referred to as globoside, was one of the earliest examples of a recognition site sensitive to structural perturbation (14). This amphipathic molecule consists of an outer globotetraose (GalNAcß13Gal14Galß14Glc) portion attached to an inner lipid called ceramide (1, 23). Isogloboside, which is also widely distributed on human blood cell surfaces, has an isoglobotetraose (GalNAcß13Gal13Galß14Glc) unit instead. Evidence has shown that these glycolipids play significant roles as receptors in adhesion of human embryonic carcinoma cells (36), pathogenesis of urinary tract infections (17, 37), and human parvovirus B19 infection (4, 15). The globo series oligosaccharides, which are found in the lipopolysaccharide structures of some pathogenic bacteria, such as Haemophilus influenzae (11) and Neisseria gonorrhoeae (8), also participate in the invasion of mammalian cells by these pathogens. Therefore, efficient synthesis of globotetraose and isoglobotetraose and their analogues as potential inhibitors of these cell-cell interactions is of growing interest for experimental and therapeutic applications.

The enzymatic synthesis of globotetraose with N-acetylgalactosaminyltransferase and the corresponding sugar nucleotide, UDP-N-acetylgalactosamine (UDP-GalNAc), has been described previously (13). However, scale-up of this process is still limited by the high cost of UDP-GalNAc and inhibition by UDP. One way to overcome these drawbacks is to use a multiple-enzyme system with in situ UDP-GalNAc regeneration from inexpensive starting materials. Since the pioneering work of Wong et al. (40) on the synthesis of N-acetyllactosamine with regeneration of UDP-galactose, several glycosylation cycles with regeneration of sugar nucleotides have been developed by using either native or recombinant enzymes. For example, UDP-glucose regeneration was used in the synthesis of trehalose (9) and N-acetyllactosamine (41), a UDP-galactose regeneration cycle was used to synthesize an -Gal epitope (7, 10), and a 6'-sialyl-N-acetyllactosamine epitope was synthesized by combining UDP-galactose and CMP-N-acetylneuraminic acid recycling systems (12).

Previously, we have described overexpression and biochemical characterization of the N-acetylgalactosaminyltransferase LgtD from H. influenzae strain Rd (33). This enzyme can catalyze introduction of an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc into an acceptor substrate in a ß(13) linkage (Fig. 1). In this study, we explored the acceptor substrate specificity of this enzyme and examined the multiple-enzyme synthesis of globotetraose and isoglobotetraose with in situ regeneration of the donor substrate UDP-GalNAc. In our system, phosphoglucosamine mutase (GlmM in Escherichia coli K-12) first converts glucosamine 6-phosphate into glucosamine 1-phosphate. Then the bifunctional UDP-N-acetylglucosamine pyrophosphorylase (GlmU in E. coli K-12) catalyzes both the acetylation of glucosamine 1-phosphate by acetyl coenzyme A (acetyl-CoA) and the uridylation of N-acetylglucosamine 1-phosphate to form UDP-N-acetylglucosamine (UDP-GlcNAc). The resulting CoA is reacetylated by phosphate acetyltransferase (PTA in E. coli K-12) with the consumption of one equivalent of acetyl phosphate. The by-product pyrophosphate is hydrolyzed by inorganic pyrophosphatase (PPA in E. coli K-12) as a driving force. After the epimerization of UDP-GlcNAc by UDP-N-acetylglucosamine C4 epimerase (WbgU) from Plesiomonas shigelloides (Kowal, unpublished data), the N-acetylgalactosaminyltransferase transfers the GalNAc residue from UDP-GalNAc to the acceptor substrates. The resulting UDP is then phosphorylated to UTP by pyruvate kinase (PykF in E. coli K-12) with the consumption of one equivalent of phosphoenolpyruvate. Overall, production of one equivalent of globotetraose or isoglobotetraose requires one equivalent each of glucosamine 6-phosphate, acetylphosphate, phosphoenolpyruvate, and acceptor substrate (globotriose [Gal14Galß14Glc] or isoglobotriose [Gal13Galß14Glc]) (Fig. 2).

View larger version (23K): [in this window] [in a new window]

FIG. 1. Formation of glycosidic bond catalyzed by ß-1,3-N-acetylgalactosaminyltransferase from H. influenzae.

View larger version (16K): [in this window] [in a new window]

FIG. 2. Multiple-enzyme system for in vitro synthesis of globotetraose and isoglobotetraose. Ac-CoA, acetyl-CoA; Ac-P, acetylphosphate; PEP, phosphoenolpyruvate; Gb3, globotriose; Iso-Gb3, isoglobotriose; Gb4, globotetraose; Iso-Gb4, isoglobotetraose.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
Restriction enzymes and T4 DNA ligase were obtained from Promega (Madison, Wis.). Vent DNA polymerase was purchased from New England Biolabs (Beverly, Mass.). Nickel-nitrilotriacetic acid (Ni-NTA) agarose, a PCR purification kit, a QIAEX II gel extraction kit, and a DNA miniprep spin kit were obtained from Qiagen (Santa Clarita, Calif.). A low-range protein standard was obtained from Bio-Rad Laboratories (Hercules, Calif.). UDP-D-[1-³H(N)]GalNAc, acetyl-CoA (acetyl-³H), isopropyl-1-ß-D-thiogalactopyranoside (IPTG), and DOWEX 1x8 resin were obtained from Sigma Chemical Co. (St. Louis, Mo.). [³H]UTP was obtained from Amersham Pharmacia Biotech. Other reagents were analytical or higher grade. All kits or enzymes were used according to the manufacturer's instructions.

Bacterial strains and plasmids.
E. coli competent strain DH5 (lacZM15 hsdR recA) was obtained from Gibco-BRL Life Technology (Carlsbad, Calif.). Plasmid vector pET15b and E. coli competent strain BL2(DE3) [F^- ompT hsdS_B (r_B^- m_B^-) gal dcm (DE3)] were obtained from Novagen Inc. (Madison, Wis.). E. coli K-12 substrain MG1655, P. shigelloides O17 strain C27, and H. influenzae type d strain RM118 (KW-20) chromosomal DNA (ATCC 51907D) were purchased from the American Type Culture Collection (Manassas, Va.).

Cloning, expression, and purification of individual enzymes.
The glmM, glmU, pta, pykF, and ppa genes were amplified by PCR from the E. coli K-12 substrain MG1655 chromosome. The wbgU gene and the lgtD gene were amplified from the P. shigelloides O17 strain C27 and H. influenzae type d strain RM118 (KW-20) chromosomes, respectively. Each PCR mixture (total volume, 50 µl) consisted of 50 ng of genomic DNA, each primer at a concentration of 200 nM, each deoxynucleoside triphosphate at a concentration of 0.2 mM, 15 mM MgCl₂, 0.5 U of Vent DNA polymerase, and 1x buffer. After the mixture was heated at 94°C for 2 min, 30 cycles consisting of 30 s at 94°C, 60 s at 55°C, and 90 s at 72°C were performed, followed by a final 10 min of elongation at 72°C. The DNA fragments obtained were digested with proper restriction enzymes and inserted into the pET15b vector. The resulting plasmids were subsequently transformed into the cloning host strain E. coli DH5 and then into expression strain BL21(DE3) with ampicillin (100 µg/ml) selection. Selected clones were characterized by restriction mapping.

For protein expression, E. coli BL21(DE3) isolates harboring the recombinant plasmids were grown in Luria-Bertani medium at 37°C. When the A₆₀₀ reached 0.8, IPTG was added to a final concentration of 0.4 mM, and expression was allowed to proceed for 8 h at 30°C. Cells were harvested by centrifugation (4,000 x g, 20 min, 4°C) and washed twice with Tris-HCl (50 mM, pH 7.0). The cell pellet was resuspended in chilled lysis buffer (50 mM Tris-HCl [pH 8.0], 0.1 M NaCl, 0.5% [wt/vol] Triton X-100, 10% [wt/vol] glycerin, 10 mM 2-mercaptoethanol) and disrupted by brief sonication (Branson Sonifier 450; VWR Scientific) on ice. The lysate was cleared by centrifugation (12,000 x g, 20 min, 4°C) and loaded at flow rate of 2 ml/min onto a Ni-NTA affinity column. After washing and elution, the fractions containing the purified enzyme were combined and dialyzed (50 mM Tris-HCl [pH 7.0], 10% [wt/vol] glycerin, 10 mM 2-mercaptoethanol) for use in activity assays and enzymatic reactions.

Enzyme assays.
In this study, 1 U of enzyme activity was defined as the amount of enzyme that produced 1 µmol of product per min at 30°C. The activity assays for the recombinant enzymes are briefly described below.

(i) N-Acetylgalactosaminyltransferase.
The N-acetylgalactosaminyltransferase reactions were performed at 30°C for 20 min in 100-µl (final volume) mixtures containing 50 mM Tris-HCl (pH 7.5), 10 mM MnCl₂, 0.1% bovine serum albumin, 1 mM dithiothreitol, 0.3 mM UDP-D-[1-³H]GalNAc (final specific activity, 1,000 cpm/nmol), 3 mM globotriose acceptor, and various amounts of purified enzyme. The acceptor was omitted in blank experiments. The reaction was terminated by adding 100 µl of ice-cold 0.1 M EDTA. Dowex 1x8-200 chloride anion-exchange resin was then added in a water suspension (0.8 ml, 1:1 [vol/vol]). After centrifugation at 10,000 x g for 2 min, supernatant (0.5 ml) was collected in a 20-ml plastic vial, and 5 ml of ScintiVerse BD was added. The vial was vortexed thoroughly before the radioactivity of the mixture was counted with a liquid scintillation counter (Beckman LS-3801).

(ii) UDP-N-acetylglucosamine C4 epimerase.
For the UDP-N-acetylglucosamine C4 epimerase reactions the 50-µl (total volume) reaction mixtures consisted of 1 mM UDP-GlcNAc, 20 mM Tris-HCl (pH 8.0), and various amounts of enzyme. The reaction mixtures were incubated at 30°C for 15 min, and the reactions were quenched by placing the mixtures in boiling water for 5 min. Samples were analyzed by capillary electrophoresis (ISCO model 3850 capillary electropherograph). A 57-cm-long bare silica column with an inside diameter of 75 µm and a window at 50 cm was used. Samples were vacuum injected for 4 s and electrophoresed at 22 kV with UV detection at 262 nm.

(iii) UDP-N-acetylglucosamine pyrophosphorylase.
The UDP-N-acetylglucosamine pyrophosphorylase activity was measured in the forward direction (formation of UDP-GlcNAc from GlcNAc-1-phosphate and UTP). Each reaction mixture contained the following components in a final volume of 100 µl: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 0.5 mM [³H]UTP, 5 mM GlcNAc-1-phosphate, and purified enzyme. After 10 min of incubation at 30°C, the reaction was terminated by boiling the mixture for 1 min, and the mixture was applied to a column of DE-52. UDP-GlcNAc was eluted with 70 mM (NH₄)HCO₃ and could be readily separated from UTP, which required a much higher concentration of bicarbonate for elution. The radioactivity of the 70 mM elution solution was counted with a liquid scintillation counter (Beckman LS-3801).

(iv) Phosphoglucosamine mutase.
The phosphoglucosamine mutase activity was measured by the method of Mengin-Lecreulx and van Heijenoort (21). The standard assay mixture (final volume, 100 µl) contained 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl₂, 1 mM glucosamine 6-phosphate, 0.4 mM [³H]acetyl-CoA, 5 mM UTP, 0.1 mM glucose 1,6-diphosphate, purified UDP-N-acetylglucosamine pyrophosphorylase, and phosphoglucosamine mutase. The mixtures were incubated at 30°C for 30 min, and the reactions were terminated by addition of 10 µl of acetic acid. Radioactive spots of acetyl-CoA and UDP-GlcNAc were separated by high-voltage electrophoresis on Whatman no. 3MM filter paper in 2% formic acid (pH 1.9) and were located by overnight autoradiography. They were cut out and collected in 20-ml plastic vials, and then 5 ml of ScintiVerse BD was added. The vials were vortexed thoroughly before the radioactivity of the mixture was counted with a liquid scintillation counter (Beckman LS-3801).

(v) Phosphate acetyltransferase.
The phosphate acetyltransferase activity was measured by a modification of an assay described by Whiteley and Pelroy (39) and Brown et al. (5), in which citrate synthase is used. The standard reaction mixture (500 µl) contained 25 mM Tris-HCl (pH 8.3), 2 mM dithiothreitol, 20 mM KCl, 4 mM acetylphosphate, 0.3 mM CoA, 5 mM NAD, 5 mM malic acid, 2 U of citrate synthase, and 3 U of malic dehydrogenase. Phosphate acetyltransferase activity was quantified by measuring the change in absorbance at 340 nm due to the formation of NADH.

(vi) Inorganic pyrophosphatase.
Inorganic pyrophosphatase catalyzes the hydrolysis of pyrophosphate. A color reagent consisting of 0.045% malachite green base and 4.2% ammonium molybdate in 4 M HCl (3:1) was used to quantitatively determine pyrophosphatase activity as described by Rumsfeld et al. (30). The assay was carried out by using a reaction mixture (300 µl) containing 50 mM Tris-HCl (pH 7.5), 3 mM MgCl₂, 160 µM sodium pyrophosphate, and purified inorganic pyrophosphatase. After incubation for 30 min at 30°C, the color reagent (600 µl) was added, and the mixture was incubated at room temperature for another 10 min. The absorbance was measured at 620 nm.

(vii) Pyruvate kinase.
The pyruvate kinase assay was carried out in a cuvette by using a 1-ml solution containing 0.1 M Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.1 M KCl, 10 mM MgCl₂, 0.2 mM NADH, 1.5 mM ADP, 60 mU of lactate dehydrogenase, and 5 mM phosphoenolpyruvate. A control contained water instead of ADP. The reaction mixtures were incubated at 30°C, and the absorbance at 340 nm was monitored.

General procedure for multiple-enzyme synthesis.
The reaction mixtures (200 ml) contained glucosamine 6-phosphate (4 mmol, 20 mM), acetylphosphate (4.4 mmol, 22 mM), CoA (0.1 mmol, 0.5 mM), glucose 1,6-diphosphate (10 µmol, 50 µM), UDP (0.1 mmol, 0.5 mM), phosphoenolpyruvate (4.4 mmol, 22 mM), KCl (50 mM), MgCl₂ (20 mM), MnCl₂ (10 mM), and bovine serum albumin (0.1%) in Tris-HCl buffer (100 mM, pH 7.5), as well as different acceptor substrates (globotriose or isoglobotriose [4 mmol, 20 mM]). The accepting substrates globotriose and isoglobotriose were prepared by galactosylation of lactose with the -1,4-galactosyltransferase from Neisseria meningitidis (42) and the -1,3-galactosyltransferase from cattle (7), respectively. All reactions were initiated by addition of ß-1,3-N-acetylgalactosaminyltransferase (5 U), UDP-N-acetylglucosamine C4 epimerase (5 U), UDP-N-acetylglucosamine pyrophosphorylase (10 U), phosphoglucosamine mutase (5 U), phosphate acetyltransferase (5 U), pyruvate kinase (10 U), and inorganic pyrophosphatase (10 U). The reaction mixtures were incubated at 25°C for 32 h. The progress of the reactions was monitored by thin-layer chromatography (2-propanol-H₂O-NH₄OH, 7:3:2 [vol/vol/vol]) performed on Baker Si250F silica gel thin-layer chromatography plates with a fluorescent indicator and by high-performance liquid chromatography (HPLC) performed with a MICROSORB NH₂ 100-Å column (4.6 by 250 mm; Varian Inc., Palo Alto, Calif.) on which the oligosaccharide peaks were recorded with a Star 9040 refractive index detector (Varian Inc.). Protein was removed by brief boiling followed by centrifugation (12,000 x g, 10 min). Then the mixture was passed through Dowex 1x8 (Cl^- form) anion-exchange resin. Oligosaccharide products were purified further by gel permeation chromatography (Bio-Gel P2; Bio-Rad) by using double-distilled water as the mobile phase. The desired fractions were pooled, lyophilized, and stored at -20°C.

Structural analysis of oligosaccharide products.
¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained by using a 400-MHz Varian Mercury-400 NMR spectrometer or a 500-MHz Varian Unity-500 NMR spectrometer with deuterated water as the solvent. Electrospray ionization mass spectrometry (ESI-MS) was performed at the mass spectrometry facility of Wayne State University. The following data were obtained.

Globotetraose (2.5 g, 89%), ß-D-N-acetylgalactosaminyl-(13)--D-galactopyranosyl-(14)-ß-D-galactopyranosyl-(14)-D-glucopyranose. ¹H NMR (500 MHz, D₂O): 5.06 (d, 0.4H, J_1,2 = 2.5 Hz, H-1), 4.73 (d, 1H, J_1",2" = 3.0 Hz, H-1"), 4.49 (d, 0.6H, J_1,2 = 7.5 Hz, H-1ß), 4.45 (d, 1H, J_1",2" = 8.5 Hz, H-1'''), 4.34 (d, 1H, J_1',2' = 7.5 Hz, H-1'), 4.21 (m, 1H), 4.08 (m, 1H) 3.86 (m, 1 H), 3.39-3.80 (m, 15.4H), 3.10 (t, 0.6H, J_1,2 = 8.5 Hz, H-2ß), 1.86 (s, 3H, NHAc). ¹³C NMR (125 MHz, D₂O): 175.37, 103.44, 100.56, 95.86, 91.94, 78.97, 77.36, 75.60, 75.08, 74.60, 74.07, 72.26, 71.63, 71.37, 71.05, 70.42, 69.11, 67.91, 67.79, 61.15, 60.53, 60.21, 52.74, 22.40. ESI-MS: 708.00 (M + H)⁺, 724.97 (M + NH₄)⁺, 729.95 (M + Na)⁺, 745.89 (M + K)⁺; 705.83 (M - H)^-.

Isoglobotetraose (2.2 g, 78%), ß-D-N-acetylgalactosaminyl-(13)--D-galactopyranosyl-(13)-ß-D-galactopyranosyl-(14)-D-glucopyranose. ¹H NMR (500 MHz, D₂O): 5.03 (d, 0.4H, J_1,2 = 3.6 Hz, H-1), 4.91 (d, 1H, J_1",2" = 4.1 Hz, H-1"), 4.47 (d, 0.6H, J_1,2 = 8.1 Hz, H-1ß), 4.44 (d, 1H, J_1''',2''' = 8.1 Hz, H-1'''), 4.33 (d, 1H, J_1',2' = 8.1 Hz, H-1'), 3.98 4.06 (m, 3H), 3.38-3.86 (m, 20.4H), 3.10 (t, 0.6H, J_1,2 = J_2,3 = 8.1 Hz, H-2ß), 1.84 (s, 3H, NHAc). ¹³C NMR (125 MHz, D₂O): 175.31, 103.31, 102.88, 95.86, 95.75, 78.94, 78.50, 77.26, 75.13, 75.07, 74.88, 74.53, 73.87, 70.89, 70.47, 69.73, 69.16, 67.84, 67.39, 64.97, 61.11, 61.07, 60.89, 52.74, 22.36. ESI-MS: 730.06 (M + Na)⁺, 1437.32 (2 M + Na)⁺; 706.14 (M - H)^-.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation and characterization of individual enzymes.
The genes encoding the enzymes in the synthetic pathway were amplified by PCR and cloned into the pET15b vector. The primers used for cloning individual genes are listed in Table 1. All recombinant proteins were expressed at 30°C in E. coli BL21(DE3) with six-His tags. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that most of the proteins were in a soluble form (Fig. 3); GlmM, GlmU, PTA, PPA, and PykF represented more than 80% of the soluble protein in the cell lysate, while WbgU and LgtD represented more than 20 and 30% of the soluble protein, respectively. Compared with the wild-type strains, overexpression of the related genes significantly (30- to 780-fold) increased the activity of individual enzymes in host cells (Table 2). The recombinant enzymes were easily purified up to 90% by one-step Ni-NTA affinity chromatography before dialysis for enzymatic reactions.

View this table: [in this window] [in a new window]

TABLE 1. Oligonucleotide primers used for cloning of individual genes into the pET15b vector

View larger version (89K): [in this window] [in a new window]

FIG. 3. SDS-PAGE analysis of expression and purification of the enzymes. Cell lysates (10 µl) were subjected to SDS-12% PAGE in a Mini Protein III cell gel electrophoresis unit (Bio-Rad) at 200 V (direct current). Detection was performed with Coomassie brilliant blue. Lanes 1 and 10, low-range molecular weight markers (Bio-Rad); lane 2, cell lysate containing GlmM; lane 3, cell lysate containing PTA; lane 4, cell lysate containing GlmU; lane 5, cell lysate containing PPA; lane 6, cell lysate containing PykF; lane 7, cell lysate containing WbgU; lane 8, cell lysate containing LgtD; lane 9, E. coli BL21(DE3) cell lysate with the empty pET15b plasmid.

View this table: [in this window] [in a new window]

TABLE 2. One-step purification of individual enzymes by nickel affinity column chromatography

Acceptor substrate specificity of LgtD from H. influenzae.
The N-acetylgalactosaminyltransferase activity was tested with 11 carbohydrate compounds, including cellobiose, lactose, globotriose, and isoglobotriose and their derivatives. As shown in Fig. 4, a galactose residue at the nonreducing end is absolutely required because terminal glucose and sialyl galactose residues result in dramatically decreased acceptor activity. Although both - and ß-galactosides are active, the recombinant enzyme apparently prefers substrates with the -anomeric configuration (compare lactose with globotriose and isoglobotriose). Meanwhile, compounds with a terminal 14-linked Gal-Gal structure are better acceptors than compounds with an 13 linkage. Replacement of the reducing end glycoside with another sugar (compare lactose with N-acetyllactosamine) is acceptable. Additionally, the aglycon structure has an effect on the acceptor specificity. It seems that a hydrophobic aglycon can slightly increase the substrate activity.

View larger version (26K): [in this window] [in a new window]

FIG. 4. Acceptor substrate specificity of recombinant ß-1,3-N-acetylgalactosaminyltransferase from H. influenzae when UDP-GalNAc was used as the donor substrate. OBn, ß-benzyl; OMe, ß-methyl; LacNAc, N-acetyllactosamine.

Stepwise analysis of synthetic pathway.
The feasibility of multiple-enzyme synthesis of globotetraose was initially tested in small-scale (0.5-ml) reactions. These reactions were carried out at 25°C for 24 h by using purified enzyme preparations. As shown in Table 3, globotetraose could be synthesized when UTP was directly used as the starting material. However, the overall yield was poor due to the reported inhibitory effect of UDP on glycosyltransferase (40). Addition of pyruvate kinase not only allowed phosphoenolpyruvate to be used instead of UTP as the energy source but also eliminated feedback inhibition because the concentration of UDP was kept lower than 0.5 mM during the sugar nucleotide regeneration step. As a result, the yield was significantly increased from 39 to 95%. To reduce the high cost of acetyl-CoA, phosphate acetyltransferase was introduced in order to recycle it from acetylphosphate and a catalytic amount of CoA (0.5 mM). Finally, inorganic pyrophosphatase was added to hydrolyze the by-product pyrophosphate. We found that this could accelerate the reaction in the forward direction (Fig. 5), but it had little effect on the overall yield of the oligosaccharide products

View this table: [in this window] [in a new window]

TABLE 3. Stepwise multiple-enzyme synthesis of globotetraose

View larger version (18K): [in this window] [in a new window]

FIG. 5. Effect of inorganic pyrophosphatase on multiple-enzyme synthesis of globotetraose. The reaction was performed in a 1-ml (total volume) mixture, and the product was monitored by HPLC. Symbols: , reaction with PPA; •, reaction without PPA.

Synthesis and structural analysis of globotetraose and isoglobotetraose.
Ni-NTA affinity chromatography-purified recombinant enzymes were employed for preparative synthesis of globotetraose and isoglobotetraose. Time course studies revealed that the reactions reached saturation within 24 h (Fig. 6). A higher yield was obtained with globotriose (89%) than with isoglobotriose (78%) in the multiple-enzyme system, which is consistent with the study of acceptor substrate specificity of N-acetylgalactosaminyltransferase from H. influenzae. Oligosaccharide products were collected and purified to allow structure analysis by ESI-MS and NMR spectroscopy. The products were repeatedly dissolved in D₂O and lyophilized before ¹H and ¹³C NMR spectra were recorded at 298 K in a 5-mm tube.

View larger version (18K): [in this window] [in a new window]

FIG. 6. Time course of reactions performed by the multiple-enzyme system. The reactions were carried out as described in Materials and Methods. The products were monitored by HPLC. Symbols: •, globotriose used as acceptor; , isoglobotriose used as acceptor.

The assignments for the various signals of oligosaccharide products were based on the one-dimensional and two-dimensional NMR data, including distortionless enhancement by polarization transfer, double quantum filtered correlated spectroscopy (COSY), heteronuclear multiple quantum coherence, and heteronuclear multiple bond correlation data. In the one-dimensional ¹H NMR, ¹³C NMR, and double quantum filtered COSY spectra (data not shown), signals for a newly introduced GalNAc residue were found when the spectra were compared with the spectra of the starting materials, globotriose (42) and isoglobotriose (7). The chemical shifts of the new anomeric hydrogen at 4.45/4.47 ppm and the N-acetyl group at 1.87/1.84 ppm, which were as long as the chemical shifts of 22.40/22.36 and 175.37/175.31 ppm for carbon in the N-acetyl group, clearly indicated that a GalNAc residue was attached to both anomers of the acceptor molecule. A coupling constant of 8.5/8.1 Hz for the H-1 resonance and the chemical shift of 103.49/103.31 ppm for the C-1 resonance of GalNAc showed that this residue was in ß-anomeric configuration. The ß-anomeric configuration of the GalNAc residue was also evident from the cross peaks between the anomeric proton and the anomeric carbon of GalNAc in the heteronuclear multiple quantum coherence spectrum (data not shown) and the cross peaks between the anomeric proton of GalNAc (H-1, 4.54) and the H-3 and H-4 ( 3.80) protons of the accepting Gal residue in the nuclear Overhauser effect spectroscopy spectrum of globotetraose (data not shown). The significant downfield increments in the chemical shifts of the C-3" resonance of Gal ( = 9.7 ppm) of the acceptor, but not in the other resonances, indicated that the GalNAc residue had been introduced into C-3 of Gal. This linkage was further confirmed by the cross peaks between the anomeric carbon of acetylglucosamine and the H-3 ( 3.50) of the Gal residue in the heteronuclear multiple bond correlation spectrum (data not shown). Therefore, the assigned structures of the products were globotetraose (GalNAcß13Gal14Galß14GlcOH) and isoglobotetraose (GalNAcß13Gal13Galß14GlcOH).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The chemical synthesis of globotetraose (25, 43) and some of its derivatives (3, 20, 26) has been described previously. In these reactions, the saccharide building blocks must be selectively protected and then coupled and finally deprotected to obtain the desired stereochemistry and regiochemistry in the products. The multiple protection-deprotection steps and the tedious purification at each stage of the synthesis normally result in lower rates of conversion (less than 70%). Therefore, most of these reactions are only on the milligram scale. Biocatalytic approaches in which isolated enzymes, especially Leloir glycosyltransferases, are used are powerful and complementary alternatives to chemical synthesis of carbohydrates and glycoconjugates (18, 19, 27). So far, a large number of mammalian glycosyltransferases have been cloned and employed in oligosaccharide synthesis; these enzymes include bovine ß-1,4-galactosyltransferase (7, 24, 28) and -1,3-galactosyltransferase (7, 10) and human -2,3-sialyltransferase (31), -1,3-fucosyltransferase (2), and blood group A/B glycosyltransferases (32). However, the utility of these enzymes in large-scale synthesis of glycoconjugates is partially limited by the high cost of eukaryotic cell culture and by the low level of protein expression. Most bacterial glycosyltransferases, on the other hand, are easily expressed at high levels in cheaper prokaryotic expression systems. Moreover, bacterial glycosyltransferases seem to have broader acceptor substrate specificities, thereby offering a tremendous advantage over mammalian enzymes in chemoenzymatic synthesis of oligosaccharides and their derivatives. The results of the present study provide another good example of this. The ß-1,3-N-acetylgalactosaminyltransferase from H. influenzae can accept a wide range of substrates, including the globo (Gal14Galß14Glc),isoglobo (Gal13Galß14Glc), and N-acetyllactosamine (Galß14GlcNAc) series oligosaccharides. Hence, the efficient synthesis of oligosaccharides with these core structures by using this enzyme should facilitate research related to potential therapeutic applications of these molecules.

The availability of new glycosyltransferases has increased the demand for sugar nucleotides in the production of oligosaccharides and glycoconjugates. Enzymatic synthesis of sugar nucleotides is more attractive than chemical methods because the yields are high and no organic solvents are used. UDP-GalNAc is synthesized in vivo by epimerization of UDP-GlcNAc. The reaction is catalyzed by UDP-Glc C4 epimerases in mammalian cells and by UDP-GlcNAc C4 epimerases in yeasts and bacteria. However, these enzymes have been utilized only for small-scale in vitro synthesis because of the unfavorable equilibrium constant (K_eq, 0.3) and the difficulty of separating UDP-GalNAc from UDP-GlcNAc (29). In situ UDP-GalNAc regeneration is therefore a better choice for large-scale production of oligosaccharides with GalNAc residues. To our knowledge, the preparative synthesis of globotetraose and isoglobotetraose described in this paper is the first application of UDP-GalNAc regeneration combined with bacterial GalNAc transferase for the production of glycoconjugates. Apparently, this is a cost-efficient way to synthesize these compounds considering the high price of UDP-GalNAc ($892.50 per 100 mg from Sigma). Purification of all recombinant enzymes might be considered a major drawback of such a multiple-enzyme system. However, it seems that this problem could be solved by the so-called "superbead" technology, in which oligosaccharides are produced with multiple enzymes immobilized directly on a nickel affinity column (6).

In summary, here we describe an efficient route for enzymatic synthesis of oligosaccharides with regeneration of the donor substrate UDP-GalNAc. Since the ß-1,3-N-acetylgalactosaminyltransferase from H. influenzae has a broad acceptor substrate specificity, both globotetraose and isoglobotetraose were synthesized with high yields. This approach might provide a practical solution for large-scale production of both natural and unnatural oligosaccharides with a GalNAcß1,3Gal moiety.

 
* Corresponding author. Mailing address: Department of Chemistry, Wayne State University, Detroit, MI 48202. Phone: (313) 993-6759. Fax: (313 577-9241. E-mail: pwang{at}chem.wayne.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Bailly, P., F. Piller, B. Gillard, A. Veyrieres, K. Marcus, and J. P. Cartron. 1992. Biosynthesis of the blood group P^K and P₁ antigens by human kidney microsomes. Carbohydr. Res. 228:277-287.[CrossRef][Medline]
2
2. Baisch, G., R. Öhrlein, M. Streiff, and F. Kolbinger. 1998. Enzymatic synthesis of sialyl-Lewis^a-libraries with two non-natural monosaccharide units. Bioorg. Med. Chem. Lett. 8:755-758.[CrossRef][Medline]
3
3. Bhattacharyya, S., B. G. Magnusson, U. Wellmar, and U. J. Nilsson. 2001. The p-methoxybenzyl ether as an in situ-removable carbohydrate-protecting group: a simple one-pot synthesis of the globotetraose tetrasaccharide. J. Chem. Soc. Perkin Trans. 8:886-890.
4
4. Brown, K. E., S. M. Anderson, and N. S. Young. 1993. Erythrocyte P antigen: cellular receptor for B19 parvovirus. Science 262:114-117.[Abstract/Free Full Text]
5
5. Brown, T. D. K., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J. Gen. Microbiol. 102:327-336.[Abstract/Free Full Text]
6
6. Chen, X., J. Fang, J. Zhang, Z.-Y. Liu, P. Andreana, P. Kowal, and P. G. Wang. 2001. Sugar nucleotide regeneration beads (superbeads): a versatile tool for practical synthesis of oligosaccharides. J. Am. Chem. Soc. 123:2081-2082.[CrossRef][Medline]
7
7. Fang, J.-W, J. Li, X. Chen, Y.-N. Zhang, J.-Q. Wang, Z.-M. Guo, W. Zhang, L. Yu, K. Brew, and P. G. Wang. 1998. Highly efficient chemoenzymatic synthesis of -galactosyl epitopes with a recombinant (13)-galactosyltransferase. J. Am. Chem. Soc. 120:6635-6638.[CrossRef]
8
8. Gotschlich, E. C. 1994. Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J. Exp. Med. 180:2181-2190.[Abstract/Free Full Text]
9
9. Haynie, S. L., and G. M. Whitesides. 1990. Enzyme-catalyzed organic synthesis of sucrose and trehalose with in situ regeneration of UDP-glucose. Appl. Biochem. Biotechnol. 23:155-170.[Medline]
10
10. Hokke, C. H., A. Zervosen, L. Elling, D. H. Joziasse, and D. H. van den Eijnden. 1996. One-pot enzymatic synthesis of the Gal alpha 13Gal beta 14GlcNAc sequence with in situ UDP-Gal regeneration. Glycoconjugate J. 13:687-692.[CrossRef][Medline]
11
11. Hood, D. W., A. D. Cox, W. W. Wakarchuk, M. Schur, E. K. Schweda, S. L. Walsh, M. E. Deadman, A. A. Martin, E. R. Moxon, and J. C. Richards. 2001. Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118). Glycobiology 11:957-967.[Abstract/Free Full Text]
12
12. Ichikawa, Y., L. C. J. Liu, G. J. Shen, and C. H. Wong. 1991. A highly efficient multienzyme system for the one-step synthesis of a sialyl trisaccharide: in situ generation of sialic acid and N-acetyllactosamine coupled with regeneration of UDP-glucose, UDP-galactose and CMP-sialic acid. J. Am. Chem. Soc. 113:6300-6302.[CrossRef]
13
13. Johnson, K. F. 1999. Synthesis of oligosaccharides by bacterial enzymes. Glycoconjugate J. 16:141-146.[CrossRef][Medline]
14
14. Jones, D. H., C. A. Lingwood, K. R. Barber, and C. W. Grant. 1997. Globoside as a membrane receptor: a consideration of oligosaccharide communication with the hydrophobic domain. Biochemistry 36:8539-8547.[CrossRef][Medline]
15
15. Jordan, J. A. 2001. Diagnosing human parvovirus B19 infection: guidelines for test selection. Mol. Diagn. 6:307-312.[CrossRef][Medline]
16
16. Josse, J. 1966. Constitutive inorganic pyrophosphatase of Escherichia coli. 1. Purification and catalytic properties. J. Biol. Chem. 241:1938-1947.[Abstract/Free Full Text]
17
17. Karlsson, K.-A. 1989. Animal glycosphingolipids as membrane attachment sites for bacteria. Annu. Rev. Biochem. 58:309-350.[CrossRef][Medline]
18
18. Khan, S. H., and R. A. O'Neill. 1996. Modern methods in carbohydrate synthesis. Harwood Academic Publishers, Amsterdam, The Netherlands.
19
19. Koeller, K. M., and C. H. Wong. 2000. Synthesis of complex carbohydrates and glycoconjugates: enzyme-based and programmable one-pot strategies. Chem. Rev. 100:4465-4494.[CrossRef][Medline]
20
20. Leontein, K., M. Nilsson, and T. Norberg. 1985. Synthesis of the methyl and 1-octyl glycosides of the P-antigen tetrasaccharide (globotetraose). Carbohydr. Res. 144:231-240.[CrossRef][Medline]
21
21. Mengin-Lecreulx, D., and J. van Heijenoort. 1996. Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli. J. Biol. Chem. 271:32-39.[Abstract/Free Full Text]
22
22. Mengin-Lecreulx, D., and J. van Heijenoort. 1993. Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. J. Bacteriol. 175:6150-6157.[Abstract/Free Full Text]
23
23. Naiki, M., and D. M. Marcus. 1975. An immunochemical study of the human blood group P₁, P, and P^k glycosphingolipid antigens. Biochemistry 14:4837-4841.[CrossRef][Medline]
24
24. Niggemann, J., J. P. Kamerling, and J. F. G. Vliegenthart. 1998. ß1,4-Galactosyltransferase-catalyzed synthesis of the branched tetrasaccharide repeating unit of Streptococcus pneumoniae type 14. Bioorg. Med. Chem. 6:1605-1612.[CrossRef][Medline]
25
25. Nilsson, U., A. K. Ray, and G. Magnusson. 1994. Synthesis of the globotetraose tetrasaccharide and terminal tri- and di-saccharide fragments. Carbohydr. Res. 252:117-136.[Medline]
26
26. Nilsson, U., A. Wendler, and G. Magnusson. 1994. Synthesis of the 2"-hydroxy, 4"-deoxy and 4"-epi analogues of beta-D-GalNAc-(13)-alpha-D-Gal-(14)-beta-D-Gal, the terminal trisaccharide of globotetraose. Acta Chem. Scand. 48:356-361.[Medline]
27
27. Palcic, M. M. 1999. Biocatalytic synthesis of oligosaccharides. Curr. Opin. Biotechnol. 10:616-624.[CrossRef][Medline]
28
28. Palcic, M. M., H. Li, D. Zanini, R. S. Bhella, and R. Roy. 1997. Chemoenzymatic synthesis of dendritic sialyl Lewis^x. Carbohydr. Res. 305:433-442.[CrossRef][Medline]
29
29. Piller. F., A. E. Eckhardt, and R. L. Hill. 1982. The preparation of UDP-N-acetylgalactosamine from UDP-N-acetylglucosamine employing UDP-N-acetylglucosamine-4-epimerase. Anal. Biochem. 127:171-177.[CrossRef][Medline]
30
30. Rumsfeld, J., K. Ziegelbauer, and F. Spaltmann. 2000. High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example. Protein Expr. Purif. 18:303-309.[CrossRef][Medline]
31
31. Seitz, O., and C.-H. Wong. 1997. Chemoenzymatic solution- and solid-phase synthesis of O-glycopeptides of the mucin domain of MAdCAM-1. A general route to O-LacNAc, O-sialyl-LacNAc, and O-sialyl-Lewis-X peptides. J. Am. Chem. Soc. 119:8766-8776.[CrossRef]
32
32. Seto, N. O. L., C. A. Compston, S. V. Evans, D. R. Bundle, S. A. Narang, and M. M. Palcic. 1999. Donor substrate specificity of recombinant blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli. Eur. J. Biochem. 259:770-775.[Medline]
33
33. Shao, J., J. Zhang, P. Kowal, Y. Lu, and P. G. Wang. 2002. Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd. Biochem. Biophys. Res. Commun. 295:1-8.[CrossRef][Medline]
34
34. Shimizu, M., T. Suzuki, K. Y. Kameda, and Y. Abiko. 1969. Phosphotransacetylase of Escherichia coli B, purification and properties. Biochim. Biophys. Acta 191:550-558.[Medline]
35
35. Somani, B. L., G. Valentini, and M. Malcovati. 1977. Purification and molecular properties of the AMP-activated pyruvate kinase from Escherichia coli. Biochim. Biophys. Acta 482:52-63.[Medline]
36
36. Song, Y., D. A. Withers, and S. Hakomori. 1998. Globoside-dependent adhesion of human embryonal carcinoma cells, based on carbohydrate-carbohydrate interaction, initiates signal transduction and induces enhanced activity of transcription factors AP1 and CREB. J. Biol. Chem. 273:2517-2525.[Abstract/Free Full Text]
37
37. Stapleton, A. E., M. R. Stroud, S. I. Hakomori, and W. E. Stamm. 1998. The globoseries glycosphingolipid sialosyl galactosyl globoside is found in urinary tract tissues and is a preferred binding receptor in vitro for uropathogenic Escherichia coli expressing pap-encoded adhesins. Infect. Immun. 66:3856-3861.[Abstract/Free Full Text]
38
38. Wang, L., S. Huskic, A. Cisterne, D. Rothemund, and P. R. Reeves. 2002. The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene. J. Bacteriol. 184:2620-2625.[Abstract/Free Full Text]
39
39. Whiteley, H. R., and R. A. Pelroy. 1972. Purification and properties of phosphotransacetylase from Veillonella alcalescens. J. Biol. Chem. 247:1911-1917.[Abstract/Free Full Text]
40
40. Wong, C.-H., S. L. Haynie, and G. M. Whitesides. 1982. Enzyme-catalyzed synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose. J. Org. Chem. 47:5416-5418.[CrossRef]
41
41. Zervosen, A., and L. Elling. 1996. A novel three-enzyme reaction cycle for the synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose. J. Am. Chem. Soc. 118:1836-1840.[CrossRef]
42
42. Zhang, J. B., P. Kowal, J. W. Fang, P. Andreana, and P. G. Wang. 2002. Efficient chemoenzymatic synthesis of globotriose and its derivatives with a recombinant (14)-galactosyltransferase. Carbohydr. Res. 337:969-976.[CrossRef][Medline]
43
43. Zou, W., J. R. Brisson, S. Larocque, R. L. Gardner, and H. J. Jennings. 1999. Synthesis and NMR assignments of galactosylgloboside and its beta-GalNAc-1,4-alpha-D-Gal-linked positional isomer in a conjugatable form. Carbohydr. Res. 315:251-261.[CrossRef][Medline]

---

Applied and Environmental Microbiology, November 2002, p. 5634-5640, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5634-5640.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J., Jarrell, H. C., Lau, P. C., Berghuis, A. M., Young, N. M., Wakarchuk, W. W. (2005). A Single Bifunctional UDP-GlcNAc/Glc 4-Epimerase Supports the Synthesis of Three Cell Surface Glycoconjugates in Campylobacter jejuni. J. Biol. Chem. 280: 4792-4802 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shao, J. Articles by Wang, P. G. Search for Related Content PubMed PubMed Citation Articles by Shao, J. Articles by Wang, P. G. Agricola Articles by Shao, J. Articles by Wang, P. G.