Monica M. Palcic

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of β(1–4)galactosyltransferase, α(1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.

The structural basis for specificity in human ABO(H) blood group biosynthesis

The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8–1.32 Å resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.

Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: Critical role of the 4″-hydroxyl group in β-d-GlcpNAc-(1 → 2)-α-d-Manp(1 → 6)-β-d-Glcp-OR

The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6′ group of oligosaccharides terminating in the sequence β-d-GlcpNAc-(1 → 2)-α-d-Manp(1 → 6)-β-d-Glcp (or Manp)-OR (5, R  (CH2)7CH3) to yield the sequence β-d-GlcpNAc-(1 → 2)-[β-d-GlcpNAc-(1 → 6)]-α-d-Manp-(1 → 6)-β-d-Glcp (or Manp)-OR. Biosynthetically, if β-(1 → 4)-galactosyltransferase acts first on 5, the product β-d-Galp-(1 → 4)-β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-β-d-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6′ group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end d-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.

Acceptor Specificity of Different Length Constructs of Human Recombinant α1,3/4-Fucosyltransferases REPLACEMENT OF THE STEM REGION AND THE TRANSMEMBRANE DOMAIN OF FUCOSYLTRANSFERASE V BY PROTEIN A RESULTS IN AN ENZYME WITH GDP-FUCOSE HYDROLYZING ACTIVITY

The acceptor specificity of recombinant full-length, membrane-bound fucosyltransferases, expressed in COS-7 cells, and soluble, protein-A chimeric forms of α1,3-fucosyltransferase (Fuc-T) III, Fuc-TIV, and Fuc-TV was analyzed toward a broad panel of oligosaccharide, glycolipid, and glycoprotein substrates. Our results on the full-length enzymes confirm and extend previous studies. However, chimeric Fuc-Ts showed increased activity toward glycoproteins, whereas chimeric Fuc-TIII and Fuc-TV had a decreased activity with glycosphingolipids, compared to the full-length enzymes. Unexpectedly, chimeric Fuc-TV exhibited a GDP-fucose hydrolyzing activity. In substrates with multiple acceptor sites, the preferred site of fucosylation was identified. Fuc-TIII and Fuc-TV catalyzed fucose transfer exclusively to OH-3 of glucose in lacto- N-neotetraose and lacto- N-tetraose, respectively, as was demonstrated by 1H NMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc6Cer, whereas Fuc-TV preferred the proximal GlcNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI3NeuAcnLc6Cer resulted in products with the sialyl-LewisXepitope as well as the VIM-2 structure. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. All three Fuc-Ts had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 or C-4 of GlcNAc.

Biocatalytic synthesis of oligosaccharides.

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.

Cloning and Heterologous Expression of an α1,3-Fucosyltransferase Gene from the Gastric PathogenHelicobacter pylori

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel α1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian α1,3/1,4-fucosyltransferases. The ∼52-kDa protein encoded byHpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to α1,3-fucosyltransferase activity but neither α1,4-fucosyltransferase nor α1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40–45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken α1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial α1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic α1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.

Carbohydrate recognition by Clostridium difficile toxin A

Clostridium difficile TcdA is a large toxin that binds carbohydrates on intestinal epithelial cells. A 2-Å resolution cocrystal structure reveals two molecules of α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH2)8CO2CH3 binding in an extended conformation to TcdA. Residues forming key contacts with the trisaccharides are conserved in all seven putative binding sites in TcdA, suggesting a mode of multivalent binding that may be exploited for the rational design of novel therapeutics.

Recent structures, evolution and mechanisms of glycosyltransferases.

Cellular glycome assembly requires the coordinated action of a large number of glycosyltransferases that catalyse the transfer of a sugar residue from a donor to specific acceptor molecules. This enzyme family is very ancient, encompassing all three domains of life. There has been considerable recent progress in structural glycobiology with the determination of crystal structures of several important glycosyltransferase members, showing novel folds and variations around a common α/β scaffold. Structural, kinetic and inhibitor data have led to the emergence of various scenarios with respect to their evolutionary history and reaction mechanisms thus highlighting the different solutions that nature has selected to catalyse glycosyl transfer.

Glycosyltransferases as biocatalysts

Glycosyltransferases are useful synthetic tools for the preparation of natural oligosaccharides, glycoconjugates and their analogues. High expression levels of recombinant enzymes have allowed their use in multi-step reactions, on mg to multi-gram scales. Since glycosyltransferases are tolerant with respect to utilizing modified donors and acceptor substrates they can be used to prepare oligosaccharide analogues and for diversification of natural products. New sources of enzymes are continually discovered as genomes are sequenced and they are annotated in the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Glycosyltransferase mutagenesis, domain swapping and metabolic pathway engineering to change reaction specificity and product diversification are increasingly successful due to advances in structure-function studies and high throughput screening methods.

Enzymic synthesis of oligosaccharides terminating in the tumor-associated sialyl-Lewis-a determinant

The isomeric sialyl-Lea-terminating pentasaccharide derivatives, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]-beta- D-GlcpNAc-(1----3)-beta-D-Galp-O(CH2)8COOMe and alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]- beta-D-GlcpNAc-(1----6)-beta-D-Galp-O(CH2)8COOMe, have been prepared by the action in sequence of a porcine submaxillary (2----3)-alpha-sialyltransferase and a human-milk (1----3/4)-alpha-fucosyltransferase on the chemically synthesized trisaccharides beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)- and -(1----6)-beta-D-Galp- O(CH2)8COOMe, respectively.