Biosynthesis of the O antigen of pathogenic Escherichia coli O157:H7. Characterization of α1,4-Fuc-transferase WbdO.

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcß1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in ß1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-ß-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcß1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcß1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.

Biosynthesis of the Pseudomonas aeruginosa common polysaccharide antigen by D-Rhamnosyltransferases WbpX and WbpY.

The Gram-negative bacterium Pseudomonas aeruginosa simultaneously expresses two O-antigenic glycoforms. While the O-specific antigen (OSA) is variable in composition, the common polysaccharide antigen (CPA) is highly conserved and is composed of a homopolymer of D-rhamnose (D-Rha) in trisaccharide repeating units [D-Rhaα1-2-D-Rhaα1-3-D-Rhaɑ1-3]n. We have previously reported that α3-D-Rha-transferase WbpZ transfers a D-Rha residue from GDP-D-Rha to D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl. Genes encoding two more D-Rha-transferases are found in the O antigen gene cluster (wbpX and wbpY). In this study we showed that WbpX and WbpY recombinantly expressed in E. coli differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family. NMR spectroscopic analysis of the WbpY reaction product showed that WbpY transferred one D-Rha residue in α1-3 linkage to synthetic D-Rhaα1-3-D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl acceptor. WbpX synthesized several products that contained D-Rha in both α1-2 and α1-3 linkages. Mass spectrometry indicated that the mixture of WbpX and WbpY efficiently catalyzed the synthesis of D-Rha oligomers in a non-processive mechanism. Since O antigens are virulence factors, these findings open the door to advancing technology for antibacterial drug discovery and vaccine development.

Mucins as anti-cancer targets: perspectives of the glycobiologist.

Mucins are highly O-glycosylated glycoproteins that carry a heterogenous variety of O-glycan structures. Tumor cells tend to overexpress specific mucins, such as the cell surface mucins MUC1 and MUC4 that are engaged in signaling and cell growth, and exhibit abnormal glycosylation. In particular, the Tn and T antigens and their sialylated forms are common in cancer mucins. We review herein methods chosen to use cancer-associated glycans and mucins as targets for the design of anti-cancer immunotherapies. Mucin peptides from the glycosylated and transmembrane domains have been combined with immune-stimulating adjuvants in a wide variety of approaches to produce anti-tumor antibodies and vaccines. These mucin conjugates have been tested on cancer cells in vitro and in mice with significant successes in stimulating anti-tumor responses. The clinical trials in humans, however, have shown limited success in extending survival. It seems critical that the individual-specific epitope expression of cancer mucins is considered in future therapies to result in lasting anti-tumor responses.

The wclY gene of Escherichia coli serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity.

The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcß1-3-L-Rhaα1-4-D-Glcα1-4-D-Galß1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galß1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.

Inhibition of bacterial growth and galactosyltransferase activity of WbwC by α, ω-bis(3-alkyl-1H-imidazolium)alkane salts: Effect of varying carbon content.

A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents.

Role of Glycans on Key Cell Surface Receptors That Regulate Cell Proliferation and Cell Death.

Cells undergo proliferation and apoptosis, migration and differentiation via a number of cell surface receptors, most of which are heavily glycosylated. This review discusses receptor glycosylation and the known roles of glycans on the functions of receptors expressed in diverse cell types. We included growth factor receptors that have an intracellular tyrosine kinase domain, growth factor receptors that have a serine/threonine kinase domain, and cell-death-inducing receptors. N- and O-glycans have a wide range of functions including roles in receptor conformation, ligand binding, oligomerization, and activation of signaling cascades. A better understanding of these functions will enable control of cell survival and cell death in diseases such as cancer and in immune responses.