CLONING & EXPRESSION OF NOVEL GLYCOSYLTRANSFERASES

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Enterotoxigenic E. coli expressing the K88 fimbrial adhesin causes severe intestinal infections in newborn and weaned piglets. Recognition of receptors on the surface of intestinal epithelial cells by K88 fimbrial adhesins, which prevents the removal of bacteria by intestinal peristalsis, is an early step in diarrheic pathogenesis. The three variants of the K88 adhesin (K88ab, K88ac, K88ad) appear to have different receptor specificities. It has been established that some pigs lack intestinal receptors for K88 adhesins and that these animals are resistant to infection by K88+ E. coli. Both glycoproteins and glycolipids have been implicated as K88 adhesin receptors. The aim of the current study was to identify and characterize glycoconjugate receptors for the K88ad adhesin. A neutral glycosphingolipid reactive with the K88 adhesin was isolated and purified from porcine intestinal brush border membranes. Preliminary assessment using anti-carbohydrate monoclonal ant ibodies suggested the presence of a terminal Lewis X epitope on the glycan. A very small aliquot (~ 7 (g) of the glycosphingolipid was submitted first for 1H-NMR analysis, but the results were inconclusive, due to the presence of multiple components in mixture. The sample was analyzed for monosaccharide composition by preparing the TMS derivatives of the methylglycosides followed by GC/MS analysis. TMS methylglycosides were prepared from the sample by methanolysis in 1 N HCl in MeOH, followed by N-acetylation with pyridine and acetic anhydride. The sample was then treated with Tri-Sil. GC/MS analysis was performed using a Hewlett-Packard 5890 GC/5970 MSD with a DB-5 column. Monosaccharides were identified by their retention times and electrospray ionization mass spectra in comparison to standards. The presence of sugars characteristic for neolacto-series glycosphingolipids was detected along with a small amount of fucose, but it was again concluded that the sample was a mixture, with the Lewis X structure belonging to a minor component. In an alternative approach, a set of purified standard glycosphingolipids of confirmed structure (from a library of natural and semi-synthetic compounds available in our laboratory at the CCRC) were sent to the collaborators for direct testing of the binding specificity of the K88 adhesin by HPTLC overlay staining. It was found that neolacto-tetra-and hexaglycosylceramides (nLc4Cer and nLc6Cer) bound the adhesin with a high degree of specificity, although some cross-reaction was observed with the isomeric lacto-tetraosylceramide (Lc4Cer). Binding was not observed with glycosphingolipids bearing Lewis X, Lewis a or histo-blood group H determinants. A manuscript describing this work has been submitted for publication. Currently, another crude preparation of glycosphingolipids from porcine intestinal brush border membranes is being fractionated at the CCRC in order to determine if the putative ligand can indeed be isolated from this natural source.