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K99 lectin fromEscherichia coliwas purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay.

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J9:191–97] was partially purified from culturedSilene albacells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of ‘unconjugated N-glycans’ in a suspension medium of culturedSilene albacells.

In this study it was shown that the O101 lipopolysaccharide isolated from Escherichia coli B41 did not contain an O-specific polysaccharide and that its sugar moiety is probably restricted to the core oligosaccharide. It is characterized by the presence of galactose, glucose, N-acetylglucosamine, heptose and 3-deoxy-d-manno-2-octulosonic acid and the fatty acid composition is typical of an Enterobacteriaceae lipopolysaccharide. Methylation analysis indicated terminal non-reducing galactose and glucose and also 1,2-linked glucose which is a substitution pattern typical of an E. coli lipopolysaccharide core oligosaccharide. The obtained structural information is sufficient to explain the previously observed interactions between the O101 lipopolysaccharide and the K99 lectin.