Glycan engineering of cells and proteins

Cell surface carbohydrates are involved in several biological processes including the binding of cells, microbes (viruses, bacteria and bacterial toxins) and antibodies to cell surfaces. The sequencing of the genomes of several species, including humans, and the subsequent identification of glycosyltransferase genes, have resulted in the elucidation of the specificity in vitro of most glycosyltransferases.

By transfecting host cells carrying known glycan phenotypes with expression plasmids encoding glycosyltransferase genes of known specificity, the glycosylation of the cell surface of transfected cells and proteins expressed in them can be modified, tailored and used as a tool to assess binding specificity of carbohydrate-binding proteins. We have used this technology to elucidate the binding specificity of viruses, bacterial adhesins and toxins.

Antibodies we have investigated include blood group ABO antibodies, and antibodies specific for α-Gal and NeuGc. ABO antibodies are of relevance in transfusion medicine and in ABO incompatible organ transplantation, and antibodies with the latter two specificities may cause rejection or immune-mediated disease of xenogeneic tissue, for example bioprosthetic heart valves. We are also interested in the biophysical surface properties of recombinant mucin-type proteins.

Our experimental expertise lies in the making of stable cell lines with defined glycosylation phenotypes and may involve expressing as many as five different recombinant genes. Large scale cell culture, protein purification, glycan release and glycan characterization by liquid chromatography - mass spectrometry are important experimental procedures in our arsenal of methods.