THE VARIANT-SPECIFIC SURFACE PROTEIN OF GIARDIA, VSP4A1, IS A GLYCOSYLATED AND PALMITOYLATED PROTEIN

The variant-specific surface proteins (VSPs) of the ancient protist Gi ardia duodenalis (syn.: Giardia intestinalis, Giardia lamblia) are cys teine- and threonine-rich polypeptides that can vary considerably in s equence and size. In the present study, we have purified a VSP (VSP4A1 , formerly called CRISP-90) from a cloned Giardia isolate, derived fro m a sheep, by Triton X-114 phase partitioning and anion-exchange chrom atography. Analysis of the purified VSP4A1 showed that this protein is posttranslationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositio nal analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling o f VSP4A1 with galactosyltransferase/ UDP-[H-3]Gal. The glycans were re leased by beta-elimination, suggesting that they are O-linked to the p rotein. Bio-Gel P4 chromatography of the released galactosylated glyca ns and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus, These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of sh ort O-linked glycan. Compositional analysis and radiolabelling experim ents also indicated that VSP4A1 is modified with covalently linked pal mitate (1 mol/mol of protein), Hydroxylamine treatment at neutral pH o f [H-3]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modif ication appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.