The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein.
The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis (syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CR1SP-90) from a cloned Giardia isolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. 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) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by beta-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans 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 short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of[3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.
Papanastasiou
P
P
Institute of Parasitology, University of Zürich, Switzerland.
McConville
M J
MJ
Ralton
J
J
Köhler
P
P
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D016829:Giardia lamblia / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006867:Hydrolases; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008565:Membrane Proteins; D011134:Polysaccharides; D015800:Protozoan Proteins; D019308:Palmitic Acid; D006867:Hydrolases; C100894:atrazine chlorohydrolase