Structural features affecting variant surface glycoprotein expression in Trypanosoma brucei.
The glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of Trypanosoma brucei is the most abundant GPI-anchored protein expressed on any cell, and is an essential virulence factor. To determine what structural features affect efficient expression of VSG, we made a series of mutations in two VSGs. Inserting 18 amino acids, between the amino- and carboxy-terminal domains, reduced the expression of VSG 221 to about 3% of the wild-type level. When this insertion was combined with deletion of the single carboxy-terminal subdomain, expression was reduced a further three-fold. In VSG 117, which contains two carboxy-terminal subdomains, point mutation of the intervening N-glycosylation site reduced expression about 15-fold. Deleting the most carboxy-terminal subdomain and intervening region, including the N-glycosylation site, reduced expression to 15-20% of wild type VSG, and deletion of both subdomains reduced expression to <1%. Despite their low abundance, all VSG mutants were GPI anchored on the cell surface. Our results suggest that, for a protein to be efficiently displayed on the surface of bloodstream-form T. brucei, it is essential that it contains the conserved structural motifs of a T. brucei VSG. Serum resistance-associated protein (SRA), which confers human infectivity on T. brucei, strongly resembles a VSG deletion mutant. Expression of three epitope-tagged versions of SRA in T. brucei conferred total resistance to human serum. SRA possesses a canonical GPI signal sequence, but we were unable to obtain unequivocal evidence for the presence of a GPI anchor. SRA was not released during osmotic lysis, indicating that it is not GPI anchored on the cell surface.
Wang
Jun
J
Laboratory of Molecular Parasitology, The Rockefeller University, Box 185, 1230 York Avenue, New York, NY 10021-6399, USA.
Böhme
Ulrike
U
Cross
George A M
GA
D000595:Amino Acid Sequence; D000818:Animals; D017353:Gene Deletion; D005786:Gene Expression Regulation; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D015800:Protozoan Proteins; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry* / Q000378:metabolism*
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C403774:serum resistance associated protein, Trypanosoma brucei