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.
Laboratory of Molecular Parasitology, The Rockefeller University, Box 185, 1230 York Avenue, New York, NY 10021-6399, USA.
Jun
J
Wang
Ulrike
U
Böhme
George A M
GA
Cross
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