Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides.
The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.
Bangs
J D
JD
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Doering
T L
TL
Englund
P T
PT
Hart
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D006017:Glycolipids / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006031:Glycosylation; D008563:Membrane Lipids / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D006017:Glycolipids; D008563:Membrane Lipids; D009844:Oligosaccharides; D014643:Variant Surface Glycoproteins, Trypanosoma; D006026:Glycoside Hydrolases