Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor.
The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.
D000818:Animals; D050260:Carbohydrate Metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology; D014643:Variant Surface Glycoproteins, Trypanosoma
D006017:Glycolipids; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D009227:Myristic Acids; D010716:Phosphatidylinositols; D014643:Variant Surface Glycoproteins, Trypanosoma; D019814:Myristic Acid; D014415:Tunicamycin; D003513:Cycloheximide; D008715:Methionine