Trypanosoma brucei brucei: inhibition of glycosylation of the major variable surface coat glycoprotein by tunicamycin

Abstract

Trypanosoma brucei brucei incorporates D-[3H]mannose into protein in vitro in a medium we describe here. The label appears entirely in glycoproteins with approximately 90% in the major variable surface coat glycoprotein (VSCG). Incorporation is linear for 60 min and usually continues for an additional 30 min although at a decreased rate. In the same medium incorporation of L-[14C]serine is linear for 90 min. Incorporation of [3H]mannose is completely inhibited by tunicamycin at concentrations above 100 ng/ml, indicating that the label is being added as part of an N-linked oligosaccharide. This is reflected by a 5% decrease in the apparent molecular weight of VSCG on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Cycloheximide inhibits incorporation of both mannose and serine, although the rates and extent of inhibition differ. Based on the effects of tunicamycin or cycloheximide on incorporation of either precursor, we suggest that N-linked glycosylation occurs subsequent to synthesis of the VSCG polypeptide.