Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant

Abstract

ABSTRACT Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [ 3 H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man 9 GlcNAc 2 , whereas that from ConA 1-1 is Man 7 GlcNAc 2 . The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man 4 GlcNAc 2 modified with a terminal N -acetyllactosamine group, which is poorly recognized by ConA.