Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.
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 [3H]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 Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.
Acosta-Serrano
Alvaro
A
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
O'Rear
Jessica
J
Quellhorst
George
G
Lee
Soo Hee
SH
Hwa
Kuo-Yuan
KY
Krag
Sharon S
SS
Englund
Paul T
PT
D000818:Animals; D003208:Concanavalin A / Q000494:pharmacology; D004286:Dolichols / Q000032:analysis / Q000096:biosynthesis; D004351:Drug Resistance / Q000235:genetics; D006017:Glycolipids / Q000096:biosynthesis; D006031:Glycosylation; D045782:Hemiterpenes; D009154:Mutation; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000439:Pentanols / Q000032:analysis / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism*; D014353:Trypanosomiasis, African / Q000188:drug therapy
D004286:Dolichols; D006017:Glycolipids; D045782:Hemiterpenes; D009844:Oligosaccharides; D000439:Pentanols; C477742:mannosyl(6)-N-acetyl(2)glucose; D003208:Concanavalin A; C009034:prenol; D010088:Oxidoreductases; C106822:polyprenol reductase