Characterization of a Leishmania mexicana mutant defective in synthesis of free and protein-linked GPI glycolipids.
The cell surface of the promastigote stage of the protozoan parasite, Leishmania mexicana is coated by a number of glycosylphosphatidylinositol (GPI)-anchored proteins, a GPI-anchored lipophosphoglycan (LPG) and an abundant class of free GPIs, termed glycoinositolphospholipids (GIPLs). We have developed a new screen for isolating L. mexicana mutants that are defective in GPI biosynthesis, involving concanavalin A selection of a parental strain with a modified surface coat. One mutant was isolated that lacked the major GIPL species and mature GPI-protein anchor precursors, but synthesized normal levels of LPG anchor precursors. Based on analysis of apolar GIPLs that accumulate in this mutant and in vivo and in vitro synthesized GPIs, this mutant was found to have a defect in the addition of an alpha1-6 linked mannose to the common precursor, Man(1)GlcN-PI. The apolar GIPLs were transported to the cell surface with the same kinetics as mature GIPLs. However, non-anchored isoforms of the major GPI-anchored protein, gp63, were either slowly secreted (with a t(1/2) of 2 h) or retained within the endoplasmic reticulum, respectively. These findings suggest that common enzymes are involved in the synthesis of GIPLs and protein anchors and have implications for understanding how the biosynthesis of the major surface components of these parasites is regulated.
Naderer
Thomas
T
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Vic. 3010, Australia.
McConville
Malcolm J
MJ
D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D003208:Concanavalin A / Q000378:metabolism; D006017:Glycolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D007894:Leishmania mexicana / Q000235:genetics* / Q000378:metabolism*; D008018:Life Cycle Stages; D008358:Mannose / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010743:Phospholipids; D015800:Protozoan Proteins; C115336:glycoinositolphospholipid, Trypanosoma; D003208:Concanavalin A; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D008358:Mannose