A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways.
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
Mensa-Wilmot
K
K
Department of Zoology, University of Georgia, Athens 30602.
LeBowitz
J H
JH
Chang
K P
KP
al-Qahtani
A
A
McGwire
B S
BS
Tucker
S
S
Morris
J C
JC
D000818:Animals; D002240:Carbohydrate Sequence; D006028:Glycosphingolipids / Q000378:metabolism; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; C008290:lipophosphonoglycan; D010727:Phosphoric Diester Hydrolases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase