title: GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways.
abstract: 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 (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. 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. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
authors.affiliation: Department of Zoology, University of Georgia, Athens 30602.
authors.forename: K
authors.initials: K
authors.lastname: Mensa-Wilmot
authors.forename: J H
authors.initials: JH
authors.lastname: LeBowitz
authors.forename: K P
authors.initials: KP
authors.lastname: Chang
authors.forename: A
authors.initials: A
authors.lastname: al-Qahtani
authors.forename: B S
authors.initials: BS
authors.lastname: McGwire
authors.forename: S
authors.initials: S
authors.lastname: Tucker
authors.forename: J C
authors.initials: JC
authors.lastname: Morris
mesh_terms: D000818:Animals; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000737:chemistry; D006457:Hemoglobinuria, Paroxysmal / Q000378:metabolism; D018320:Leishmania major / Q000235:genetics / Q000378:metabolism*; D008322:Mammals; D008666:Metalloendopeptidases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000096:biosynthesis*; D013601:T-Lymphocytes / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D010738:Type C Phospholipases / Q000096:biosynthesis*
chemical_list: D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; D015800:Protozoan Proteins; D010738:Type C Phospholipases; D008666:Metalloendopeptidases