A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

Abstract

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 (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 ,,ol0-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 comparianentalized 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(lat)Man(lot4)GlcN-(lat)-myo-inositol-l-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-l-negative T-lymphoma are discussed. LYCOSYLPHOSPHATIDYLINOSITOL (GP1) t anchors attach a diverse group of macromolecules to membranes in eukaryotes (see 19, 22 for recent reviews). The plasma membrane of the protozoan parasite Trypmwsoma brucei is covered with about 107 molecules of the variant surface glycoprotein (VSG), a GPI-anchored molePlease address all correspondence to Dr. K. Mensa-Wilmot, Department of Zoology, University of Georgia, Athens, Georgia 30602.1. Abbreviations used in this paper: CRD, cross-reacting determinant; dol-P-Man, dolichol-phosphoryl-mannose; eGPI-PLC, recombinant glycosylphosphatidylinositol phospholipase C; EtN, ethanolamine; gp63, 63-kD GPI-anchored glycoprotein of Leishmania parasites; GIPL, glycoinositol phospholipid; GPI, glycosylphosphatidylinositol; GPI-PLC, glycosylphosphatidylinositol phospholipnse C; Gall, Galactofuranose; GleN, glucosamine; GIcNAC, N-acetyl glucosamine; LP-1, putative protein-GPI precursor (possibly EtN-phospho-ManrGlcN-PI); LP-2, putative protein-GPl precursor; LPG, lipophosphoglycan; Man, mannose; mfVSG, membraneform VSG; PI, phosphatidylinositol; PNH, paroxysmal nocturnal hemoglobinuria; UDP-GIcNAc, uridine 5'-diphospho N-acetylglucosamine; VSG, variant surface glycoprotein of Trypanosoma brucei.cule. The VSG GPI consists of dimyristoylphosphatidylinositol l...

Leishmania mexicanaMutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

“…We have been unable to detect any secretion of gp63 from ⌬GPI8, suggesting that this protein is being degraded intracellularly. In contrast, depletion of intracellular levels of GPI protein anchor precursors by ectopic expression of T. brucei GPI-PLC in L. major resulted in the secretion of a hydrophilic form of gp63 (Mensa-Wilmot et al, 1994). Similarly, cleavage of the signal peptide without anchor addition has been reported in both mammalian and trypanosomal in vitro systems and resulted in the release of processed, but GPI-free, protein (Maxwell et al, 1995a;Ramalingam et al, 1996;Sharma et al, 1999).…”

“…Interestingly, targeted deletion of six of the seven L. major gp63 genes did not affect growth in vitro or prevent the formation of disease in mice (Joshi et al, 1998). An alternative approach to obtaining Leishmania parasites lacking GPI-anchored proteins exploited the finding that ectopic expression of T. brucei GPI-PLC in Leishmania results in the selective degradation of protein anchor GPI precursors (Mensa-Wilmot et al, 1994;Ilgoutz et al, 1999b;Mensa-Wilmot et al, 1999). However, this approach does not result in the complete removal of all GPI-anchored proteins and may also lead to the partial degradation of non-protein-linked GPIs (Mensa-Wilmot et al., 1999) and, therefore, is unable to provide definitive data on the specific functions of GPI-anchored proteins.…”

Endosomes, Glycosomes, and Glycosylphosphatidylinositol Catabolism in Leishmania major

“…GPI-PLCp from T. brucei was highly suitable as the in vivo probe of GPIs because heterologous expression of the enzyme in L. major causes a deficiency of (free) protein-GPI without affecting polysaccharide-GPI metabolism (21,46). L. major expressing GPI-PLCp or cysteine mutants of the enzyme were analyzed for two reasons.…”

“…gp63) that should have been GPIanchored. These proteins enter the ER because of their Nterminal signal peptide but lack signals for retention in the cell (21,22). Polysaccharide-GPIs are not cleaved in vivo by GPIPLCp in L. major (21).…”

“…Heterologous (stable) expression of GPI-PLCp in L. major confers a GPI-negative phenotype on the cells by depleting intermediates of protein-GPI biosynthesis (21). The shortage of prefabricated GPIs for anchoring polypeptides to membranes leads to constitutive secretion of major cell surface polypeptides (e.g.…”