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A Glycosylphosphatidylinositol (GPI)-Negative Phenotype Produced in Leishmania major by GPI Phospholipase C from Trypanosoma brucei: Topography of Two GPI Pathways

Kojo Mensa-Wilmot, Jonathan H. LeBowitz, Kwang-Poo Chang, Ahmed Al-Qahtani, Bradford S. McGwire, Samantha Tucker and James C. Morris
The Journal of Cell Biology
Vol. 124, No. 6 (Mar., 1994), pp. 935-947
Stable URL: http://www.jstor.org/stable/1616097
Page Count: 13
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A Glycosylphosphatidylinositol (GPI)-Negative Phenotype Produced in Leishmania major by GPI Phospholipase C from Trypanosoma brucei: Topography of Two GPI Pathways
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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 ∼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(1α6)Man(1α4)GlcN-(1α6)-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.

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