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 compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. 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
Subject(s)
Humans , Endoplasmic Reticulum , Phosphatidylinositols/biosynthesis , Glycolipids/biosynthesis , Hemoglobinuria, Paroxysmal/metabolism , Leishmania tropica , Trypanosoma brucei brucei , Type C Phospholipases , Cell Line , Phosphatidylinositols/metabolism , Glycolipids/metabolism , Mammals , Variant Surface Glycoproteins, TrypanosomaABSTRACT
The genetic lesion in paroxysmal nocturnal hemoglobinuria (PNH) cells resides in a DNA element that 1) encodes a product required for assembly of GlcNAc-inositol phospholipid and 2) is commonly affected in different patients. In this study, three alternative mRNA transcripts (1600, 1200 and 950 bp) that derive from this genetic element in normal cells were characterized. The 1200-bp transcript was found to arise from splicing out of 374 bp of exonic sequence extending from positions 407-780. The 950-bp transcript was found to arise from removal of this and 284 bp of additional exonic sequence beginning further upstream at position 123. Analyses of transcripts expressed in Epstein-Barr virus (EBV)-transformed B lymphocytes prepared from two PNH patients showed that both failed to express normal 1600-bp transcripts. One expressed truncated transcripts of 1000 and 800 bp generated by an alternate splice which utilized a downstream signal in place of the normal intronic splice signal. The other expressed a 1600 bp-transcript with multiple nucleotide changes but normal 1200- and 950- bp "spliced" transcripts