Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23.
In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidylinositol-specific phospholipase C(PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPI-core glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A2 (PLA2) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolamine-PO4-6-Man alpha1-2-Man alpha1-6-Man alpha1-4-GlcN alpha1-6-PI) bearing beta-linked N-acetylgalactosamine residue(s).
Tomavo
S
S
Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps- Universitaet Marburg, Germany.
Dubremetz
J F
JF
Schwarz
R T
RT
D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D004075:Diglycerides / Q000032:analysis; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007294:Inositol / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014122:Toxoplasma / Q000378:metabolism*; D014316:Tritium; D014709:Vero Cells
D000953:Antigens, Protozoan; D004075:Diglycerides; D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D009227:Myristic Acids; D010169:Palmitic Acids; D015800:Protozoan Proteins; D019814:Myristic Acid; D014316:Tritium; D019308:Palmitic Acid; D007294:Inositol; D019856:Ethanolamine