Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1.
We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.
Seeber
F
F
Fachbereich Biologie/Parasitologie, Philipps-Universitat Marburg, Marburg, Germany.
Dubremetz
J F
JF
Boothroyd
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000276:immunology; D014122:Toxoplasma / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma