ANALYSIS OF TOXOPLASMA-GONDII STABLY TRANSFECTED WITH A TRANSMEMBRANEVARIANT OF ITS MAJOR SURFACE PROTEIN, SAG1

We have genetically engineered Toxoplasma gondii so that its major sur face antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM ) instead of its natural GPI anchor (SAG1-GPI) in order to initiate st udies to address the function of this protein anchor in parasitic prot ozoa 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 t he surface of the parasite, indicating that the GPI anchor does not de termine its localization nor overall three-dimensional structure. No s ignificant difference was seen in any aspect of the growth of the SAG1 -TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does no t 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 SAG 1-GPI suggesting a stabilizing function of the glycolipid anchor again st degradation and/or membrane release, Antibodies to SAG1 are shed fr om SAG1-TM parasites as they invade, just as they are stripped from SA G1-GPI bearing parasites, The stripping, therefore, is unlikely to be driven by the action of lipases.