The SAG5 locus of Toxoplasma gondii encodes three novel proteins belonging to the SAG1 family of surface antigens

Abstract We have identified three novel Toxoplasma gondii proteins showing close structural similarity to molecules of the SAG1 family, a group of glycosylphosphatidylinositol-anchored surface antigens expressed by the invasive stages of T. gondii . The novel proteins, denominated SAG5A, SAG5B and SAG5C, are encoded by tandemly arrayed and tightly clustered genes containing no introns. The 367 amino acid-long SAG5B and SAG5C are 97.5% identical to each other, whereas SAG5A (362 amino acids) consists of a C-terminal domain sharing 98% identity with SAG5B and SAG5C, and an N-terminal domain whose identity to the other SAG5 polypeptides is only 42%. Expression analysis of the T. gondii strains RH (virulent) and 76K (avirulent) showed that all members of the SAG5 cluster are transcribed in T. gondii tachyzoites and bradyzoites. However, immunoblot studies on the RH strain revealed that the synthesis of SAG5A does not occur in tachyzoites and is possibly controlled at the post-transcriptional level. On the contrary, SAG5B and SAG5C were detected by immunoblot in tachyzoite lysates and found to migrate in the 40–45 kDa range under reducing conditions or at approximately 34 kDa under unreduced conditions. Triton X-114 partitioning of tachyzoite protein lysates treated with phosphatidylinositol-specific phospholipase C indicated that SAG5B and SAG5C are glycosylphosphatidylinositol-anchored membrane-associated molecules. Consistently, immunofluorescence analysis of transformed tachyzoites over-expressing SAG5B or SAG5C showed that these molecules are targeted to the parasite surface. The characterisation of the SAG5 locus sheds further light on the complex repertoire of SAG1-related genes in T. gondii , that now comprises 14 highly homologous members and five distantly related genes belonging to the SAG2 family.