Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii

Abstract

The monoclonal antibody (mAb) TG17.179 recognizes an excreted-secreted antigen (ESA) of 28.5 kDa named Gra 2, which is stored in the dense granules of Toxoplasma cells and secreted into the parasitophorous vacuole after host cell invasion. Screening of an expression cDNA library with TG17.179 led to the isolation of several clones, the longest one (clone L) being of 1030 bp. Clone L cDNA was found to be homologous to a previously described composite cDNA encoding a P28 protein of Toxoplasma gondii. Characterization of one genomic clone indicates that the complete GRA 2 gene is about 1.3 kb in length, including an intron of 241 bp. Northern blot and primer extension analyses confirmed the size of the mature messenger (1.1 kb). Amino acid partial sequencing of the native antigen purified by HPLC and metabolic radiolabelings of ESAs perfectly matched the primary amino acid structure deduced from the clone L cDNA. This primary translation product consists of an 185 amino acid polypeptide (19.8 kDa) including a 23 amino acid signal sequence. The presence of many serine and threonine residues may indicate an O-glycosylation. The predicted mature polypeptide shows an internal helical domain with 2 amphipathic alpha-helices. These might be involved in the association of Gra 2 with the membranous network within the parasitophorous vacuole.