CHARACTERIZATION OF A DENSE GRANULE ANTIGEN OF TOXOPLASMA-GONDII (GRA6) ASSOCIATED TO THE NETWORK OF THE PARASITOPHOROUS VACUOLE

This work describes the molecular characterization of GRA6, a novel To xoplasma gondii dense granule antigen of 32 kDa. cDNA clones encoding this protein were isolated using a rat serum directed against an HPLC fraction enriched in the protein GRAS. Cross-reactivity between GRAS a nd GRA6 was demonstrated by production of sera against the recombinant GRAS protein. A serum against a recombinant fragment of GRA6 which do es not react with GRAS allowed the localization of this antigen at the subcellular level. GRA6 is detected in the dense granules of tachyzoi tes, and in the parasitophorous vacuole, closely associated to the net work. The gene encoding GRA6 and its flanking regions were completely sequenced from cDNA and genomic inserts. Primer extension experiments demonstrated that the cap site of the GRA6 gene was located 37 bp upst ream of the 5' end of the longest cDNA insert (1600 bp). The GRA6 gene potentially encodes a 230-amino-acid polypeptide, does not contain an y introns and seems to be present as a single copy in the genome of T. gondii. The deduced polypeptide contains two hydrophobic regions with the characteristics of transmembrane domains. The N-terminal domain d oes not fit the classical feature of a signal peptide. The central hyd rophobic domain is flanked by two hydrophilic domains which contain fo ur blocks of amino acids homologous to the GRAS protein. The C-termina l hydrophilic region comprises 24% of glycine residues, which may indi cate a structural role for GRA6 in the network.