Characterization of Toxoplasma gondii surface antigen I (SAGI) secreted from Pichia pastoris: evidence of hyper O-glycosylation

A truncated form of surface antigen I (SAGIt), the immunodominant surface a ntigen of Toxoplasmo gondii, was expressed in the methylotrophic yeast, Pic hia pastoris. The truncated protein lacked the C-terminal residues which, i n native SACI, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG It was efficiently secreted into the culture medium as three protein sp ecies having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneit y was dependent upon the composition of the medium used to grow the yeast t ransformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAGIt he terogeneity was related to the presence of O-linked oligosaccharides contai ning alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAGIt were recognized by monoclonal and human-s erum-derived antibodies, specific for the native SAGI, which suggested that the O-glycosylations had no major effect on the protein conformation. Howe ver, ELISA and Western-blot analysis with human sera showed that the O-carb ohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SA G It species or deglycosylation is required in order to use recombinant SAG I as a diagnostic reagent. Moreover, the presence of carbohydrates, not fou nd on the native protein, suggests that addition of unnatural glycan struct ures by P. pastoris is a potential drawback that should be considered when using this expression system.