Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation.
A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, 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 SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.
Letourneur
O
O
bioMérieux, R&D, Unité Immunoessais, Chemin de l'Orme, 69280 Marcy l'Etoile, France. odile_letourneur@eu.biomerieux.com
Gervasi
G
G
Gaïa
S
S
Pagès
J
J
Watelet
B
B
Jolivet
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid / Q000379:methods; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000276:immunology*
D000953:Antigens, Protozoan; D017931:DNA Primers; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C047168:SAG1 antigen, Toxoplasma