Isolation and partial characterization of an aggregation-competent stage-specific antigen prepared by phenol/water extraction from Dictyostelium discoideum

Abstract

The phenol/water extract of aggregation-competent cells of Dictyostelium discoideum contained a stage-specific antigen which was undetected in the extract of growth-phase cells. The anti cell homogenate serum which was absorbed with the extract of the growth-phase cells (anti H serum) formed a single arc with the phenol/water extract of the aggregation-competent cells by immunoelectrophoresis. However, the extract from growth-phase cells or cells treated with tunicamycin, a selective inhibitor of the glycosylation of N-glycosyl protein, did not react with the anti H serum. The antigen showed an affinity for concanavalin A and was partially purified by gel filtration on Sepharose 4B. [ 3H]Mannose-labeled antigen was purified from the metabolically labeled extract by immunoaffinity chromatography on anti H IgG-conjugated Sepharose, and showed a single radioactive peak with a molecular weight of 68 000 on analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The immunodeterminant groups of the antigen were partially characterized by radioimmunoassay. The reactivity of the antigen against anti H serum decreased significantly after treatment with exo-α-mannosidase. Pronase or exo- β- N-acetylhexosaminidase treatment slightly decreased the reactivity; however, neuraminidase, β-galactosidase, α- l-fucosidase or mild alkaline treatment had no effect. These results indicate that the antigenic reactivity of the stage-specific antigen mainly depends upon α-mannosyl residues at the non-reducing terminal of the asparagine-linked oligosaccharide moiety.