Cross-reactivity of contact site A to antibody produced against a stage-specific antigen from a phenol/water extract of Dictyostelium discoideum.

The stage-specific antigen, gp68 (Hirano, T., Yamada, H., & Miyazaki, T. (1983) Biochim. Biophys. Acta 742, 224-234), was purified from a phenol/water extract of aggregation-competent cells of Dictyostelium discoideum by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Anti-gp68 was produced against purified gp68 which was determined to be homogeneous by silver staining on analysis by SDS-polyacrylamide gel electrophoresis. The cross-reactivity of anti-gp68 against cellular antigens was estimated by immuno-affinity chromatography on anti-gp68 immunoglobulin G (IgG)/Sepharose. When the whole cell lysate was applied to this affinity column, three major proteins, with molecular weights of 80,000, 68,000, and 56,000, were obtained in the absorbed fraction. When the butanol extract, which was enriched in contact site A, an adhesion mediating glycoprotein, was applied to the same column, two major proteins with molecular weights of 80,000 (corresponding to contact site A) and 56,000 were obtained in the absorbed fraction but, however, gp68 was negligible. Reversely, when the phenol/water extract was applied to anti-contact site A-IgG/Sepharose, only gp68 was obtained in the absobed fraction. Moreover, contact site A was seen to compete with [3H]mannose-labeled gp68 in a competition radioimmunoassay using anti-gp68 serum. The effect of Pronase or exo-alpha-mannosidase digestion on the antigenic activity of gp68 was examined by radioimmunoassaying. The results indicated that the alpha-mannosyl residue of the non-reducing terminal in the carbohydrate moiety of gp68 was a major immunodeterminant. However, the polypeptide chain did not participate in the antigenic reactivity against anti-gp68. Both anti-gp68 and anti-contact site A agglutinated heat killed-yeast cells. Also, both anti-sera inhibited EDTA-stable cell adhesion of aggregation-competent cells in the presence of Fab from goat anti-rabbit IgG. These results indicate that gp68 and contact site A have a common antigenic determinant against anti-gp68, and that the target antigen of anti-gp68 was somehow involved in cell adhesion.