Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

Abstract

We have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp8O) of Dictyostelium discoideum. gp8O has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp8O. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp8O affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp8O. Although gp8O itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.

“…A few of these antigenic proteins are present in vegetative cells and exhaustive absorption of this serum with vegetative cells removes some but not all immunological reactivity to gp80 (11). However, the exhaustively absorbed serum is no longer found to block EDTA-resistant intercellular adhesion (11).…”

“…Rabbit serum prepared against purified gp8O reacts not only with gp8O but with several other proteins, apparently due to the presence of shared antigenic determinants (11,12). A few of these antigenic proteins are present in vegetative cells and exhaustive absorption of this serum with vegetative cells removes some but not all immunological reactivity to gp80 (11).…”

“…A few of these antigenic proteins are present in vegetative cells and exhaustive absorption of this serum with vegetative cells removes some but not all immunological reactivity to gp80 (11). However, the exhaustively absorbed serum is no longer found to block EDTA-resistant intercellular adhesion (11). These results raised the possibility that some determinants on gp8O might not be critical to the mechanism of adhesion, whereas others are clearly crucial as shown by the ability of purified gp8O alone to neutralize the adhesion-blocking activity of the antiserum.…”

“…However, the exhaustively absorbed serum is no longer found to block EDTA-resistant intercellular adhesion (11). These results raised the possibility that some determinants on gp8O might not be critical to the mechanism of adhesion, whereas others are clearly crucial as shown by the ability of purified gp8O alone to neutralize the adhesion-blocking activity of the antiserum.Monoclonal antibodies have been raised against gp8O (11,14,15 described (11). Amoebae were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to 10-3 to 10-4 survival and were immediately plated into microtest plates at about 1 cell per well as described previously (6).…”

“…Monoclonal antibodies have been raised against gp8O (11,14,15 described (11). Amoebae were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to 10-3 to 10-4 survival and were immediately plated into microtest plates at about 1 cell per well as described previously (6).…”