Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Abstract.

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development.