Monoclonal antibodies directed against the sexual binding site of Chlamydomonas eugametos gametes.
Monoclonal antibodies were raised against the mt- sexual agglutinin of Chlamydomonas eugametos gametes. Those that blocked the agglutination site were selected. They were divided into two classes dependent upon whether they gave a weak (class A) or clear positive (class B) reaction with mt- flagellar membranes in an ELISA and an indirect immunofluorescence test using glutaraldehyde-fixed mt- gametes. Class A antibodies were shown to be specific for the agglutinin in an extract of mt- gametes, based on results from immunoblotting, immunoprecipitation, affinity chromatography, and the absence of a reaction with nonagglutinable cells. Surprisingly, class A mAbs also recognized two mt+ glycoproteins, one of which is the mt+ agglutinin. Class B antibodies were shown to bind to several glycoproteins in both mt- and mt+ gametes, including the mt- agglutinin. Fab fragments from class A mAbs blocked the sexual agglutination process, but those from class B did not, even though the parent antibody did. We conclude that the class A epitope lies in or close to the agglutination site of the mt- agglutinin, whereas the class B epitope lies elsewhere on the molecule. We also conclude that the mt- agglutinin is the only component on the mt- flagellar surface directly involved in agglutination. Class A mAbs were found to elicit several reactions displayed by the mt+ agglutinin. They bound to the mt- agglutinin on gamete flagella and induced most of the reactions typical of sexual agglutination, with the exception of flagellar tip activation. None of these reactions was induced by Fab fragments. High concentrations of class A mAbs completely repressed the sexual competence of live mt- gametes, but low concentrations stimulated cell fusion.
Homan
W
W
Department of Plant Physiology, University of Amsterdam, The Netherlands.
Musgrave
A
A
de Nobel
H
H
Wagter
R
R
de Wit
D
D
Kolk
A
A
van den Ende
H
H
D000371:Agglutination; D000373:Agglutinins / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002459:Cell Fusion; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000648:ultrastructure; D002696:Chlamydomonas / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005407:Flagella / Q000276:immunology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000276:immunology; D006825:Hybridomas; D007118:Immunoassay
D000373:Agglutinins; D000911:Antibodies, Monoclonal; D006023:Glycoproteins