Chlamydomonas agglutinin conjugated to agarose beads as an in vitro probe of adhesion.
Flagellar sexual agglutinins are responsible for the primary recognition and adhesion events of mating in Chlamydomonas reinhardi which culminate in zygotic union of plus and minus gametes. Recent studies in this laboratory have shown the plus agglutinin to be an extremely large (greater than 10(6) D) and asymmetric glycoprotein containing a high proportion of hydroxyproline and serine residues [14, 27, 28]. This paper reports an improved method for in vitro investigations of the adhesive nature of this molecule. Purified agglutinin is covalently attached to an insoluble (Affi-gel 15 agarose bead) support and shown to retain potent agglutination activity when presented to living minus gametes, which rapidly and extensively adhere to the coated bead surface by their flagella. The specificity of the response is documented by the lack of interaction of plus gametes with the immobilized plus agglutinin (IA+). Using this simple yet sensitive bioassay, we have subjected IA+ beads to various enzymatic, chemical and physical treatments and assessed the effects on agglutinin activity. These studies reveal that Chlamydomonas plus agglutinin is sensitive to thermolysin or trypsin digestion, alkaline borohydride reduction, periodate oxidation, thiol reduction and heating at 65 degrees C, but unaffected by treatment with chymotrypsin, endo- or exoglycosidases, or incubation with isolated minus agglutinin. The implications of these results for agglutinin structure and possible functional interactions in initial recognition/adhesion events are discussed.
D000268:Adhesiveness; D000371:Agglutination; D001681:Biological Assay; D002696:Chlamydomonas / Q000032:analysis / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006358:Hot Temperature; D000072235:Mating Factor; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000494:pharmacology; D010455:Peptides / Q000032:analysis / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D010455:Peptides; D013439:Sulfhydryl Reagents; D000072235:Mating Factor; D006026:Glycoside Hydrolases; D010447:Peptide Hydrolases