The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.
The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D003166:Complement Activating Enzymes / Q000276:immunology / Q000378:metabolism; D011415:Complement Factor B / Q000378:metabolism; D011416:Complement Factor D / Q000276:immunology / Q000378:metabolism; D003170:Complement Pathway, Alternative; D006461:Hemolysis; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D009842:Oligopeptides / Q000378:metabolism; D011817:Rabbits
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D003166:Complement Activating Enzymes; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B