A calcium-binding monoclonal antibody that recognizes a non-calcium-binding epitope in the short consensus repeat units (SCRs) of complement C1r.
C1r is a Ca(2+)-binding serine protease that interacts with two other plasma proteins, C1q and C1s, to form C1, the first component of the complement cascade. A monoclonal antibody, BG6, has been produced which binds to C1r only in the presence of Ca2+, requiring 3-5 microM Ca2+ for half-maximal binding. The antibody reacts with native and heat-denatured C1r, and with zymogen C1r, but does not cross-react with C1s or C1q. BG6 did not significantly affect the esterolytic activity of C1r toward a synthetic thioester substrate nor the hemolytic activity of C1 reconstituted from subcomponents in the presence of the antibody. A tryptic fragment of C1r which consists of the C-terminal gamma region of the A chain disulfide-linked to the B chain (gamma B) binds in a Ca(2+)-dependent manner to BG6-Sepharose. Western blotting experiments have further localized the epitope to the gamma region of the A chain, which is composed of two short consensus repeat (SCR) units. The N-terminal alpha region contains the only previously determined Ca(2+)-binding site in the C1r molecule. Equilibrium dialysis experiments confirmed that C1r-gamma B does not bind Ca2+, and showed that antibody BG6 and the gamma B/BG6 complex do bind Ca2+. Thus, the Ca(2+)-dependent nature of this interaction is due exclusively to binding of the metal ion to the antibody. Equilibrium dialysis and immunoblotting have further localized the Ca(2+)-binding site to the Fab fragment of BG6, indicating that the metal-induced conformational change residues in or near the variable region of the IgG. BG6 may set a precedent for the preparation of Ca(2+)-dependent antibodies to non-Ca(2+)-binding epitopes in other proteins.
Biochemistry Laboratory, American Red Cross Biomedical Research and Development, Rockville, MD 20855.
S L
SL
Ward
K C
KC
Ingham
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D015923:Complement C1r / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D000939:Epitopes; D010446:Peptide Fragments; D015923:Complement C1r; D002118:Calcium