Probing functional sites on complement protein B with monoclonal antibodies. Evidence for C3b-binding sites on Ba.
We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D003166:Complement Activating Enzymes / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism*; D011415:Complement Factor B / Q000276:immunology* / Q000378:metabolism; D004792:Enzyme Precursors / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D000939:Epitopes; D010446:Peptide Fragments; D003179:Complement C3b; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B