Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb.
Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.
D000911:Antibodies, Monoclonal / Q000502:physiology; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003167:Complement Activation; D050577:Complement C3-C5 Convertases / Q000276:immunology / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism; D003180:Complement C3b Inactivator Proteins / Q000502:physiology; D011415:Complement Factor B / Q000378:metabolism; D017242:Complement Factor H; D003170:Complement Pathway, Alternative; D004347:Drug Interactions; D006461:Hemolysis; D006801:Humans; D010447:Peptide Hydrolases / Q000502:physiology*; D011414:Properdin / Q000502:physiology
D000911:Antibodies, Monoclonal; C493495:CFH protein, human; D003180:Complement C3b Inactivator Proteins; D011414:Properdin; D003179:Complement C3b; D017242:Complement Factor H; D003166:Complement Activating Enzymes; D010447:Peptide Hydrolases; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B