Structural and functional studies on the human C3b/C4b receptor (CR1) purified by affinity chromatography using a monoclonal antibody.
A procedure was devised that has several advantages over previously described methods to purify CR1 from both erythrocytes (E) and the HL-60 promyelocytic cell line. Using a monoclonal antibody immunoaffinity column, CR1 was purified to homogeneity as assessed by silver staining and 2-D gel analysis. Protein purified by this method comigrates on SDS-PAGE with 125I surface-labeled CR1 isolated by immunoprecipitation or iC3-Sepharose affinity chromatograhy and can be specifically immunoblotted with a second monoclonal anti-CR1 antibody. Employing this method, CR1 can be purified to homogeneity in amounts adequate for both functional studies and biochemical microanalysis. Purified E CR1 is functionally active as assessed by its ability to specifically rebind to an iC3-Sepharose affinity column, act as a cofactor for I-mediated cleavage of C3b to C3c and C3d, g and to accelerate decay of both the classical and alternative pathway C3 convertases. Its specific activity is similar to that of CR1 purified by a method not employing the potentially denaturing washing and eluting conditions of immunoaffinity chromatography. The pIs of the two major E CR1 allotypes are both approximately 7.1. Using pooled human E CR1, an amino acid composition was derived which revealed a relatively high proline content. This has also been found in two functionally related and genetically linked complement-regulatory proteins, H and C4-binding protein, NH2-terminal sequencing of E CR1 and HL-60 CR1 was unsuccessful indicating that the NH2-terminus is blocked.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D003179:Complement C3b / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism*; D015935:Complement C4b; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011951:Receptors, Complement / Q000302:isolation & purification / Q000378:metabolism*; D017463:Receptors, Complement 3b
D000911:Antibodies, Monoclonal; D003181:Complement C4; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D003179:Complement C3b; D015935:Complement C4b