Functional studies on the secreted form of human C4 (C4s), two incompletely processed two-subunit C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and pro-C4.
The functional properties of the secreted form of C4 (C4s), which has a Mr approximately 5000 greater than the predominant C4 molecule found in plasma (C4p), two incompletely processed two-chain C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and the extracellular C4 precursor (designated pro-C4(E)] were evaluated. All four molecules are secreted in parallel by a human hepatoma-derived cell line (Hep G2). Secretion of hemolytically active C4 is linear up to approximately 12 hr, peaks at 24 hr, and then progressively decreases over the next 48 hr. This loss of C4s functional activity parallels the proteolytic conversion of C4s to C4bs. To compare the hemolytic efficiencies of C4s and C4p, a solid-phase competitive radioimmunoassay was developed to permit measurement of the small quantities of C4 antigen in these cultures. The hemolytic efficiencies of C4s and C4p were similar. These results indicate that extracellular processing of C4s to C4p does not modulate the hemolytic activity of the molecule. Consistent with their ability to bind methylamine, both the alpha s-chain and the alpha - gamma subunit undergo denaturation-induced autolysis. The extracellular and intracellular pro-C4 molecules are also sensitive to autolytic cleavage. Interestingly, the beta - alpha subunit is resistant to autolysis. In experiments in which C4s and C4p were cleaved by C1-s to C4bs, C4(beta - alpha + gamma), C4(beta + alpha - gamma), and pro-C4(E) were resistant to C1-s cleavage and thus hemolytically inactive relative to C4s. These data indicate that processing of C4 to a three-chain structure is required to provide the proper conformation for efficient activation by C1.
D000818:Animals; D001329:Autolysis; D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003166:Complement Activating Enzymes / Q000378:metabolism; D003173:Complement C1s; D003181:Complement C4 / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism* / Q000502:physiology; D011489:Protein Denaturation; D011498:Protein Precursors / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology
D003181:Complement C4; D010446:Peptide Fragments; D011498:Protein Precursors; D003166:Complement Activating Enzymes; D003173:Complement C1s