Identification and structural characterization of two incompletely processed forms of the fourth component of human complement.

Abstract

A B S T R A C T Immunoprecipitates of human C4 fromEDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the a-chains of the secreted (C45) and predominant plasma (C4P) forms of C4, two additional molecules with apparent molecular weights of -168,000 (p168) and '125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of -5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or a2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C45 and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14±4.8 and 21±9.2% (mean±SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4'. Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the ,B-and a-chains and between p125 and the a-and -y-chains. Partial NIH2-terminal sequencing revealed that the ,-chain was NH2-terminal in p168 and that the a-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent unThis work was presented, in part, at

C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells

“…90, 70 and ^0 kD, corresponding to pro-C4 [361 and C4 i:, {i and y chains, were identified in the GMC culture supernatant (F^ig, 2a). The band of ^^ 160 kD precipitated by anli-C4 probably represents an s fi intermediate of C4 [36], The eKtraeellular C4 ot. (i and y chains were more strongly detected in IF^N-y-trealed cells compared with untreaied control eells (Fig, 2a), Kinetic studies indicated ihat there wa^ an initial increase ir.…”

Purification and Characterisation of the Fourth Component of Bovine Complement

“…Trypsin Digestion, Radiolabelling with ,4C Methylamine and Fluorography C4 protein, immunoprecipitated from bovine plasma, was reacted with l4C methylamine hydro chloride as described by Chan and Atkinson [17]. This protein was then subjected to SDS-PAGE, or first partially digested with trypsin and then electrophoresed.…”

Counterregulatory effects of interferon-gamma and endotoxin on expression of the human C4 genes.

“…3). Radiolabeled C4 a and A chains and an a-/3 intermediate (27) were detected in the extracellular fluid of IFN-y-activated monocytes. The C4 y chain was also detected in other experiments (data not shown).…”