Precursor complement protein (pro-C4) is converted in vitro to native C4 by plasmin

Abstract

The fourth component of complement (C4) is a constituent of the classical serum complement pathway, one of the principle effectors of inflammatory reactions. A single chain protein (molecular weight (MW) 200,000), presumed to be the precursor of C4 (pro-C4), was first identified as a product of cell-free translation on polysomes from guinea pig liver homogenate¹. Pro-C4 has since been detected in human^2,3 and guinea pig⁴ plasma and intracellularly in macrophage⁵ and liver cultures¹. Native C4 is composed of three polypeptide chains of unequal size and linked by disulphide bridges—the α, β, and γ chains which have MWs of approximately 95,000, 75,000 and 31,000 respectively (ref. 6). Kinetic studies of C4 biosynthesis in tissue culture, a comparison of peptide maps from tryptic digests⁷ and identity of the amino-terminal segments of guinea pig pro-C4 with β chain of guinea pig and human C4 (ref. 8) suggested that pro-C4 was a precursor of C4. However, conversion of pro-C4 to native C4 has not been demonstrated directly. We now report that the addition of plasmin to radiolabelled pro-C4 (in cell lysates of guinea pig macrophages) generated a protein with the characteristic size and subunit structure of native C4, establishing the precursor–product relationship between these proteins. These data also suggest that a plasmin-like enzyme is responsible for post-translational conversion of this procomplement (pro-C4) protein to C4 in vivo. Another enzyme, the C1s subunit of the first component of complement, cleaved pro-C4 at a single site, distinct from the plasmin cleavage sites, to generate fragments of approximate MWs 112,000 and 91,000. This experiment indicated that β-α-γ is the sequence of subunits in pro-C4.