CHEMICAL AND FUNCTIONAL-CHARACTERIZATION OF A FRAGMENT OF C1S CONTAINING THE EPIDERMAL GROWTH-FACTOR HOMOLOGY REGION

CĪs, one of the three subcomponents of CĪ, the first component of complement, is a serine protease comprising two disulfide-linked chains, the B chain, containing the catalytic site, and the A chain, involved in Ca2+ binding and Ca2+-dependent interaction(s) with the other CĪ subcomponents. In an attempt to identify the regions responsible for the latter functions, CĪs was submitted to limited proteolysis with plasmin, a treatment that split the A chain into three major fragments, α1,α2, and γ. Fragment α2, which comprised the epidermal growth factor-like (EGF-like) region of CĪs, was heterogeneous, starting at serine 97 or phenylalanine 105 and ending at lysine 195. This fragment was reduced and alkylated and then digested with elastase, and three peptides covering positions 131–135, 131–139, and 131–140 were characterized by amino acid analysis, Edman degradation, and mass spectrometry, showing that position 134 of CĪs is occupied partly by an asparagine (47%) and partly by an eryfArO-β-hydroxyasparagine, in contrast with the homologous position (150) of CĪ r which only contains evyrftro-β-hydroxyasparagine. As measured by equilibrium dialysis, native α2, like the other plasmin-cleavage fragments, did not retain the ability of intact CĪs to bind Ca2+. In the same way, plasmin cleavage abolished the ability of Cïs to dimerize or to associate with CĪr in the presence of Ca2+. In contrast, both al and the N-terminal αl fragment, starting at serine 24 of the A chain, were able to compete significantly with intact CĪs for the formation of the Ca2+-dependent CĪs-CĪr-CĪr-CĪs tetramer. These data indicate that the EGF homologous region of CĪs does not contain all the structural elements required for Ca2+ binding and Ca2+-dependent protein-protein interaction but suggest that these elements are contributed by both the al and α2 regions. © 1990, American Chemical Society. All rights reserved.