Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator

Abstract

One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the α'-chain was radioactively labelled with iodo[2-14C]acetic acid. The α'-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. C4c was reduced and alkylated and the two α'-chain fragments of C4c were separated. The molecular weights, amino acid analyses and carbohydrate content of the three α'-chain fragments were determined. C4d has a mol.wt. of 44.500 and a carbohydrate content of 6%. The two α'-chain fragments of C4c have mol.wts. of 25,000 (α3) and 12.000 (α4) and carbohydrate contents of 10 and 22% respectively. The N-terminal amino acid sequences of C4d, the α3 and the α4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b α'-chain, the 25.000-mol.wt. fragment (α3) was shown to be derived from the N-terminal part of the α'-chain. C-Terminal analyses were done on the α'-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the α3 fragment. The C-terminal residue of the α'-chain and of the α4 fragment could not be identified. The order of the three fragments of the α'-chain is therefore: α3(25.000) - C4d(44.500) - α4(12.000). The specificity of C3bINA is for an Arg-Xaa peptide bond.