Human complement component C1-s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

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Abstract
Human C1s proenzyme (Mr 83,000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromtography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C.hivin.1 with self-activated C.hivin.1r. After reduction and S-carboxamidomethylation the heavy chain of C.hivin.1s (Mr 57,000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C.hivin.1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C.hivin.1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the .alpha.2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C.hivin.1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C.hivin.1s.