title: Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation.
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 chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s 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. C1s 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 C1s 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 C1s.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003166:Complement Activating Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D003167:Complement Activation; D015923:Complement C1r; D003173:Complement C1s; D003488:Cyanogen Bromide; D004789:Enzyme Activation; D004792:Enzyme Precursors; D006801:Humans; D006868:Hydrolysis; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis
chemical_list: D000596:Amino Acids; D004792:Enzyme Precursors; D010446:Peptide Fragments; D003166:Complement Activating Enzymes; D015923:Complement C1r; D003173:Complement C1s; D003488:Cyanogen Bromide