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

Abstract

Human Cls proenzyme (Mr 83000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent Cls by ion-exchange chromatography on DEAE-Sephacel. Single-chain Cls proenzyme was activated to two-chain Cis with self-activated Cir. After reduction and S-carboxamidomethylation the heavy chain of Cis (Mr 57000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of Cis 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. Cis heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the a2 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 Cls proenzyme a fragment was isolated which overlaps the Cis 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 Cis.

Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains.

“…This tetramer in turn interacts with the single recognition subunit Clq (25). A partial amino acid sequence of the Cls heavy chain has been published (26), which shows high homology with the amino acid sequence of Clr heavy chain. Recently, a nearly full-length cDNA clone for Cls has been produced (27); although neither the nucleotide sequence nor the deduced amino acid sequence was given, it was stated that, like Cir, Cls has an EGF-like domain with an asparagine in the same position as that which is hydroxylated in Cir.…”

Neutron scattering study of the (γ‐B) catalytic domains of complement proteases Cl̄r and Cl̄s

“…The catalytic subunit of human Cl, the first component of the classical pathway of complement, is a Ca2+-dependent tetrameric association (Cls-Clr-Clr-Cls) of two serine proteases, Clr and Cls, which are sequentially activated upon binding of C 1 to activators [l-3]. Activation occurs in both cases through cleavage of a single Arg-Ile bond [4,5], that converts single chain proenzymes into active enzymes (Cir, cis) comprising two disulphide-linked chains, A (N-terminal) and B (Cterminal). Each monomeric protease is thought to be organized in at least two functional domains or regions: (i) an interaction region (cr) corresponding to the Nterminal half of the A chain, responsible for Ca2+-dependent C 1 r-C 1 s interaction; (ii) a catalytic domain, comprising the intact B chain and the C-terminal 7 region of the A chain, connected to each other through a single disulphide bridge (Fig.…”

Analysis of the N‐linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry

“…To our knowledge, however, no case of a single Olinked pentose residue has ever been described. The hypothesis that the extra mass arises from heterogeneity at the sequence level appears unlikely, considering that no such heterogeneity has ever been detected by sequence analyses performed at the cDNA [4,5] or protein [21] levels. Further analyses will be required to identify and locate this minor modification.…”