Genomic and cDNA cloning of the human C1 inhibitor

Abstract

Amino acid sequencing of trypsin fragments of C1 inhibitor gave regions of low codon degeneracy that were used for oligonucleotide probes. Human liver cDNA libraries gave clones containing most of the protein sequence, showing that the inhibitory domain belongs to the 'serpin' class of protein inhibitors. Fragments of these cDNA clones were used to probe human genomic cosmid libraries. The genomic sequence was found to be about 17 x lo3 base pairs, with a coding sequence of approximately 1800 base pairs containing introns at amino acid positions -6, 162, 207,275, 321, 395, and one in the 5' non-coding region. There is very little similarity of intron position amongst the serpin genes. All but one of the intron positions in the C1 inhibitor structural gene correspond to surface residues if C1 inhibitor is considered to have a structure similar to the cleaved form of al-antiproteinase. The serine and threonine residues in the N-terminal 100 amino acids of the sequence thought to carry complex carbohydrates are found in a single exon.The first component of complement possesses serine proteinase activity in subcomponents C l r and Cls [l], which is inhibited by the plasma protein known as the C1 inhibitor. This inhibitor is a protein molecule of M , 95 000 which contains about 35% carbohydrate and which forms a stoichiometric complex with the serine proteinase it inhibits [2]. It also inhibits other plasma serine proteinases such as factors XIa and XIIa, plasmin and kallikrein.The functional importance of C1 inhibitor is emphasised by the occurrence of the condition known as hereditary angioneurotic oedema in patients having a genetic abnormality of this molecule. The disease can occur in two main forms: type I, in which the molecule is not expressed by the defective gene, and type 11, in which a molecule of abnormal structure is produced which is immunochemically positive but functionally negative [3].Difficulties of purifying adequate quantities of C1 inhibitor in homogeneous form [4], probably because of the large and variable amount of carbohydrate which contains some sialic acid, have inhibited conventional amino acid sequence studies. Until recently there was remarkably little published amino acid sequence work on C1 inhibitor: the N-terminal 40 residues had been determined [5], and a few residues were known in the 'active-site' region [6]. Sequencing of cDNA clones by Tosi et al. [7] has shown recently that the C-terminal domain is a molecule of the serpin class of proteinase inhibitors. Extensive amino acid sequence results combined with cDNA seauence have recentlv been reDorted bv Bock et al.coding for C1 inhibitor has enabled us to determine the amino acid sequence of the inhibitor protein and to screen human genomic libraries for its gene. Probes prepared from fragments of cDNA clones have allowed us to identify the coding regions of the genomic sequence. DNA sequencing of the genomic clones and comparison with the cDNA sequence has given the location of the intron-exon junctions in the structural gene....

Molecular cloning of silkworm (Bombyx mori) antichymotrypsin

“…ITBA, baboon a-1-antitrypsin (Kurachi et al, 1981); CBGH, corticosteroid-binding globulin (Hammond et al, 1987); TBGH, thyroxine-binding globulin (Flink et al. 1986) ; PCIH, protein-C inhibitor (Suzuki et al, 1987); NEXH, human protease nexin (Gloor et al, 1986); NEXR, rat protease nexin (Sornmer et al, 1987); PAIl, plasminogen-activator inhibitor 1 (Pannekoek et al, 1986); RORF, rabbit ORF (Upton et al, 1986) ; OVCH, ovalbumin (McReynolds et al, 1978); DYCH, chicken gene Y (Heilig et al, 1982); PAI2, plasminogen-activator inhibitor 2 (Ye et al, 1987); AT3H, antithrombin 111 (Chandra et al, 1983b); SEVV, vaccinia-virus serpin (Pickup et al, 1986); LU2H, heparin-cofactor I1 (Blinder et al, 1988); AGTH, human angiotensinogen (Kageyama et al, 1984); AGTR, rat angiotensinogen (Ohkubo et al, 1983); A2AP, a-2-antiplasmin (Holmes et al, 1987); CIIH, C1-inhibitor (Carter et al, 1988). Other designations are the same as those used in Fig.…”

Nonsense mutations affect C1 inhibitor messenger RNA levels in patients with type I hereditary angioneurotic edema.

“…It consists of eight exons interrupted by introns that contain a high density of repetitive DNA sequences (Alu repeats), thus making the Cl INH gene susceptible to rearrangements (4). Inherited deficiency of Cl INH is known as hereditary angioneurotic edema (HANE).…”

“…kb long with a coding region of -1,800 bp (2)(3)(4). It consists of eight exons interrupted by introns that contain a high density of repetitive DNA sequences (Alu repeats), thus making the Cl INH gene susceptible to rearrangements (4).…”

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

“…Introduction C1 inhibitor (Cl inh),' a highly glycosylated plasma protein is a member of the serpin family of protease inhibitors (1)(2)(3) P1 residue, i.e., the reactive center residue of the serpins, which is exposed on a loop (4,5), is recognized by the substrate binding site of the target protease. Proteolytic attack on the peptidyl bond carboxy-terminal to the P1 residue ensures the formation of a very stable complex between the inhibitor and the target protease.…”