Human inhibitor of the first component of complement , Cl : Characterization of cDNA clones and localization of the gene to chromosome 11 ( protease inhibitors / serpins ) ALVIN

C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (Cl) by inactivation of the serine protease subcomponents, COr and Cls. C1 inhibitor cDNA clones have been isolated, and one of these (pClINH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with ail-antitrypsin and a1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of Bamffl-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11. protease inhibitors, the available sequence data do not yet confirm a relationship. Hereditary angioneurotic edema is a dominantly inherited disease caused by apparent heterozygous deficiency of C1 inhibitor (16-18). There are two forms of the disease, types I and II (19). Type I is characterized by diminished plasma levels of an apparently normal C1 inhibitor protein. In type II hereditary angioneurotic edema, a dysfunctional C1 inhibitor protein that is unable to inhibit C1 activity is present at levels varying from 30% to 400% of normal, as measured antigenically. To determine the structural basis of C1 inhibitor function, to define its evolutionary relationship to other protease inhibitors, and to analyze the molecular genetic basis of hereditary angioneurotic edema, we have begun to examine the C1 inhibitor gene. In this report, we have characterized a C1 inhibitor cDNA clone that represents approximately half the coding sequence of the protein, and we have compared the sequence of this clone with the analogous regions of other protease inhibitors. In addition, we provide evidence that the human C1 inhibitor gene is on chromosome 11. C1 inhibitor regulates the activity of the first component of complement (Cl) by inhibition of the proteolytic activity of its subcomponents Clr and Cls. This prevents the activation ofC4 and C2 by Cls, thereby providing an important control over classical pathway activation. In addition to Cir and Cls, C1 inhibitor also inhibits several other serine proteinases including plasmin, kallikrein, and coagulation factors XIa and XIIa (1-4). Although each ofthese other enzymes is inhibited by other protease inhibitors, Clr and Cls are inhibited only by C1 inhibitor, which functions in a manner similar to the other plasma protease inhibitors in that it forms an apparent covalent complex with Clr and Cls in an equimolar ratio (5-7). This results in the dissociation of macromolecular C1, with the release of Clr-Cls-(C1 inhibitor)2 complexes (8-10). C1 inhibitor is the most heavily glycosylated plasma protein, containing about 35% carbohydrate by weight (11, 12). Its carbohydrate composition suggests that it has a number of 0-glycosidic-linked oligosaccharide units (12). It consists of a single polypeptide chain with an apparent Mr of 105,000 as determined by NaDodSO4/PAGE (5, 11-14). The amino-terminal amino acid sequence (40 residues) has been reported, as has the sequence (12 residues) surrounding the reactive center (12, 15). Although the mechanism of protease inhibition by C1 inhibitor is analogous to that of other plasma MATERIALS AND METHODS Protein and Peptide Isolation. C1 inhibitor was isolated from normal human plasma as described (12). Digestion with CNBr was done by incubation of salt-free reduced and alkylated protein with CNBr (100-fold molar excess over total methionine) in 70% formic acid. Peptides were separated by initial gel filtration on Sepharose CL-6B (2.5 x 170 cm) equilibrated in 0.2 M Tris/5 mM EDTA/6 M guanidine HCl, pH 8.0. The pool of smaller peptides from this column was fractionated by HPLC on an Aquapore RP300 column (Brownlee Labs, Santa Clara, CA) equilibrated in 0.1% CF3COOH and developed with a linear acetonitrile gradient to 70%. Amino Acid Sequence Analysis. Automated Edman degradations were performed with a Beckman 890C sequencer modified with a cold trap. A 0.1 M Quadrol program was used (20). Conversion was performed with methanolic HCl (1 part acetyl chloride and 7 parts methanol at 65°C for 10 min). Phenylthiohydantoin derivatives were identified by HPLC using a Zorbax ODS column (DuPont Instruments) equilibrated in 0.01 M sodium acetate, pH 5.5/20% acetonitrile and developed with an acetonitrile gradient (21). Repetitive yields in all sequencing runs ranged from 92% to 95%. Synthetic Oligonucleotides. Six C1 inhibitor CNBr peptides were subjected to sequence analysis (Table 1). One of these (CB5) was the amino-terminal peptide (12), while another Abbreviations: C1, first component of complement: bp, base pair(s); kb, kilobase(s). 3161 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3162 Biochemistry: Davis et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Amino-terminal sequences of C1 inhibitor CNBr peptides CB1: Leu-Phe-Val-Glu-Pro-Ile-Leu-Glu-Val-Xaa-Leu CB2: Ala-Phe-Ser-Pro-Phe-Ser-Ile-Ala-Ser-Leu-Leu-Xaa-Gln-Val-Leu-Leu-Gly-Ala-Gly-Asn-Ala-Glu-Ala-Xaa-