The Functional Integrity of the Serpin Domain of C1-inhibitor Depends on the Unique N-terminal Domain, as Revealed by a Pathological Mutant

Abstract

C1-inhibitor (C1-InhThe autosomal dominant disease hereditary angioedema (HAE) 1 is caused by functional deficiency of C1-inhibitor (C1-Inh) (1). C1-Inh is the major inhibitor of C1s and C1r of the classical pathway of complement and also an inhibitor of the contact system proteases, factor XIIa, kallikrein, and factor XIa (2-9). A lack of this inhibitor may result in recurrent episodes of acute, local, circumscribed edema of the skin or mucosa. The most serious and potentially life-threatening manifestation of the disease is laryngeal edema (10). HAE is inherited as an autosomal dominant trait; affected individuals are heterozygous. Based on the relative levels of functional and antigenic C1-Inh, two types of HAE have traditionally been described. In type I (Ϯ 85% of HAE patients), defective expression of one allele results in low antigenic and functional levels. In type II (Ϯ15% of HAE patients), levels of functional C1-Inh are low, but C1-Inh antigen levels are normal because of the presence of a dysfunctional mutant protein. Although heterozygosity would suggest functional C1-Inh levels of around 50% in HAE, these levels are lower than 50%, typically 5-30%, which is ascribed to increased C1 activation and C1-Inh consumption (10, 11). However, the description of low levels of non-functional C1-Inh mutants in patients with type I HAE has demonstrated that the distinction between types I and II HAE is not absolute (12). For example, low levels of dysfunctional mutant protein may occur in cases where the mutation also leads to defective protein secretion (12, 13). Many different mutations can lead to dysfunctional C1-Inh, as has recently been reviewed (14). C1-Inh is a human plasma glycoprotein and a member of the serine protease inhibitor (serpin) family to which antithrombin III (ATIII), plasminogen activator inhibitor 1, and ␣1-antitrypsin also belong. Serpins are suicide inhibitors that function as a set mousetrap by forming highly stable complexes with their cognate proteases. In its active form, a serpin has a metastable structure with a flexible reactive site loop that protrudes from the core of the molecule, the central ␤-sheet. The key residues P1-P1Ј form the reactive site, a pseudosubstrate for the target protease. Upon proteolytic attack on this bond, the reactive site loop is pulled into the central ␤-sheet, inducing a dramatic conformational change that results in the formation of covalent complexes between inhibitor and protease (15, 16). Recent crystallographic studies support the idea that during complex for- 1 The abbreviations used are: HAE, hereditary angioedema; C1, complement factor 1; C1-Inh, C1 esterase inhibitor, inhibitor of C1, coagulation factors XIIa, XIa, and kallikrein; WT, wild type; C1-Inh 76 , C1-Inh 98 , and C1-Inh 115 , C1-Inh deletion mutants starting at amino acid 76, 98, and 115, respectively; ⌬3 C1-Inh, C1-Inh mutant lacking amino acids 62-116; ATIII, antithrombin III; mAB, monoclonal antibody; RII, mAb reacting with all forms of C1-Inh; Kok-12, mAb reacting with cleaved...

Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

“…The expression yields ranged from 3-10 mg/l and, therefore, were comparable to that reported previously for the production of full-length C1 inhibitor using the same expression system (16). In keeping with the report by Wolff et al (16) and in contrast with the highly heterogeneous material produced in P. pastoris (12,19), the C1 inhibitor serpin domain expressed in the current study was homogeneous in terms of size, as shown by SDS-PAGE and MALDI mass spectrometry analyses, indicating relative homogeneity of the N-linked oligosaccharides. In this regard, the mass spectrometry analyses performed on the recombinant proteins are consistent with the occurrence of short, oligomannose carbohydrates comprising two N-acetylglucosamine residues and three to four mannose residues (calculated mass = 893-1055), in keeping with previous analyses (16).…”

“…1). The N-terminal extension of C1 inhibitor does not seem to affect protease inhibition (11,12), and its functional role remains elusive. However, interaction of C1 inhibitor with selectins on endothelial cells is known to be mediated by tetrasaccharides located in this area (9).…”

C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

“…This truncation removes the nonconserved part of C1-inh and leaves most of the biological activities essentially unchanged (11,12) (Fig. 1A).…”

“…Sequencing analysis revealed 14 potential O-glycosylation sites (9), seven of which had been verified by carbohydrate analysis (10). Most of the sugars are present in the N-terminal domain and do not affect proteinase inhibition (11,12), but affinity to endotoxins and selectins depends on the N-glycans.…”

Cysteine Proteinase from Streptococcus pyogenes Enables Evasion of Innate Immunity via Degradation of Complement Factors

“…Also, a part of the C1-INH amino acid sequence of Asp 84 to Thr 138 corresponded to the sequence that is lost in patients with C1-INH deficiency (Fig. 3C) (23). The second cleavage site between Val 127 and Thr 128 was also included in the sequence.…”