Serpin Domain Research Articles

C1 inhibitor: molecular and clinical aspects

C1 inhibitor (C1-INH) is a serine protease inhibitor (serpins) that inactivates several different proteases in the complement, contact, coagulation, and fibrinolytic systems. By its C-terminal part (serpin domain), characterized by three beta-sheets and an exposed mobile reactive loop, C1-INH binds, and blocks the activity of its target proteases. The N-terminal end (nonserpin domain) confers to C1-INH the capacity to bind lipopolysaccharides and E-selectin. Owing to this moiety, C1-INH intervenes in regulation of the inflammatory reaction. The heterozygous deficiency of C1-INH results in hereditary angioedema (HAE). The clinical picture of HAE is characterized by bouts of local increase in vascular permeability. Depending on the affected site, patients suffer from disfiguring subcutaneous edema, abdominal pain, vomiting and/or diarrhoea for edema of the gastrointestinal mucosa, dysphagia, and dysphonia up to asphyxia for edema of the pharynx and larynx. Apart from its genetic deficiency, there are several pathological conditions such as ischemia-reperfusion, septic shock, capillary leak syndrome, and pancreatitis, in which C1-INH has been reported to either play a pathogenic role or be a potential therapeutic tool. These potential applications were identified long ago, but controlled studies have not been performed to confirm pilot experiences. Recombinant C1-INH, produced in transgenic animals, has recently been produced for treatment of HAE, and clinical trials are in progress. We can expect that the introduction of this new product, along with the existing plasma derivative, will renew interest in exploiting C1-INH as a therapeutic agent.

Potentiation of C1 esterase inhibitor by StcE, a metalloprotease secreted by Escherichia coli O157:H7.

The complement system is an essential component of host defense against pathogens. Previous research in our laboratory identified StcE, a metalloprotease secreted by Escherichia coli O157:H7 that cleaves the serpin C1 esterase inhibitor (C1-INH), a major regulator of the classical complement cascade. Analyses of StcE-treated C1-INH activity revealed that surprisingly, StcE enhanced the ability of C1-INH to inhibit the classical complement-mediated lysis of sheep erythrocytes. StcE directly interacts with both cells and C1-INH, thereby binding C1-INH to the cell surface. This suggests that the augmented activity of StcE-treated C1-INH is due to the increased concentration of C1-INH at the sites of potential lytic complex formation. Indeed, removal of StcE abolishes the ability of C1-INH to bind erythrocyte surfaces, whereas the proteolysis of C1-INH is unnecessary to potentiate its inhibitory activity. Physical analyses showed that StcE interacts with C1-INH within its aminoterminal domain, allowing the unaffected serpin domain to interact with its targets. In addition, StcE-treated C1-INH provides significantly increased serum resistance to E. coli K-12 over native C1-INH. These data suggest that by recruiting C1-INH to cell surfaces, StcE may protect both E. coli O157:H7 and the host cells to which the bacterium adheres from complement-mediated lysis and potentially damaging inflammatory events.

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma.

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.

Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion.

C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, is an inhibitor of proteases in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the most heavily glycosylated plasma protein, containing 13 definitively identified glycosylation sites as well as an additional 7 potential glycosylation sites. C1INH consists of two distinct domains: a serpin domain and an amino-terminal domain. The serpin domain retains all the protease-inhibitory function, while the amino-terminal domain bears most of the glycosylation sites. The present studies test the hypothesis that plasma C1INH bears sialyl Lewis(x)-related moieties and therefore binds to selectin adhesion molecules. We demonstrated that plasma C1INH does express sialyl Lewis(x)-related moieties on its N-glycan as detected using mAb HECA-452 and CSLEX1. The data also show that plasma C1INH can bind to P- and E-selectins by FACS and immunoprecipitation experiments. In a tissue culture model of endothelial-leukocyte adhesion, C1INH showed inhibition in a dose-dependent manner. Significant inhibition (>50%) was achieved at a concentration of 250 micro g/ml or higher. This discovery may suggest that C1INH plays a role in the endothelial-leukocyte interaction during inflammation. It may also provide another example of the multifaceted anti-inflammatory effects of C1INH in various animal models and human diseases.

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

C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.

Structural/Functional Characterization of the α2-Plasmin Inhibitor C-Terminal Peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.

Structural and Functional Aspects of C1-Inhibitor

C1-Inh is a serpin that inhibits serine proteases from the complement and the coagulation pathway. C1-Inh consists of a serpin domain and a unique N-terminal domain and is heavily glycosylated. Non-functional mutants of C1-Inh can give insight into the inhibitory mechanism of C1-Inh. This review describes a novel 3D model of C1-Inh, based on a newly developed homology modelling method. This model gives insight into a possible potentiation mechanism of C1-Inh and based on this model the essential residues for efficient inhibition by C1-Inh are discussed.

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop–sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into β-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from β-sheet A.

Role of the distal hinge region of C1-inhibitor in the regulation of C1s activity

A synthetic peptide corresponding to residues 448–459 of C1-inhibitor (C1-inh) binds to C1s, is a non-competitive inhibitor of C1s activity and prevents formation of an SDS-stable C1s–C1-inh complex. Substitutions of residues Q452, Q453 or F455 in this peptide resulted in loss of C1s binding and inhibitory activity of the peptide. NMR analysis of the peptide showed an area of well-defined structure from E450 to F455. The side chains of Q452, Q453 and Q455 were exposed to the solvent and therefore available for C1s binding. The defined structure in the peptide is compatible with our computer model of the serpin domain of C1-inh.

The sequence of a cDNA encoding functional murine C1-inhibitor protein

The murine C1-inhibitor protein is 482 amino acids long. It consists of an N-terminal domain of 118 amino acids rich in proline and threonine and a serpin domain. The N-terminal domain has 39% identity with the corresponding regions of human and bovine C1 inhibitor.

Functional analysis of the serpin domain of C1 inhibitor.

To analyze the role of the heavily glycosylated amino-terminal domain of C1 inhibitor in protease inhibitory activity, two truncated C1 inhibitor molecules were constructed. The abilities of the recombinant truncated inhibitors to complex with target proteases were compared with that of the wild-type recombinant protein. One recombinant truncated molecule consisted of amino acid residues 76 to 478 (C-serp(76)) and the other of residues 98 to 478 (C-serp(98)). The recombinant proteins were each expressed in similar quantities. The thermal denaturation profiles of the two truncated proteins were similar to that of the wild-type protein. Identical binding of C1s, C1r, kallikrein, and beta factor XIIa was observed with the three molecules. Furthermore, the truncated molecules also effectively inhibited C1 activity in hemolytic assays. These studies therefore clearly demonstrate that the amino-terminal domain of C1 inhibitor does not influence complex formation with target proteases.

Two-domain structure of the native and reactive centre cleaved forms of C [formula omitted] inhibitor of human complement by neutron scattering

The C 1 ̄ inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C 1 ̄ inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight ( M r) is calculated as 71,100. Neutron scattering gives an M r of 76,000 (± 4000) and a matchpoint of 41.8 to 42.3% 2H 2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration R G were biphasic. Neutron contrast variation of C 1 ̄ inhibitor in H 2O- 2H 2O mixtures gave an overall radius of gyration R G at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional R G of 1.43 nm. The reactive centre cleaved form of C 1 ̄ inhibitor has the same M r and structure as the native molecule. The length of C 1 ̄ inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, α, is large at 59 × 10 −5 from the R G data and 28 × 10 −5 from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C 1 ̄ inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of α 1 antitrypsin (7 nm × 3 nm × 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s 20, w 0 for CT inhibitor using the hydrodynamic sphere approach showed that a two-domain head and-tail structure with an M r of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s 20, w 0 of 3.7 S. Possible roles of the N-terminal domain in the function of C 1 ̄ inhibitor are discussed.

Genomic organization of the human amyloid beta-protein precursor gene

Amyloid β-protein (BP) deposited in Alzheimer brains is a cleavage product of a larger precursor (BPP). The BPP gene encodes three types of mRNA generated by alternative splicing, two of which contain the sequence encoding Kunitz-type serine-protease inhibitor (serpin). To investigate the regulatory mechanisms of BPP synthesis at the gene level, we isolated 36 genomic DNA clones covering all the exons of the human BPP gene. This gene consists of 18 exons and spans more than 170 kb. BP is encoded by the 16th and the 17th exons and the serpin domain by the 7th exon. Sequence analysis showed that the 7th and 8th introns lack a typical branchpoint for splicing. This might relate to the alternative splicing. The promoter of the BPP gene has some characteristics of those of housekeeping genes and contains a number of possible methylation sites. The methylation status of the promoter was analyzed by Southern blotting but no alteration was observed among tissues and between control and Alzheimer brains. We also tested the roles of two possible activator protein-1-binding sites and a possible heat-shock element found within the promoter. Northern blotting showed that the transcription of the BPP gene was apparently induced by 12-O- tetradecanoylphorbol-13- acetate (phorbol derivative) in HeLa cells.