The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.     

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.     

Electrophoretic analysis of the "cross-class" interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases

We investigated the "cross-class" interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen-1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen-1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen-1 formed sodium dodecyl sulfate (SDS)- and heat-stable complexes with chymotrypsin, rSCC antigen-1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS- and heat-stable complex. The cleavage was observed between the Gly353-Ser354 bond, indicating that rSCC antigen-1 interacts with cysteine proteinases not at the predicted reactive site P1-P1' portion (Ser354-Ser355), but at the Gly353-Ser354 of the P2-P1 portion. These findings promote understanding of the "suicide inhibition" mechanism of SCC antigen-1 against cysteine proteinases.     

An ester bond linking a fragment of a serine proteinase to its serpin inhibitor

Most known members of the serpin superfamily are serine proteinase inhibitors. Serpins are therefore important regulators of blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix. Serpins form SDS-resistant complexes of 1:1 stoichiometry with their target proteinases by reaction of their P1-P1' peptide bond with the active site of the proteinases. The nature of the interactions responsible for the high stability of the complexes is a controversial issue. We subjected the complex between the serine proteinase urokinase-type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor-1 (PAI-1) to proteolytic digestion under nondenaturing conditions. The complex could be degraded to a fragment containing two disulfide-linked peptides from uPA, one of which included the active site Ser, while PAI-1 was left undegraded. By further proteolytic digestion after denaturation and reduction, it was also possible to degrade the PAI-1 moiety, and we isolated a fragment containing 10 amino acids from uPA, encompassing the active site Ser, and 6 amino acids from PAI-1, including the P1 Arg. Characterization of the fragment gave results fully in agreement with the hypothesis that it contained an ester bond between the hydroxyl group of the active site Ser and the carboxyl group of the P1 Arg. These data for the first time provide direct evidence that serine proteinases are entrapped at an acyl intermediate stage in serine proteinase-serpin complexes.     

Evolutionary relationships among the serpins

The serpins are a widely distributed group of serine proteinase inhibitors found in plants, birds, mammals and viruses. Despite the great evolutionary divergence of these organisms, their serpins are highly conserved, both in sequence and structurally. Amino acid sequences were aligned by a combination of automatic algorithms and by consideration of conserved structural elements in those serpins for which crystal structures exist. The program HOMED was used which allowed the alignment of amino acids to be simultaneously converted into the equivalently aligned nucleotide sequences. The aligned amino acids were used as the basis for superposition of the four known three-dimensional structures for which coordinates are available and compared with an optimal three-dimensional superposition in order to estimate the reliability of the sequence alignment. Phylogenetic relationships implied by these nucleotide sequence alignments were determined by the method of maximum parsimony. The proposed gene tree suggested that as much diversity existed between the plant serpin and mammalian serpins as was present among mammalian serpins and provided further evidence that the architecture of serpin molecules is highly constrained.     

A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.     

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.     

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

BACKGROUND: Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS: The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS: We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.     

Heat-induced conversion of ovalbumin into a proteinase inhibitor

Ovalbumin is a member of the serine proteinase inhibitor (serpin) family but is unable to inhibit proteinases. Here we show that heating transforms it into inhibitory ovalbumin (I-ovalbumin), a potent reversible competitive inhibitor of human neutrophil elastase (Ki = 5 nM) and cathepsin G (Ki = 60 nM) and bovine chymotrypsin (Ki = 30 nM). I-ovalbumin also inhibits bovine trypsin, porcine elastase and alpha-lytic proteinase with Ki values in the micromolar range. Thus, I-ovalbumin differs from active serpins by its inability to form irreversible complexes with proteinases. I-ovalbumin is unusually thermostable: it does not undergo any structural transition between 45 degrees C and 120 degrees C as tested by differential scanning calorimetry, and it retains full inhibitory capacity after heating at 120 degrees C. It has 8% less alpha-helices and 9% more beta-sheet structures than native ovalbumin, as shown by circular dichroism. Our results show that the primary sequence of ovalbumin contains the information required for enabling the first step of the serpin-proteinase interaction to occur, i.e. the formation of the Michaelis-like reversible complex, but does not contain the information needed for stabilizing this initial complex.     

Isolation and some molecular properties of a trypsin-like enzyme from larvae of European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

A one-step high-yielding procedure is presented for the purification of a trypsin-like proteinase from Ostrinia nubilalis larvae, consisting of benzamidine-sepharose affinity chromatography. The purified enzyme was homogeneous as judged by SDS-PAGE. The enzyme presents a molecular mass of 24 650 Da, a maximum pH activity profile of 9.5, a remarkable thermal stability and an optimum temperature of about 53 degrees C Km values determined using N alpha-benzoyl-DL-arginine-ethylester and N alpha-benzoyl-DL-arginine-p-nitro-anilide were 3.2 x 10(-5) M and 4.1 x 10(-4) M respectively. The proteinase was inhibited by some typical serine proteinase inhibitors such as N alpha-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitors, benzamidine and phenylmethylsulfonyl fluoride. In particular, it was competitively inhibited by benzamidine with a Ki of 1.2 x 10(-5) M, whereas it was not affected by cysteine proteinases inhibitors. Comparative analysis of the amino acid composition and N-terminal sequence of O. nubilalis proteinase confirmed that this enzyme is very similar to other serine proteinases from lepidopteran larvae.     

Human neutrophil proteinase 3: mapping of the substrate binding site using peptidyl thiobenzyl esters

A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steady-state kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active site: (a) the preferred P1 residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valine or alanine (in decreasing order of preference); (b) the enzyme has an extended active site; and (c) its active site is similar to that of the related serine proteinases leukocyte elastase and leukocyte cathepsin G.     

Organization of serpin gene-1 from Manduca sexta. Evolution of a family of alternate exons encoding the reactive site loop

Manduca sexta serpin gene-1 encodes a family of serpins whose amino acid sequences are identical in their amino-terminal 336 residues but variable in their carboxyl-terminal 39-46 residues, which includes the reactive site loop (Jiang, H., Wang, Y., and Kanost, M. R. (1994) J. Biol. Chem. 269, 55-58). Here, we report the gene's complete nucleotide sequence and exon-intron structure. A unique characteristic of this gene is its exon 9, which is present in 12 alternate forms between exons 8 and 10. Isolation and characterization of cDNA clones containing exons 9C, 9H, and 9I, which were not found previously, indicate that all 12 alternate forms of exon 9 can be utilized to generate 12 different serpins. The splicing pathway apparently allows inclusion of only one exon 9 per molecule of mature serpin-1 mRNA. Analysis of exon-intron border sequences reveals unique features that may be involved in regulation of RNA splicing. The exon 9 region has apparently evolved through rounds of exon duplication and sequence divergence. The exons near the center of the region may have evolved recently, whereas the outermost exons are the most ancient. Exons 9G and 9H were duplicated as a pair from exons 9E and 9F, an event that may have occurred more than once in the history of this gene.     

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.     

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.     

Characterization of a human alpha1-antitrypsin variant that is as stable as ovalbumin

The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human alpha1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of alpha1-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type alpha1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 alpha1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 alpha1-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.     

Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.     

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.     

A serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and ELANH2 which have a common structure almost identical to the 18q21 ovalbumin serpin genes

The human genes encoding the "ovalbumin" subgroup of closely related serine proteinase inhibitors (serpins) are located at 18q21.3 and 6p25. Those at 6p25 include proteinase inhibitor 6 (PI-6; gene symbol PI6), proteinase inhibitor 9 (PI-9; gene symbol PI9) and monocyte neutrophil elastase inhibitor (M/NEI; gene symbol ELANH2). Here we describe the fine mapping of these genes to a 200-kb region of chromosome 6 that includes the markers WI-8835 and D6S1338, and the establishment of the gene order: tel-PI6-PI9-ELANH2-cen. PI6 and ELANH2 are transcribed towards the telomere, and structural analysis shows that PI6 and PI9 are organized identically, having seven exons and six introns. PI6 and PI9 are almost identical in structure to the ovalbumin serpin genes at 18q21.3. The 18q21.3 genes have an extra exon and intron, otherwise all the other exon/intron boundaries are conserved between the two groups. These results represent the first detailed map of the chromosome 6 serpin gene cluster, and demonstrate that although they are very closely related, the 6p25 and 18q21-->q23 ovalbumin serpin genes form two structurally distinct groups. These findings do not support a previously proposed model for evolution of the clusters which invoked an inter-chromosomal duplication of the entire 6p25 group to 18q21.3.