Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 degrees C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI-I, CmPI-II and CmPI-III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI-I and CmPI-II was confirmed, while CmPI-III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI-I and CmPI-II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI-I (6 amino acids) and CmPI-II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI-II. Both inhibitors, CmPI-I and CmPI-II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (Ki) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI-I and CmPI-II are the first human neutrophil elastase inhibitors described in a mollusk.     

A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.     

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.     

Recombinant production, purification and biochemical characterization of domain 6 of LEKTI: a temporary Kazal-type-related serine proteinase inhibitor

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor which is of pathophysiological relevance for skin diseases and atopy. Domains 2 and 15 of LEKTI contain six cysteine residues and match the Kazal-type inhibitor motif almost exactly. The other 13 domains seem to be Kazal-type derived but lack the cysteines in positions 3 and 6 usually conserved within this family of inhibitors. Here, we report the recombinant production and comprehensive biochemical characterization of the 7.7 kDa LEKTI domain 6 (LD-6). Testing a selected number of different serine proteinases, we show that both native and recombinant LD-6 exhibit a significant but temporary inhibitory activity on trypsin. Furthermore, the relation of LEKTI domain 6 to Kazal-type inhibitors is confirmed by determining its disulfide bond pattern (1-4/2-3) and its P(1) site located after the second Cys residue of LD-6. The established strategy for the recombinant production of LEKTI domain 6 will enable further investigation of its mode of action and its physiological role.     

Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.     

Biochemical characterization of alpha-ketooxadiazole inhibitors of elastases.

A series of alpha-ketooxadiazole compounds was prepared and evaluated in vitro as potential inhibitors of human neutrophil elastase (HNE), proteinase-3 (PR-3), and porcine pancreatic elastase (PPE). Several compounds have been found to be very potent, fast, reversible, and selective inhibitors of HNE with Ki values below 100 pM. The highest kon value exceeded 10(7) M(-1) s(-1). Some alpha-ketooxadiazoles were also very effective against PR-3 and PPE with Ki values in the range of 5(-10) nM and 0.1(-2) nM, respectively. The two rings, 1,2,4- and 1,3,4-oxadiazole, are amenable to substitutions, extending the P' side of the inhibitor and allowing additional binding interactions at S' subsites of the enzyme. Nonpeptidic HNE inhibitors containing the oxadiazole heterocycle displayed promising oral bioavailability.     

Inhibition of serine proteinases plasmin,trypsin, subtilisin A,cathepsin G,and elastase by LEKTI: a kinetic analysis

The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K(i)) of 27 +/- 5, 49 +/- 3, 67 +/- 6, 317 +/-36, and 849 +/- 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

Infestin,a thrombin inhibitor presents in Triatoma infestans midgut,a Chagas' disease vector: gene cloning,expression and characterization of the inhibitor

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).     

BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae

BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K(i) 0.4 and 0.3nM) and subtilisin A (K(i) 1.4nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K(i) 50nM) and BmSI-6 (K(i) 2.2nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.     

Structural and functional study of an Anemonia elastase inhibitor, a "nonclassical" Kazal-type inhibitor from Anemonia sulcata

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.     

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.     

Boophilus microplus tick larvae, a rich source of Kunitz type serine proteinase inhibitors

Serine proteinase inhibitors from Boophilus microplus tick larvae (BmTIs) were purified by affinity chromatography on a trypsin-Sepharose column. BmTIs presented molecular weight between M(r) 6200 and 18,400 and inhibitory activity for trypsin, HuPK (human plasma kallikrein) and neutrophil elastase. Using ion exchange chromatography, BmTIs were separated in several protein pools named BmTI-A to BmTI-F and BmTI-1 to BmTI-7. All BmTI forms presented inhibitory activity for trypsin with apparent dissociation constants (K(i)) in the nM range. In this work, we describe the purification of BmTI-D, BmTI-2, and BmTI-3. These three inhibitors affected neutrophil elastase and HuPK with K(i) also in nM range. BmTI-D proved to be the best HuPK inhibitor, while BmTI-3 was more efficient for neutrophil elastase with dissociation constants (K(i)) of 12 and 0.5 nM, respectively. BmTI-D, BmTI-2, and BmTI-3 N-terminal amino acid sequences allowed us to include them into the BPTI-Kunitz type serine proteinase inhibitor family. BmTIs purified on trypsin-Sepharose were also used in a bovine immunization assay, resulting in antibody (anti-BmTIs) production.     

Tri-domain bifunctional inhibitor of metallocarboxypeptidases A and serine proteases isolated from marine annelid Sabellastarte magnifica

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar K(i) values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.     

Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris

Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.     

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.     

Homologous proteins with different folds: the three-dimensional structures of domains 1 and 6 of the multiple Kazal-type inhibitor LEKTI

We have determined the solution structures of recombinant domain 1 and native domain 6 of the multi-domain Kazal-type serine proteinase inhibitor LEKTI using multi-dimensional NMR spectroscopy. While two of the 15 potential inhibitory LEKTI domains contain three disulfide bonds typical of Kazal-type inhibitors, the remaining 13 domains have only two of these disulfide bridges. Therefore, they may represent a novel type of serine proteinase inhibitor. The first and the sixth LEKTI domain, which have been isolated from human blood ultrafiltrate, belong to this group. In spite of sharing the same disulfide pattern and a sequence identity of about 35% from the first to the fourth cysteine, the two proteins show different structures in this region. The three-dimensional structure of domain 6 consists of two helices and a beta-hairpin structure, and closely resembles the three-dimensional fold of classical Kazal-type serine proteinase inhibitors including the inhibitory binding loop. Domain 6 has been shown to be an efficient, but non-permanent serine proteinase inhibitor. The backbone geometry of its canonical loop is not as well defined as the remaining structural elements, providing a possible explanation for its non-permanent inhibitory activity. We conclude that domain 6 belongs to a subfamily of classical Kazal-type inhibitors, as the third disulfide bond and a third beta-strand are missing. The three-dimensional structure of domain 1 shows three helices and a beta-hairpin, but the central part of the structure differs remarkably from that of domain 6. The sequence adopting hairpin structure in domain 6 exhibits helical conformation in domain 1, and none of the residues within the putative P3 to P3' stretch features backbone angles that resemble those of the canonical loop of known proteinase inhibitors. No proteinase has been found to be inhibited by domain 1. We conclude that domain 1 adopts a new protein fold and is no canonical serine proteinase inhibitor.     

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.