Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Studies on a leukocyte elastase inhibitor present in the culture medium of inflamed synovial tissue

The spent medium of cultured inflamed synovial tissue contains a potent inhibitor of leukocyte elastase. This leukocyte elastase inhibitor has no effect on leukocyte cathepsin G and pancreatic elastase is only marginally affected. The inhibitor is a glycoprotein, stable to heat, acid and reductive alkylation. Pretreatment of the inhibitor with either trypsin or chymotrypsin results in its inactivation.     

Bronchial leukocyte proteinase inhibitor: hydrodynamic properties and interaction with alpha 2-macroglobulin-bound elastase

Bronchial leukocyte proteinase inhibitor (BLPI) is an 11.7 kDa, acid-stable protein found in mucous secretions, which inhibits neutrophil elastase. The Stoke's radius of BLPI calculated from sedimentation equilibrium and sedimentation velocity centrifugation data was in good agreement with the value determined by gel filtration. These data indicate that BLPI exists in a compact globular conformation at both neutral and acidic pH. BLPI, due to its small compact size, can inhibit neutrophil elastase after the enzyme has been complexed with alpha 2-macroglobulin (A-2-M) but alpha 1-proteinase inhibitor failed to inactivate A-2-M-bound elastase. The apparent association rates of BLPI and Eglin C with A-2-M-bound elastase were found to be 6.3 X 10(2) M-1s-1 and 1.1 X 10(3) M-1s-1, respectively. These apparent association rates decreased 168-fold for BLPI and 909-fold for Eglin C, relative to the association rates of these inhibitors with free elastase. These changes probably result from a combination of effects, such as inhibitor accessibility to the enzyme and/or reaction rate, but regardless of the mechanism these data suggest that BLPI may function to control both free and A-2-M-bound elastase.     

The degradation of human lung elastin by neutrophil proteinases

Human lung elastin has been isolated by both a degradative and nondegradative procedure and the products obtained found to have amino acid compositions comparable to published results. These elastin preparations, when utilized as substrates for various mammalian proteinases, were solubilized by porcine elastase at a rate six times faster than human leukocyte elastase. Leukocyte cathepsin G also solubilized lung elastin but only at 12% of the rate of the leukocyte elastase. In all cases the elastin prepared by nondegradative techniques proved to be the best substrate in these studies. The differences in the rate of digestion of elastin of the two elastolytic proteinases was readily attributed to the specificity differences of each enzyme as judged by carboxyterminal analysis of solubilized elastin peptides. The plasma proteinase inhibitors, alpha-1-proteinase inhibitor and alpha-2-macroglobulin abolished the elastolytic activity of both leukocyte enzymes, while alpha-1-antichymotrypsin specifically inactivated cathespsin G. Two synthetic inhibitors, Me-O-Suc-Ala-Ala-Pro-Val-CH2Cl (for elastase and Z-Gly-Leu-Phe-CH2Cl (for cathepsin G) were equally effective in abolishing the elastolytic activity of the two neutrophil enzymes. However, inhibition of leukocyte elastase by alpha-1-proteinase inhibitor was significantly suppressed if the enzyme was preincubated with elastin prior to addition of the inhibitor.     

Crystallization and preliminary diffraction studies of porcine pancreatic elastase in complex with a novel inhibitor

Porcine pancreatic elastase (PPE) was crystallized in complex with a novel inhibitor at pH 5 and X-ray diffraction data were collected at a synchrotron source to 1.66 A. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 50.25 A, b = 57.94 A and c = 74.69 A. PPE is often used as model for drug target, due to its structural homology with the important therapeutic target human leukocyte elastase (HLE). Elastase is a serine protease that belongs to the chymotrypsin family, which has the ability to degrade elastin, an important component in connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases.     

Studies on the interaction between leukocyte elastase, antileukoproteinase and the plasma proteinase inhibitors alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The dominating inhibitor of leukocyte elastase in human respiratory tract secretions is a low molecular mass inhibitor, designated antileukoproteinase. An equimolar antileukoproteinase-elastase complex was produced and subjected to gel filtration after differing time intervals and was found to be stable. On addition to human serum, however, elastase dissociated from antileukoproteinase and formed a complex with alpha 1-proteinase inhibitor. A small amount of elastase was also found bound to alpha 2-macroglobulin. Antileukoproteinase was capable of inhibiting elastase bound to alpha 2-macroglobulin. This inhibition was more complete and more rapid when the alpha 2-macroglobulin-elastase complex was in a molar ratio of 1:1 than in a ratio of 1:2.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.     

Inhibition by elastase inhibitors of the formyl Met Leu Phe-induced chemotaxis of rat polymorphonuclear leukocytes

Rat leukocyte elastase has been purified successively by AH-Sepharose Kappa-elastin affinity chromatography and by ion exchange chromatography on a carboxymethyl Sephadex resin. It has great similarities with human leukocyte elastase in its molecular weight, substrate specificity and inhibitory profile. The effect of rat leukocyte elastase inhibitors in influencing the chemotactic response of rat PMN to fMetLeuPhe has been compared to that of other proteinase inhibitors. The results indicated that oleoyl (Ala)2ProValCH2Cl, a specific inhibitor of human and rat leukocyte elastases and Eglin C, which also inhibits human and rat cathepsin G, are among the powerful inhibitors of rat PMN chemotaxis induced by the formyl oligopeptide. This suggests that these neutral proteinases, in addition to their known participation in connective tissue catabolism, could play a role in PMN locomotion and chemotaxis.     

An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center

Horse leukocyte elastase inhibitor rapidly forms stable, equimolar complexes with both human leukocyte elastase and cathepsin G, porcine pancreatic elastase, and bovine alpha-chymotrypsin. Formation of the inhibitor-pancreatic elastase complex results in peptide bond cleavage at the reactive site of the inhibitor so that a small peptide fragment representing the carboxyl-terminal sequence of the inhibitor is released. Sequence analysis of both this peptide, as well as that of an overlapping peptide obtained by enzymatic inactivation of native inhibitor with either Staphylococcus aureus metalloproteinase, Pseudomonas aeruginosa elastase, or cathepsin B, yields data which indicate that the reactive site encompasses a P1-P1' Ala-Met sequence. However, unlike the human endothelial plasminogen activator inhibitor, which also has a Met residue in the P1' position, oxidation of the horse inhibitor only slightly reduces its association rate constant with either of the elastolytic enzymes tested or with chymotrypsin. Comparison of the amino acid sequence at or near the reactive site of the horse inhibitor (P2-P18') with members of the serpin superfamily of proteinase inhibitors indicates that it not only belongs in this class but also represents the first example of a functionally active intracellular serpin.     

NMR and enzymatic investigation of the interaction between elastase and sodium trifluoroacetate.

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.     

Effect of oxidation of methionine in a peptide substrate for human elastases: a model for inactivation of alpha 1-protease inhibitor.

Human α₁-protease inhibitor which is an important plasma protein, contains a methionine residue at its reactive site. A model synthetic peptide substrate, succinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine p-nitroanilide, has been employed to study the effect of oxidation of methionine on the rate of hydrolysis of this substrate by human elastases. The methionine sulfoxide derivative obtained by mild oxidation of this substrate is hydrolyzed by pancreatic elastase 2 and leukocyte elastase at rates that are 5% and 0.3% of the rates measured for hydrolysis of the parent compound by the respective enzymes. These results suggest that oxidation of the active site methionine residue of human α₁-protease inhibitor may decrease the rate of reaction of pancreatic or leukocyte elastase with this inhibitor.     

Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence

A potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and porcine pancreatic elastase (EC 3.4.21.36) was purified to homogeneity from human horny layers. It inhibits human leukocyte elastase and porcine pancreatic elastase in a 1:1 molar ratio and shows equilibrium dissociation constants of 6 x 10(-10) M and 1 x 10(-9) M, respectively. Inhibition of plasmin, trypsin, alpha-chymotrypsin, and cathepsin G was not observed. This inhibitor proved to be an acid stable basic peptide with an isoelectric point of 9.7. The complete amino acid sequence appears to be unique with 38% homology to the C-terminal half of antileukoprotease. The sequence shows that the inhibitor is composed of 57 amino acids and predicts a Mr of 7017. The high affinity as well as the apparent specificity for elastases suggests a functional role in preventing elastase-mediated tissue proteolysis. It is suggested that the term "elafin" be used to designate this inhibitor.     

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.     

Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin

The addition of either cathepsin-G or leukocyte elastase to endotoxin-stimulated human peripheral blood monocytes decreased the immunoreactive tumor necrosis factor (TNF) detected in culture supernatants in a concentration-dependent manner. Both enzymes also induced a loss of supernatant cytolytic activity as determined on the WEHI-164 target cell line. Incubation of recombinant human TNF and lymphotoxin (LT) with either cathepsin-G or leukocyte elastase resulted in a loss of cytokine bioactivity. Examination of enzyme-treated recombinant cytokines by gel electrophoresis revealed that cathepsin-G cleaved LT into a 12.6-kDa fragment and leukocyte elastase fragmented LT into a 14.1-kDa product. On Western blots cathepsin-G and leukocyte elastase degraded TNF into 11- and 7.6-kDa fragments, respectively. Incubating leukocyte elastase with plasma elastase inhibitor alpha-1-antitrypsin prevented the loss of recombinant TNF bioactivity and blocked the degradation of this cytokine. This study suggests that two of the most abundant neutrophil proteases, cathepsin-G and leukocyte elastase, may be important regulators of TNF and LT bioactivity.     

分泌性白细胞蛋白酶抑制因子在肿瘤中的作用

分泌性白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor, SLPI)是一个可抑制多种丝氨酸蛋白酶活性的阳离子蛋白质。SLPI羧基端具有抑制糜蛋白酶、胰弹性蛋白酶等抗蛋白酶活性，氨基端的功能尚不清楚，可能具有抗菌、抗真菌、抗病毒、抗炎和免疫调节等活性。近年来研究发现，SLPI在有些癌症，如卵巢癌、肺癌、胃癌、结肠癌中表达升高，但在有些癌症如乳腺癌、前列腺癌、口腔癌中表达降低。目前，尚未完全了解SLPI在调控致癌效应中的作用。本文就SLPI在肿瘤及抗肿瘤中的可能作用及其机制进行综述，为SLPI在抗肿瘤中的应用提供新思路。     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.     

Partial purification and characterization of mouse peritoneal exudative macrophage elastase

Mouse peritoneal exudate macrophage elastase can be significantly purified with 60% recovery of the starting activity by affinity chromatography against SDS-treated alpha-elastin covalently linked to agarose beads. The enzyme has an apparent Mr of 26 500 based on SDS-acrylamide gel electrophoresis. Molecular sieving chromatography on Sephadex gel gives a Mr for macrophage elastase of 21 000--28 000. The enzyme is not inhibited by chloromethyl ketone inactivators specific for pancreatic and leukocyte elastase nor by phenylmethylsulfonyl fluoride. Macrophage elastase also does not bind to tritiated diisopropylphosphorofluoridate. The enzyme is inhibited by EDTA and thus appears to be a metallo-protease. Macrophage elastase is resistant to human alpha 1-proteinase inhibitor and to human and mouse alpha 2-macroglobulin. In view of its lack of susceptibility to these endogenous serum proteinase inhibitors, macrophage elastase may play an important role in physiological and pathological remodeling of connective tissues.     

白细胞弹性蛋白酶抑制剂

人白细胞弹性蛋白酶广泛参与体内的组织损伤反应,与炎症反应、自身免疫、肿瘤形成和转移等有密切关系。目前人白细胞弹性蛋白酶抑制剂已成为新药开发的热点,具有很高的应用价值,其中西维来司钠已经成功进入市场。本文对近几年发现的人白细胞弹性蛋白酶抑制剂的结构及其作用进行综述。