The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus

The interaction of three proteinases (seryl, cysteinyl, and metallo-) from Staphylococcus aureus with human plasma alpha 1-proteinase inhibitor has been investigated. As expected, none of the enzymes was inactivated by this protein, each, instead causing the conversion of the native inhibitor into an inactive form of decreased molecular weight. Amino-terminal sequence analysis indicated that inhibitor inactivation had occurred by peptide bond cleavage near the reactive center of this protein. When the inhibitor was modified by this treatment, it became resistant to both pH and temperature denaturation and, in contrast to the intact denatured protein, did not undergo further proteolytic degradation. This process of inactivation of alpha 1-proteinase inhibitor by pathogenic proteinases could result in a deregulation of its target enzyme, neutrophil elastase, and, therefore, may be important in the consumption of some plasma proteins by this enzyme during septicemia.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase.     

Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases

The rates of interaction of a number of serine proteinases with a mutant form of alpha 1-proteinase inhibitor (referred to as alpha 1-proteinase inhibitor (Pittsburgh)), in which a methionine-358 to arginine-358 mutation has occurred, have been determined. An approximately 6,000-fold increase in the second order association rate constant with human thrombin was observed (48 M-1 X s-1 for the normal protein to 3.1 X 10(5) M-1 X s-1 for the arginine mutant), confirming previously observed data using bovine thrombin (Owen, M.C., Brennan, S.O., Lewis, J.H. & Carrell, R.W. (1983) New England J. Med. 309, 694-698). However, substantial increases in the rates of association with other trypsin-like enzymes were also noted, indicating that the replacement of methionine by a basic residue affects all serine proteinases with this kind of specificity. There was a marked decrease in the rates of interaction of the Pittsburgh mutant with both human neutrophil elastase and porcine pancreatic elastase, the inhibitor being converted into lower molecular mass fragments after interaction with either enzyme. Butanedione caused a substantial loss in the inhibitory activity of the arginine mutant, while having no effect on the normal protein. These data, when compared to those previously reported for differences in reaction rates between normal and oxidized alpha 1-proteinase inhibitor (Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, 3931-3934), are consistent with the interpretation that the amino acid in the P1-position at the reactive site of this protein has a marked effect on determining its primary specificity.     

Mutants of plasminogen activator inhibitor-1 designed to inhibit neutrophil elastase and cathepsin G are more effective in vivo than their endogenous inhibitors

Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.     

Recombinant DNA-derived forms of human alpha 1-proteinase inhibitor. Studies on the alanine 358 and cysteine 358 substituted mutants

The specificity and reactivity of human alpha 1-proteinase inhibitor has been investigated by in vitro mutagenesis of the reactive site P1 methionine 358 residue to alanine 358 and cysteine 358. A comparison of the second-order association rates of both uncharged mutants with 9 serine proteinases indicated that each reacted similarly to either the normal plasma inhibitor or to a mutant containing valine in this position (Travis, J., Owen, M., George, P., Carrell, R., Rosenberg, S., Hallewell, R. A., and Barr, P. J. (1985) J. Biol. Chem. 260, 4384-4389) when tested against either neutrophil or pancreatic elastase. However, oxidation, carboxymethylation, or aminoethylation of the cysteine mutant to yield a charged P1 residue resulted in a significant decrease in association rates with both elastolytic enzymes, and aminoethylation created an excellent trypsin and plasmin inhibitor. These results indicate that the specificity of alpha 1-proteinase inhibitor is determined in a general manner by the class of amino acid residue in the P1 position. Substitution within the same category, such as from valine to alanine or cysteine among the aliphatic hydrophobic residues, has little effect on association rates with the elastolytic enzymes tested. However, alteration from an uncharged to a charged residue may cause considerable changes in both inhibitor specificity and reactivity as noted here with the cysteine derivatives and also previously with a natural variant in which methionine 358 to arginine 358 conversion resulted in the production of a potent thrombin inhibitor (Owen, M. C., Brennan, S. O., Lewis, J. H., and Carrell, R. W. (1983) N. Engl. J. Med. 309, 694-698).     

Kinetic and chemical evidence for the inability of oxidized alpha 1-proteinase inhibitor to protect lung elastin from elastolytic degradation

The oxidation of human alpha 1-proteinase inhibitor results in the conversion of this protein into a form which cannot protect lung elastin from degradation by elastolytic proteinases. Data indicate that this is primarily because of the lowering of the association rate between the modified inhibitor and neutrophil elastase, as well as in a change in Ki from near 10(-14) to near 10(-10)M. This is consistent with the hypothesis that oxidation of alpha 1-proteinase inhibitor in the lung by cigarette smoke results in a lowering of the protection of this organ from elastolytic degradation.     

The primary role of the P1 residue (ser359) of alpha-1-proteinase inhibitor

The replacement of ser359 with ala359 at the P1 position in human alpha-1-proteinase inhibitor results in the production of a variant protein containing 15% of the inhibitory activity of the normal inhibitor. Separation of active from inactive inhibitor on anhydrochymotrypsin-sepharose yields a form which has a second order association rate with neutrophil elastase which is approximately one half that for the native protein. These data indicate that the P1 residue is not of primary importance during the interaction of proteinases with alpha-1-proteinase inhibitor. Since substitution of alanine for serine causes the formation, primarily, of inactive inhibitor the major function of ser359 probably involves proper folding to give a functionally active inhibitory conformation.     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

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.     

Proteolytic inactivation of alpha-1-anti-chymotrypsin. Sites of cleavage and generation of chemotactic activity.

The effect of several microbial and mammalian proteinases on the inhibitory activity of human plasma alpha-1-anti-chymotrypsin (alpha-1-Achy) has been tested. Most of these enzymes caused rapid inactivation of the inhibitor by cleavage at single sites within the reactive-site loop between P5 Lys and P3' Leu, with additional cleavages also being detected in some cases near the NH2 terminus of the native protein. In contrast, two of the enzymes tested failed to inactivate alpha-1-Achy, although they could cause removal of peptides near the NH2 terminus. Studies of neutrophil chemotaxis revealed that native or NH2-terminally truncated alpha-1-Achy was not stimulatory. However, testing of two enzymatically inactivated forms of the inhibitor (alpha-1-Achy), cleaved at widely different positions within the reactive-site loop, indicated that they had become potent chemoattractants at concentrations within the nanomolar range. Competition studies using proteolytically inactivated alpha-1-proteinase inhibitor suggested that the chemotactic activity of the two inactivated serpins was probably mediated by the same mechanism. The physiological relevance of this chemotactic activity is underscored by the results of plasma elimination studies which indicate that alpha-1-Achy is eliminated at approximately the same rate as native alpha-1-Achy, thus prolonging chemotactic stimuli within the tissues.     

Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.     

Free radicals inactivate human neutrophil elastase and its inhibitors with comparable efficiency

Free radicals produced in a Fenton reaction (H202/Cu), modelling some xenobiotic and cell-mediated inflammatory affronts, efficiently inactivated the elastase-inhibitor eglin, but equally, human neutrophil elastase itself. Elastase activity was not regenerated from proteinase/inhibitor complexes during radical attack. Three different elastase inhibitors, eglin, secretory leukocyte proteinase inhibitor and alpha-1-proteinase inhibitor were all similarly sensitive to inactivation. Unlike certain oxidants which can selectively inactivate alpha-1-proteinase inhibitor, free radicals may influence comparably the availability of both proteinase inhibitors and their targets.     

Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity

Hydrogen peroxide is a component of cigarette smoke known to be essential for inactivation of alpha(1)-antitrypsin, the primary inhibitor of neutrophil elastase. To establish the molecular basis of the inactivation of alpha(1)-antitrypsin, we determined the sites oxidized by hydrogen peroxide. Two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation was known to cause loss of anti-elastase activity. The other, methionine 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-elastase activity, an effect not previously recognized. The equal susceptibility of methionine 358 and methionine 351 to oxidation was confirmed by mass spectrometry. To verify this finding, we produced recombinant alpha(1)-antitrypsins in which one or both of the susceptible methionines were mutated to valine. M351V and M358V were not as rapidly inactivated as wild-type alpha1-antitrypsin, but only the double mutant M351V/M358V was markedly resistant to oxidative inactivation. We suggest that inactivation of alpha(1)-antitrypsin by oxidation of either methionine 351 or 358 provides a mechanism for regulation of its activity at sites of inflammation.     

Purification and characterization of a novel cysteine proteinase (periodontain) from Porphyromonas gingivalis. Evidence for a role in the inactivation of human alpha1-proteinase inhibitor

Periodontal disease is characterized by inflammation of the periodontium manifested by recruitment of neutrophils, which can degranulate, releasing powerful proteinases responsible for destruction of connective tissues, and eventual loss of tooth attachment. Although the presence of host proteinase inhibitors (serpins) should minimize tissue damage by endogenous proteinases, this is not seen clinically, and it has been speculated that proteolytic inactivation of serpins may contribute to progression of the disease. A major pathogen associated with periodontal disease is the Gram-negative anaerobe Porphyromonas gingivalis, and in this report, we describe a novel proteinase that has been isolated from culture supernatants of this organism that is capable of inactivating the human serpin, alpha1-proteinase inhibitor, the primary endogenous regulator of human neutrophil elastase. This new enzyme, referred to as periodontain, belongs to the cysteine proteinase family based on inhibition studies and exists as a 75-kDa heterodimer. Furthermore, periodontain shares significant homology to streptopain, a proteinase from Streptococcus pyogenes, and prtT, a putative proteinase from P. gingivalis. Clearly, the presence of this enzyme, which rapidly inactivates alpha1-proteinase inhibitor, could result in elevated levels of human neutrophil elastase clinically detected in periodontal disease and should be considered as a potential virulence factor for P. gingivalis.     

Cigarette smoke decreases the rate constant for the association of elastase with alpha 1-proteinase inhibitor by a non-oxidative mechanism

This paper describes a non-oxidative impairment of the biological function of alpha 1-proteinase inhibitor by cigarette smoke. Aqueous solutions of cigarette smoke are able to decrease the rate constant kass for the inhibition of porcine pancreatic elastase by human plasma alpha 1-proteinase inhibitor. The value of kass decreases linearly with the concentration of smoke (from 2.2 X 10(5) M-1 s-1 to 0.6 X 10(5) M-1 s-1). This effect is not due to an oxidation of the inhibitor. When pancreatic elastase is reacted with elastin in the presence of alpha 1-proteinase inhibitor and cigarette smoke solution, elastolysis occurs at a rate nearly identical to that observed in the absence of inhibitor. This effect is due to a smoke-induced decrease in kass. These observations may serve as a model of biological regulation of proteolysis via a change in the rate constant for a proteinase-proteinase inhibitor association. The influence of cigarette smoke on the inhibition of human neutrophil elastase by alpha 1-proteinase inhibitor could not be studied in detail because the enzyme precipitates in the presence of concentrated smoke solution.     

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by alpha 1-proteinase inhibitor and alpha 2-macroglobulin

At pH 8.0 and 25 degrees C alpha 1-proteinase inhibitor and alpha 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7.10(5) M-1.s-1 and 6.4.10(6) M-1.s-1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of alpha 1-proteinase inhibitor and alpha 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M-1.s-1. In contrast, alpha 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

Heparin strongly decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor

Heparin depresses the second-order rate constant ka for the inhibition of neutrophil elastase by alpha 1-proteinase inhibitor. High molecular mass heparin decreases ka from 1.3 x 10(7) M-1 s-1 to a limit of 4.6 x 10(4) M-1 s-1. Low molecular mass heparin is about 7-fold less effective. Dermatan sulfate and chondroitin sulfate are less efficient. Heparin preparations used in clinical care also strongly depress ka when tested at concentrations corresponding to their clinical efficacy. Heparin also decreases the ka for the elastase/eglin c and the cathepsin G/alpha 1-proteinase inhibitor systems but not that for the alpha 1-proteinase inhibitor/pancreatic elastase or trypsin pairs. These results, together with Sepharose-heparin binding studies, indicate that the ka-depressing effect of the polymer is related to its ability to form a tight complex with elastase but not with alpha 1-proteinase inhibitor. One mol of high molecular mass heparin binds 3 mol of neutrophil elastase with a Kd of 3.3 nM. Low molecular mass heparin binds elastase with a 1:1 stoichiometry and a Kd of 89 nM. For both heparins ka is lowest when elastase is fully saturated with heparin. From this we conclude that heparin decreases ka, because the heparin-elastase complex is able to slowly react with alpha 1-proteinase inhibitor and not because the inhibitor slowly dissociates the heparin-elastase complex. These findings may have important pathophysiological bearing.     

Differential inhibitory properties of dog and human alpha 1-proteinase inhibitors

Dog alpha 1-proteinase inhibitor (alpha 1-PI) was found to be an effective inhibitor of bovine chymotrypsin and also of porcine pancreatic elastase as in the case of human inhibitor. The dog inhibitor inactivated both proteinases at a molar ratio of 1:1. However, compared to the human inhibitor, dog alpha 1-PI was a relatively poor inhibitor of bovine trypsin. The association rate constants (kass) of the interactions of dog alpha 1-PI with bovine chymotrypsin and with porcine elastase were determined to be 6.9 +/- 0.3 X 10(6) M-1 s-1 and 6.4 +/- 0.1 X 10(5) M-1 s-1, respectively. These values are 1.3- and 2.7-fold higher than the corresponding values for the human inhibitor. On the other hand, kass for the dog inhibitor with bovine trypsin (2.6 +/- 0.3 X 10(4)M-1 s-1) was found to be about 5 times smaller than that of the human inhibitor.