Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Plasma protein and liver mRNA levels of two closely related murine alpha 1-protease inhibitors during the acute phase reaction

Plasma levels of alpha 1-PI(T) and alpha 1-PI(E), two closely related murine alpha 1-protease inhibitors, having affinities for trypsin and elastase, respectively, were compared to changes in specific liver mRNA levels after induction of the acute-phase reaction by subcutaneous injection of turpentine. In earlier, qualitative experiments an increase in plasma levels of alpha 1-PI(E), but not alpha 1-PI(T), during the acute-phase reaction had been shown. It is now shown that stimulation of plasma alpha 1-PI(E) levels reaches a maximum of 35-50% above baseline 12 h after induction of the acute-phase response using either a functional or immunological assay to measure protease inhibitor activity. Consistent with earlier observations, little or no change in plasma levels of alpha 1-PI(T) is seen. Determination of mRNA levels in the mouse liver specific for alpha 1-PI(E) and alpha 1-PI(T) was accomplished using a cell-free translation system followed by immunoprecipitation of the 35S-labeled protease inhibitors. The apparent Mr's of alpha 1-PI(E) and alpha 1-PI(T) synthesized in vitro are 42K and 46K, respectively. Apparent Mr's of the native proteins in plasma are 55K and 65K. Unexpectedly, mRNA levels for both alpha 1-PI(E) and alpha 1-PI(T) were found to increase after induction of the acute-phase reaction. Maximal stimulation for both mRNAs was approximately 300% and occurred 9 h after turpentine administration. Under these conditions, levels of translatable albumin mRNA in the mouse liver decreased to 40% of baseline in 6-9 h.     

Functional diversification during evolution of the murine alpha(1)-proteinase inhibitor family: role of the hypervariable reactive center loop

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors that are involved in the regulation of a number of proteolytic processes. Alpha(1)-PI, like most serpins, functions by covalent binding to, and inhibition of, target proteinases. The interaction between alpha(1)-PI and its target is directed by the so-called reactive center loop (RCL), an approximately 20 residue domain that extends out from the body of the alpha(1)-PI polypeptide and determines the inhibitor's specificity. Mice express at least seven closely related alpha(1)-PI isoforms, encoded by a family of genes clustered at the Spi1 locus on chromosome 12. The amino acid sequence of the RCL region is hypervariable among alpha(1)-PIs, a phenomenon that has been attributed to high rates of evolution driven by positive Darwinian selection. This suggests that the various isoforms are functionally diverse. To test this notion, we have compared the proteinase specificities of individual alpha(1)-PIs from each of the two mouse species. As predicted from the positive Darwinian selection hypothesis, the various alpha(1)-PIs differ in their ability to form covalent complexes with serine proteinases, such as elastase, trypsin, chymotrypsin, and cathepsin G. In addition, they differ in their binding ability to proteinases in crude snake venoms. Importantly, the RCL region of the alpha(1)-PI polypeptide is the primary determinant of isoform-specific differences in proteinase recognition, indicating that hypervariability within this region drives the functional diversification of alpha(1)-PIs during evolution. The possible physiological benefits of alpha(1)-PI diversity are discussed.     

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.     

Mouse alpha 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.     

Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants

Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor

We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

Recombinant alpha 1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a natural serine protease inhibitor. Although mainly thought to protect the airways from neutrophil elastase, alpha(1)-PI may also regulate the development of airway hyperresponsiveness (AHR), as indicated by our previous findings of an inverse relationship between lung alpha(1)-PI activity and the severity of antigen-induced AHR. Because allergic stimulation of the airways causes release of elastase, tissue kallikrein, and reactive oxygen species (ROS), all of which can reduce alpha(1)-PI activity and contribute to AHR, we hypothesized that administration of exogenous alpha(1)-PI should protect against pathophysiological airway responses caused by these agents. In untreated allergic sheep, airway challenge with elastase, xanthine/xanthine oxidase (which generates ROS), high-molecular-weight kininogen, the substrate for tissue kallikrein, and antigen resulted in bronchoconstriction. ROS and antigen also induced AHR to inhaled carbachol. Treatment with 10 mg of recombinant alpha(1)-PI (ralpha(1)-PI) blocked the bronchoconstriction caused by elastase, high-molecular-weight kininogen, and ROS, and the AHR induced by ROS and antigen. One milligram of ralpha(1)-PI was ineffective. These are the first in vivo data demonstrating the effects of ralpha(1)-PI. Our results are consistent with and extend findings obtained with human plasma-derived alpha(1)-PI and suggest that alpha(1)-PI may be important in the regulation of airway responsiveness.     

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.     

Distinct and additive effects of elastase and endotoxin on expression of alpha 1 proteinase inhibitor in mononuclear phagocytes

Expression of alpha 1 proteinase inhibitor (alpha 1-PI) in human mononuclear phagocytes may provide a local mechanism for inactivation of serine proteases at sites of tissue injury, thereby preventing incidental damage to surrounding tissue and allowing for orderly initiation of repair. We have previously shown that serine (neutrophilic or pancreatic) elastase and lipopolysaccharide (LPS) each mediate an increase in the expression of alpha 1-PI in human peripheral blood monocytes and bronchoalveolar macrophages. In this study we demonstrate that elastase and LPS have an additive positive regulatory effect on alpha 1-PI expression. Distinct pretranslational and translational mechanisms of action for elastase and LPS, respectively, account for the additive effect. The possibility that translational regulation of alpha 1-PI by LPS involves a mechanism analogous to that of the yeast gene GCN4 during amino acid starvation and that of the human ferritin gene in response to iron is discussed.     

The folding of alpha-1-proteinase inhibitor: kinetic vs equilibrium control

Previous folding studies of alpha-1-proteinase inhibitor (alpha1-PI), which regulates the activity of the serine protease human neutrophil elastase, show an intermediate state at approximately 1.5 M guanidine-HCl (Gu). For the normal form of alpha1-PI, we demonstrate the reversible formation of the same stable distribution of monomeric and polymeric intermediates after approximately 1 h in 1.5 M Gu at approximately 23 degrees C from fully folded or fully unfolded alpha1-PI at similar final total concentrations and show that the stable distribution of monomeric and polymeric intermediates conforms with the law of mass action. We attribute these observations to an apparent equilibrium among intermediates. Our CD data are compatible with the intermediates having slightly relaxed structures relative to that of fully folded alpha1-PI and, thus, with the polymeric intermediates having a loop-sheet structure. Furthermore, we observe that the rates of folding (fast and slow terms) from the intermediate state are the same as those from the fully unfolded state, thereby supporting the contention that this intermediate state is on the folding pathway. We attribute the tendency of the Z mutant protein to polymerize/aggregate to an increased rate of the monomeric intermediate to form the apparent equilibrium distribution of intermediate species relative to its rate of folding to give intact alpha1-PI.     

Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase

Mouse macrophage elastase, a metalloproteinase, catalytically inactivates human alpha 1-proteinase inhibitor (alpha 1-PI) by attacking a single peptide bond between Pro357 and Met358, resulting in Mr = 4,200 and 47,800 fragments. We show here that this proteolytically inactivated alpha 1-PI is a potent chemotactic factor for human neutrophils at a concentration of 1 nM. The chemotactic response is equivalent to that elicited by formyl-methionyl-leucyl-phenylalanine. Native alpha 1-PI does not stimulate chemotaxis. Purification of the two fragments of alpha 1-PI that result from proteolysis by macrophage elastase indicated that the Mr = 4,200 fragment is responsible for the chemotactic activity. However, the two proteolysis fragments do not dissociate from each other under physiologic conditions. Therefore, the ability of proteolytically inactivated alpha 1-PI to act as a mediator of inflammation is due to rearrangement of the alpha 1-PI molecule rather than to release of a cleavage fragment.     

Elastase is a constituent product of T cells

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.     

The murine alpha(1)-proteinase inhibitor gene family: polymorphism,chromosomal location,and structure

alpha(1)-Proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors, which function in maintaining homeostasis through regulation of numerous proteolytic processes. In laboratory mice (Mus musculus domesticus), alpha(1)-PI occurs in multiple isoforms encoded by a family of three to five genes that are polymorphic among inbred strains and that are located at the Serpina1 locus on chromosome 12. In the present study, we have characterized the alpha(1)-PI gene family of inbred mice in more detail. We show that mice express seven isoforms, all of which are encoded by genes that map to the Serpina1 locus. In addition, polymorphism at the locus is defined by three haplotypes (Serpina1(b), Serpina1(c), and Serpina1(l)) that differ with regard to both the number and identity of alpha(1)-PI genes. Finally, we present the complete sequence of an 84-kb region of Serpina1 containing a tandem repeat of two alpha(1)-PI genes.     

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.     

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.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.