Proteases from purulent sputum. Purification and properties of the elastase and chymotrypsin-like enzymes.

A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.     

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.     

Clarification of mechanism of human sputum elastase inhibition by a new inhibitor,ONO-5046, using electrospray ionization mass spectrometry

Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) to probe the nature of the covalent E-I complex was successfully applied to clarify the mechanism of human sputum elastase (HSE) inhibition by a new inhibitor, ONO-5046. The mass spectrum of the four HSE isozymes displayed their molecular ion peaks at m/z=26,018, 25,929, 25,200, and 25,054, respectively. Immediately after incubation, inactivation of HSE with ONO-5046 increased the four molecular ion peaks by approximately 84 amu, which was assigned to the mass unit of the pivaloyl moiety of ONO-5046. An additional minute of incubation of E-I complex restored the original molecular ion peaks. These observations strongly suggested that ONO-5046 inactivates HSE by a reversible 'acylation-deacylation' mechanism.     

Deleterious effect of Brij 35 on alkyl 2-pyrones and other hydrophobic inhibitors of human sputum and leucocyte elastase

Brij 35 significantly reduced the inhibitory activity of hydrophobic alkyl 2-pyrones, oleic acid and alkyl peptides towards human sputum and leucocyte elastase, whereas 4-methoxy-6-(2'-hydroxy-2'-(carbobutyloxy)-vinyl)-2-pyrone, alpha-1-proteinase inhibitor and a sulfated chitosan were unaffected. The effect of Brij 35 on elastase appeared to be irreversible, since dialysis against Brij-free buffer was not accompanied by a return to inhibitory activity by the first group of inhibitors. However, passage through an ionic-exchange column was effective in removing the detergent from the enzyme. Brij 35 is also an activator of the elastases: kcat for Boc-Ala-4-nitrophenyl ester and methylsuccinyl-Ala-Ala-Pro-Val-4-nitroanilide increased by 20% and 40%, respectively in the presence of 0.015% Brij 35. Binding of the substrates to the enzyme is unaffected, since Km is unchanged.     

Elastases from human and canine granulocytes, I. Some proteolytic and esterolytic properties.

The lysosome-like granules of human and canine granulocytes contain an enzyme with elastinolytic activity. The enzymatic behaviour of these elastases was further characterized using the protein substrates elastin-orcein and azocasein and the synthetic substrates tert.-butyloxycarbonyl-alanine p-nitrophenylester (Boc-Ala-ONp) and 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide (Suc-Ala3-NHNp) in photometric assays. The affinities of the granulocyte elastases and of porcine pancreatic elastase to these substrates are very similar, e.g. human granulocyte elastase: KM (Boc-Ala-ONp) = 0.35mM, KM (Suc-Ala3-NHNp) = 1.25mM, porcine pancreatic elastase: KM (Boc-Ala-ONp) = 0.3mM, KM (Suc-Ala3-NHNp) - 1.15mM. The most convenient substrate for the assay of human and dog granulocyte elastases and for kinetic measurements with these enzymes is Suc-Ala3-NHNp. Using this substrate, the dissociation constant of the complex of human granulocyte elastase with human alpha1-antitrypsin could be determined (Ki = 3.5 x 10(-10)M).     

Purification and characterization of elastase-specific inhibitor. Sequence homology with mucus proteinase inhibitor

Elastase-specific inhibitor (ESI) was purified from sputum of patients with chronic bronchitis and compared with mucus proteinase inhibitor (MPI, BrI) isolated, without the use of affinity chromatography on an enzyme, from non-purulent sputum of a patient with bronchial carcinoma. The N-terminal sequence of 27 residues of the latter was determined and showed serine as the only N-terminus. The partial N-terminal amino-acid sequence of ESI shows some homology with MPI, especially around the reactive site of MPI for human neutrophil elastase. This region could therefore be the reactive site of ESI. The thermodynamic and kinetic constants of the reactions of ESI with human neutrophil elastase and with porcine pancreatic elastase show that ESI is a fast-acting inhibitor.     

Investigation of the active center of rat pancreatic elastase

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.     

Isolation and characterization of rat pancreatic elastase

Proelastase has been purified to homogeneity from rat pancreatic tissue by a combination of CM-Sephadex and immobilized protease inhibitor affinity resins. Trypsin activation yields an elastolytic enzyme that possesses a specificity toward small hydrophobic residues in synthetic amide substrates, similar to those of porcine elastase 1 and canine elastase. However, the rat enzyme also rapidly hydrolyzes a substrate containing tyrosine in the P1 position. N-Terminal sequence analysis reveals that rat proelastase has an identical activation peptide with that of porcine proelastase 1 and has two conservative amino acid sequence differences from the activation peptide of canine proelastase. The sequence data established that rat proelastase corresponds to the elastase 1 mRNA clone isolated by MacDonald et al. [MacDonald, R. J., Swift, G. H., Quinto, C., Swain, W., Pictet, R. L., Nikovits, W., & Rutter, W. J. (1982) Biochemistry 21, 1453]. The sequence and substrate data obtained for rat and canine elastases suggest that there is a family of pancreatic elastases with properties similar to those of the classically described porcine elastase 1.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.     

Elastases from human and canine granulocytes, II. Interaction with protease inhibitors of animal, plant, and microbial origin.

Inhibitors of animal, plant, and microbial origin were tested against human and canine granulocytic elastases. The trypsin-chymotrypsin inhibitors from dog submandibular glands, from soybeans (Bowman-Birk) and from chickpeas show strong interaction with these proteases (Ki = 10(-8) - 10(-9)M). The trypsin-kallikrein inactivator of bovine organs (Trasylol) is not active against granulocytic elastases or against human granulocytic cathepsin G. Elastatinal, a specific inhibitor of elastases, isolated from actinomycetes (Streptomyces griseoruber), forms stable complexes with elastase from human (Ki = 6.2 X 10(-6)M) and canine granulocytes (Ki = 1.1 X 10(-6)M). A possible therapeutic application of these inhibitors for the inactivation of granulocytic proteases, which are able to degrade connective tissue in different pathological states, is discussed.     

Inhibition of human leukocyte elastase by acetyl and trifluoroacetyl oligopeptide chloromethyl ketones.

The action of human leukocyte elastase on a series of acetyl and trifluoroacetyl tri-, tetra-, and pentapeptide chloromethyl ketones has been investigated. Leukocyte and pancreatic elastases react quite differently with these irreversible inhibitors. For instance, leukocyte elastase has a much lower affinity for the compounds than pancreatic elastase. On the other hand, the inhibition rate constants of the two enzymes are not influenced in the same way by peptide chain elongation. The two elastases, however, share a common property: trifluoroacetyl tri- and tetraalanine chloromethyl ketones are more tightly bound but are less reactive than the corresponding acetylated inhibitors. This behavior is probably due to the formation of nonproductive complexes between the enzymes and the trifluoroacetylated inhibitors.     

A new inhibitor of elastase from the sea anemone (Anemonia sulcata)

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.     

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.     

Human leukocyte granule elastase: rapid isolation and characterization.

Human granulocytic elastases have been purified by a two-step procedure involving affinity chromatography of crude extracts of leukocytic granules on Sepharose-Trasylol, followed by ion-exchange chromatography on CM-cellulose to resolve the isoelastases. All of these enzymes were found to be glycoproteins with the carbohydrate content of the major form being composed essentially of only neutral sugars. The molecular weight of this form was found to be near 30 000 daltons with the other forms being slightly higher. Preliminary structural analyses indicate that all of the elastase isozymes have identical NH2-terminal sequences suggesting that the differences in mobility of the four proteins are not due to different degrees of activation from a common zymogen but, more likely, from minor changes in carbohydrate content. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase, but both are inhibited by synthetic elastase active-site directed low molecular weight compounds (Tuhy, P. M., and Powers, J. C. (1975), FEBS Lett. 50, 359) as well as by plasma alpha-1-proteinase inhibitor (formerly called alpha-1-antitrypsin). In the latter case a stable complex with mol wt of 78 000 daltons is formed indicating the formation of a 1:1 complex.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

The elastases]

Elastases are proteinases capable of solubilizing fibrous elastin. They may belong to the class of serine proteinases, cysteine proteinases and metalloproteinases. Mammalian elastases occur mainly in the pancreas and the phagocytes. Among non-mammalian elastases there is a great variety of bacterial metallo and serine elastases. The elastolytic activity varies from one elastase to another and is usually not correlated with the catalytic efficiency of these proteinases. One may measure this activity using native or labelled elastins. With pure elastases one may use synthetic substrates. There is a large number of natural (proteins) and synthetic elastase inhibitors. Elastases play a pathologic role in pulmonary emphysema, cystic fibrosis, infections, inflammation and atherosclerosis.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

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.     

Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.     

Identification and expression of an elastase homologue in <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis> with implications to the origin of vertebrate pancreas

Elastases have been identified in a variety of organisms ranging from bacteria to insects to mammals, yet little is known to date about them in amphioxus, a model animal for insights into the origin and evolution of vertebrates. In this study we demonstrate the presence of an elastase homologue, named BbElas, in Branchiostoma belcheri. The recombinant BbElas hydrolyses the elastase specific substrate N-succinyl-Ala-Ala-Ala p-nitroanilide, which can be inhibited by the serine proteinase inhibitor PMSF, the elastase-specific inhibitor elastatinal and the cysteine proteinase inhibitor PCMB. Phylogenetic analysis shows that BbElas represents the archetype of vertebrate elastases, hinting at the clues that the different isoforms of vertebrate elastases are originated from an ancestral gene like BbElas. Our results also suggest that the mid-gut in amphioxus is to homologous vertebrate pancreas, a novel proposal which deserves further study.