Specific inhibition of human granulocyte elastase with peptide aldehydes

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.     

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.     

Elastolysis of normal and partially cross-linked elastin

Rate of elastolysis by pancreatic and leukocyte elastases of normal and copper deficient porcine aortic elastins were measured using a conductimetric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if a near uniformity exists in KM, differences were observed in catalytic rates, kcat increasing approximately 40% for copper deficient elastin elastolysis by leukocyte elastase. This higher susceptibility to proteolysis may have implications for understanding turnover of elastin in tissues.     

pH dependence of salt activation of human leukocyte elastase

The effects of pH on salt stimulation of the rates of hydrolysis of three substrates by human leukocyte elastase were studied. The enzyme was most active at pH 10.5, 8.0-8.5, and 9.5 for the hydrolyses of fluorescein isothiocyanate-labeled S-carboxymethylated bovine serum albumin (FITC-CM-BSA), succinyl-L-Ala-L-Pro-L-Ala-7-methylcoumaryl-4-amide (Suc-APA-MCA), and succinyl-L-Ala3-p-nitroanilide (Suc-Ala3-pNA), respectively, in the absence of NaCl. The enzyme was activated by 0.5 M NaCl similarly at all pHs tested for the hydrolysis of Suc-Ala3-pNA, but more at neutral and alkaline pH values, respectively, for the hydrolyses of FITC-CM-BSA and Suc-APA-MCA. Thus, in the presence of 0.5 M NaCl, the enzyme was most active at pH 8.0 and 10.0 with FITC-CM-BSA and Suc-APA-MCA, respectively. In contrast, the proteolytic activity of porcine pancreatic elastase was somewhat inhibited by 0.5 M NaCl.     

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.     

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

Although protease E was isolated from human pancreas over 10 years ago [Mallory, P. A., & Travis, J. (1975) Biochemistry 14, 722-729], its amino acid sequence and relationship to the elastases have not been established. We report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. We have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. We also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1.     

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).     

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).     

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.     

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.     

Elastase-type activity of human serum. Its variation in chronic obstructive lung diseases and atherosclerosis

Human serum was found to contain enzyme activities hydrolyzing succinyl trialanine paranitroanilide and 3H-kappa-elastin Sepharose substrates. Both types of activities could be partly abolished by serine active site titrants (phenylmethanesulfonylfluoride, diisopropylphosphorofluoridate) and partly by neutral chelating agents (EDTA; 1-10-phenanthroline). The combination of phenylmethanesulfonylfluoride and EDTA gave a complete inhibition of human serum elastase-type activities indicating the presence of at least two different types of elastases (serine and metalloproteases) in human serum. In nonsmokers, the average serum elastase-type activity on succinyl trialanine paranitroanilide was found equal to 78.1 ng/ml porcine pancreatic elastase equivalents and on 3H-kappa-elastin sepharose beads equal to 688.8 ng/ml. No statistically significant differences were observed in elastase levels in the sera of individuals presenting clinical symptoms of atherosclerosis. The sera of patients suffering from chronic obstructive lung diseases contained, however, higher amounts of elastase-type activities, respectively equal to 237.2 ng/ml on succinyl trialanine paranitroanilide and 1,096 ng/ml on 3H-kappa-elastin Sepharose beads and was quantitatively significant when compared with control subjects.     

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.     

Kinetics of nitroanilide cleavage by astacin

The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.     

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.     

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

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

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

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.     

Irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotrypsin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (kobsd/[I] = 27,000 M-1 s-1) and porcine pancreatic elastase (PPE, kobsd/[I] = 11,000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-ValP(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PheP(OPh)2 reacts with chymotrypsin (146,000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was greater than 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)