A gelatin-specific protease from hamster lung-derived cell cultures

Summary Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited byp-chloromecuribenzoate,N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase. This research was supported by Program Project Grant HL-19717 from the National Heart, Lung, and Blood Institute, Grant AG 000-38-02 from the National Institute of Aging, and National Institute of Health Grant 5T32HL07035.     

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.     

Modulation of collagen production following bleomycin-induced pulmonary fibrosis in hamsters. Presence of a factor in lung that increases fibroblast prostaglandin E2 and cAMP and suppresses fibroblast proliferation and collagen production

To elucidate mechanisms involved in the regulation of lung collagen content we studied hamsters with bleomycin-induced pulmonary fibrosis. Lung collagen in this model is increased as the result of greatly increased lung collagen synthesis rates. However, collagen synthesis rates are subsequently restored to normal. Hamster lung explants from both normal and bleomycin-exposed hamsters were cultured, and the effects of explant conditioned medium (CM) on lung fibroblast (IMR-90) proliferation and collagen production in vitro were determined. Lung explant CM increased fibroblast prostaglandin (PG)E2 production and intracellular cAMP, and decreased both fibroblast proliferation and collagen production in a dose-dependent manner. Greater activity was observed with lung explant CM from bleomycin-exposed lungs. Incubation of fibroblasts with indomethacin prior to addition of CM blocked CM-mediated changes in PGE2 and cAMP and inhibited changes in fibroblast proliferation and collagen production. Exogenous PGE2 or dibutyryl cAMP also suppressed fibroblast proliferation and collagen production. The suppressive activity in lung-conditioned medium is nondialyzable, has an apparent molecular weight of 15,000-20,000 by gel filtration, and is heat-stable. It is not species-restricted since CM from hamster lung affected human and hamster lung fibroblasts similarly. Activity is present preformed in lung and bronchoalveolar lavage fluid, although bronchoalveolar macrophages produce a nondialyzable factor in culture which suppresses fibroblast proliferation. The suppressive activity identified in fibrotic lung may represent a means for limiting collagen accumulation following tumor injury.     

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.     

Purification and characterization of a novel calcium-dependent serine proteinase secreted from malignant hamster embryo fibroblast Nil2C2

A novel serine proteinase was purified from the conditioned medium of malignant hamster embryo fibroblasts, Nil2C2. The molecular weight of the purified enzyme was estimated to be 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The enzyme was split into two subunits (Mr 66,000 and 33,000) with a reducing agent. The enzyme hydrolyzed not only synthetic peptides which are susceptible to trypsin digestion but also extracellular matrix proteins such as type I and IV collagen, fibronectin and gelatin. For the digestion of these proteins, Ca2+ at millimolar concentrations was essential but Ca2+ or chelators did not affect the esterase activity for synthetic peptides. The proteolytic activity was inhibited by diisopropyl fluorophosphate (DFP) and also by phenylmethylsulfonyl fluoride. DFP was shown to bind to the 33 kDa subunit, indicating that the catalytic machinery of the enzyme is located in this subunit.     

Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Action of the Major Protease from Entamoeba histolytica on Proteins of the Extracellular Matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica , a cysteine protease of M, 27,000–29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated α2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.     

Purification and characterization of a unique alkaline elastase from Micrococcus luteus

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.     

Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors

Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.

All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro.

     

Human leucocyte neutral proteases, with special reference to collagen metabolism.

Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.     

Identification of a type V collagenolytic enzyme

A neutral metal protease has been identified which cleaves native type V collagen under conditions where pepsinized type IV collagen or the interstitial collagens are not significantly degraded. The enzyme is secreted into the media of cultured M50-76 reticulum cell sarcoma (malignant macrophages) and leiomyosarcoma tumor cells. Biosynthetically labeled type V collagen prepared from organ cultures of human amnion membrane is used for a routine assay of type V collagenolytic activity. The partially purified enzyme a) exists in a latent form requiring trypsin activation for maximum activity; b) has a molecular weight estimated by molecular sieve chromatography of approximately 80,000 daltons; c) is inhibited by EDTA but not phenylmethylsulfonyl fluoride; and d) produces specific cleavage products of both A and B collagen chains.     

A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization

The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL(-1), was observed in the medium containing soybean meal and tryptone, at pH 7 and 30 °C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7-9 and temperature 20-50 °C with optimum pH of 8 and temperature 35°C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.     

The role of glycosylation in the enzymatic conversion of procollagen to collagen: studies using tunicamycin and concanavalin A

The enzymatic conversion of chick embryo cranial bone procollagen was studied in vitro using procollagen proteases isolated from the culture medium of chick tendon fibroblasts. During the normal conversion process, chains intermediate in length between proα and α chains, as well as the COOH-terminal extension peptides, can be identified. Underglycosylated procollagen, synthesized by bones treated with an inhibitor of protein glycosylation (tunicamycin), was processed by these proteases in a manner similar to that of intact procollagen. However, medium from cells cultured with tunicamycin lacked the COOH-terminal procollagen protease activity; this did not result from a direct inhibition of the protease by the drug. Concanavalin A also inhibited the conversion of procollagen to collagen by fibroblasts in culture. In an in vitro system, Concanavalin A inhibited the COOH-terminal procollagen protease, and this inhibition was reversed by methyl-α-d-glucopyranoside. These data suggest that the COOH-terminal procollagen protease contains oligosaccharide side chains that are recognized by concanavalin A and that tunicamycin affects the secretion, activity, or activation of the enzyme.     

Partial purification and characterization of a neutral protease which cleaves type IV collagen

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.     

Degradation of fodrin by m-calpain in fibroblasts adhering to fibrillar collagen I gel

When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas micro-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, alpha-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel.     

Differential regulation of metalloproteinase production, proliferation and chemotaxis of human lung fibroblasts by PDGF, interleukin-1beta and TNF-alpha

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.     

Preclinical increase in activity of muscle microsomal trypsin-like protease in murine muscular dystrophy, C57BL/10-mdx

Preclinical alterations of protease activities in skeletal muscles from 10-day-old dystrophic mouse, C57BL/10-mdx, were examined by using 10 fluorogenic peptide substrates. Among the activities tested, only Boc-Val-Pro-Arg-MCA-hydrolyzing enzyme of the muscle microsomes showed an about 6-fold higher level of activity in mdx mouse. The increase in activity was not observed in tissues other than skeletal muscle. The enzyme had a pH optimum between 8.5 and 11.0, and was inhibited with DFP and variety of trypsin inhibitors. The enzymatic activity transiently increased at 1-2 weeks of age, the preclinical or very early stage of the disease. These results imply that the increased level of a trypsin-like protease possibly present in muscle microsomes may be closely related to the manifestation of muscular dystrophy.     

Isolation and characterisation of a chicken gelatinase (type IV collagenase).

The proform of chick gelatinase (type IV collagenase) was isolated and purified to a high specific activity of 12,071 U/mg from cultured embryonic skin fibroblasts stimulated with cytochalasin-B. The enzyme was activated in the presence of 4-aminophenylmercuric acetate with a fall in molecular weight from 66,000-58,000 on non-reducing polyacrylamide gel electrophoresis and was active over the pH range of 6.0-8.9 against a number of substrates. Further biochemical characterisation showed that the organomercurial activated form of the enzyme behaved like a typical mammalian gelatinase, actively degrading gelatin, soluble type I collagen, collagenase generated type I fragments, type IV collagen (producing 3/4 and 1/4 fragments) and type V collagen, whilst having little effect on laminin. The enzyme was inhibited by metal chelators such as EDTA and 1,10-phenanthroline, but not by inhibitors is suggested that this may be TIMP-2. An antiserum was raised to the proenzyme and was found to localise intra- and extra-cellularly in both tissue sections and cell cultures.