Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.     

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.     

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

High molecular mass type i.v. collagen-specific metalloprotease from human carcinoma tissue

A protease degrading type IV collagen was purified more than 8000-fold from human stomach carcinoma tissue. This protease degraded type IV collagen, while type I, II, III and V collagen, laminin, fibronectin, casein, albumin and hemoglobin were not affected. This enzyme had a pH optimum of pH 7.0-8.0 and was inhibited completely by EDTA and o-phenanthroline, but not by seryl, thiol and carboxyl protease inhibitors. Furthermore, the molecular mass of this enzyme was estimated to be 1 MDa by Sepharose 6B column and HPLC-gel filtration. The molecular mass and substrate specificity of this metalloprotease from human carcinoma tissue indicate it to be a new protease.     

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.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.     

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.     

Biological activity of a proteoglycan form of macrophage colony-stimulating factor and its binding to type V collagen.

Two different types of macrophage colony-stimulating factors (M-CSF) were found, one with an apparent molecular mass of 85 kDa and the other greater than 200 kDa. The high molecular mass M-CSF was identified as a proteoglycan carrying chondroitin sulfate glycosaminoglycan and was designated as the proteoglycan form of M-CSF (PG-M-CSF). In this study, we compared the biological activity of the 85-kDa M-CSF and PG-M-CSF and examined the binding properties of these two M-CSF to certain extracellular matrix proteins, i.e. types I-V collagen and fibronectin, using a modified enzyme-linked immunosorbent assay. PG-M-CSF was capable of supporting the formation of murine macrophage colonies, and pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate, did not alter its colony-stimulating activity. The specific activity of PG-M-CSF was similar to that of the 85-kDa M-CSF. The 85-kDa M-CSF had no apparent affinity for the extracellular matrix proteins examined, whereas PG-M-CSF had an appreciable binding capacity to type V collagen, but did not bind to types I, II, III, and IV collagen or to fibronectin. Pretreatment of PG-M-CSF with chondroitinase AC completely abolished the binding of the species to type V collagen. Addition of exogenous chondroitin sulfate inhibited the binding of PG-M-CSF to type V collagen in a dose-dependent manner. These data indicated that the interaction between PG-M-CSF and type V collagen was mediated by the chondroitin sulfate chain of PG-M-CSF. PG-M-CSF bound to type V collagen could stimulate the proliferation of bone marrow macrophages, indicating that the matrix protein-bound PG-M-CSF retained its biological activity. This interaction between PG-M-CSF and type V collagen implies that the role of PG-M-CSF may be distinct from that of 85-kDa M-CSF.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

Purification and characterization of tadpole back-skin collagenase with low gelatinase activity

A collagenase secreted by tadpole (Rana catesbiana) back-skin explants in culture has been purified to electrophoretic homogeneity by successive chromatography on sulfopropyl Sephadex, Sephacryl S-200, collagen Sepharose, and heparin Sepharose. The purified enzyme has a molecular weight of approximately 49,000 and an isoelectric pH of 5.0. The enzyme is more active versus soluble collagen than reconstituted fibrils and exhibits very low activity against gelatin (specific activities: Type I collagen, 7660 units/mg; Type I gelatin, 66 units/mg). The collagenase obeys simple Michaelis-Menten kinetics using soluble type I collagen (Km), 0.35 microM; Vm, 1380 units/mg, at 25 degrees C and pH 7.4) and is inhibitable by chelating agents specific for transition metals. Methylene blue catalyzes the photoinactivation of this collagenase, suggesting the presence of essential histidine, tryptophan, tyrosine, or methionine residues.     

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.     

Type-specific collagenolysis: a type V collagen-degrading enzyme from macrophages

An enzymatic activity capable of degrading type V collagen at neutral pH was found in the medium from cultured rabbit pulmonary alveolar macrophages which had been “activated” $\text{in}\text{vivo}$ by injection of complete Freund's Adjuvant. This enzyme was characterized as a metalloproteinase by virtue of its inhibition by EDTA but not by phenylmethylsulfonyl fluoride or N-ethyl maleimide. Ion-exchange chromatography on DEAE-cellulose was successful in separating the type V collagen-degrading activity from the type I collagenase which is also secreted by these cells. These observations suggest that the degradation of type V collagen is independent of the degradation of the interstitial collagens and may require the action of its own “specific collagenase”.     

Missense Mutations That Cause Bruck Syndrome Affect Enzymatic Activity, Folding, and Oligomerization of Lysyl Hydroxylase 2

Bruck syndrome is a rare autosomal recessive connective tissue disorder characterized by fragile bones, joint contractures, scoliosis, and osteoporosis. The telopeptides of bone collagen I are underhydroxylated in these patients, leading to abnormal collagen cross-linking. Three point mutations in lysyl hydroxylase (LH) 2, the enzyme responsible for the hydroxylation of collagen telopeptides, have been identified in Bruck syndrome. As none of them affects the residues known to be critical for LH activity, we studied their consequences at the molecular level by analyzing the folding and catalytic properties of the corresponding mutant recombinant polypeptides. Folding and oligomerization of the R594H and G597V mutants were abnormal, and their activity was reduced by >95% relative to the wild type. The T604I mutation did not affect the folding properties, although the mutant retained only ∼8% activity under standard assay conditions. As the reduced activity was caused by a 10-fold increase in the K_m for 2-oxoglutarate, the mutation interferes with binding of this cosubstrate. In the presence of a saturating 2-oxoglutarate concentration, the activity of the T604I mutant was ∼30% of that of the wild type. However, the T604I mutant did not generate detectable amounts of hydroxylysine in the N-terminal telopeptide of a recombinant procollagen I chain when coexpressed in insect cells. The low activity of the mutant LH2 polypeptides is in accordance with the markedly reduced extent of collagen telopeptide hydroxylation in Bruck syndrome, with consequent changes in the cross-linking of collagen fibrils and severe abnormalities in the skeletal structures.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure,expression of cDNA in E. coli and enzymatic properties

Human neutrophils can be triggered to release the collagenolytic metalloenzymes, interstitial collagenase and 92 kDa type IV collagenase/gelatinase. We have isolated and sequenced a 2.3 kb cDNA from a chronic granulocytic leukemia cDNA library that encodes for human neutrophil type IV collagenase. With the exception of one amino-acid substitution at position 280 (Arg → Gln), the deduced amino-acid sequences of neutrophil gelatinase are identical to the amino-acid sequences of the enzyme isolated from fibrosarcoma cells. Expression of the cDNA in E. coli yielded a 72 kDa protein having a gelatinolytic activity on zymogram gel. The recombinant enzyme was activated with APMA and trypsin. The activation was accompanied by a reduction in molecular weight of ≈ 10 kDa; such a reduction is characteristic of matrix metalloproteinases. The recombinant gelatinase cleaved native type V and XI collagens. Native type I collagen was not a substrate for the enzyme. These data suggest that native and recombinant 92 kDa type IV collagenase produced in E. coli have similar biochemical properties. The successful expression of the collagenase in a prokaryotic system will greatly facilitate the structure-function characterization of the enzyme and allow a more precise analysis of its physiological and pathological roles.     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts.

In the present study we investigated whether the collagen types I, III and V affect the activity of fibroblasts obtained from rabbit periosteum. The cells were cultured on plates either or not coated with different amounts of collagen type I, III or V and analyzed for their attachment, DNA synthesis and the expression and activity of matrix metalloproteinases (MMPs). Our data show that the three collagen types promoted attachment and spreading of the cells and stimulated DNA synthesis when used in relatively low concentrations. High concentrations of type V-but not of type I or III-proved to inhibit thymidine incorporation. The expression and activity of matrix metalloproteinase 1 (MMP-1; interstitial collagenase) decreased under the influence of relatively low amounts of collagen (<40 microg/well), whereas higher levels increased its release. Matrix metalloproteinase 2 (MMP-2; gelatinase A) was up-regulated by the different types of collagen; the active fraction of stromelysin-1 (MMP-3) decreased. Accordingly, the mRNA expression of MMP-1 and -3 were reduced. The expression of MMP-2 mRNA, however, proved to be unaffected. Blocking antibodies to beta(1)-integrin or echistatin increased the level of MMP-1 but had no effect on MMP-2. All parameters tested were similarly affected by type I and III collagen, whereas the effect of type V was always less. We conclude that the collagen types I, III and V provide different sets of signals for fibroblasts that differently modulate their proliferation and MMP expression.     

Type V collagen. Quantitation in normal lungs and in lungs of rats with bleomycin-induced pulmonary fibrosis

Type V collagen was first isolated in 1976; there is still controversy as to how many molecular species of type V collagen exist. Although its structural and functional roles remain unclear, reports of changes in the relative amount of type V collagen from that present in normal tissue have been reported in such diverse pathologic conditions as atherosclerotic aortas, prolapsed mitral valves, and fibrotic lungs. Methods for quantitating type V collagen relative to other collagens have consisted of solubilizing the collagen with pepsin and then analyzing the ratios of the intact chains by gel electrophoresis or by column chromatography. In tissues in which only a small percentage of the total collagen can be solubilized by pepsin, such analyses may not accurately reflect changes in the total collagen present. In this report, a method for quantitating type V collagen relative to types I and III collagens based on CNBr peptide mapping is presented. CNBr solubilizes virtually all the collagen present in any tissue. The method is applied to a model of bleomycin-induced pulmonary fibrosis in rats. It was found that type I collagen increased relative to types III and V collagens, which seemed to remain at values comparable to those observed in lungs from control (normal) rats, both in terms of newly synthesized collagen (collagen synthesized by lung minces during 4 h in culture) and total unlabeled lung collagen (collagen synthesized during the life of the animal).