Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na₂EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.     

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.     

Matrix degrading properties of sperm serine proteinase, acrosin.

The serine proteinase acrosin plays an important role in sperm penetration of the zona pellucida. In the present study we investigated the effect of the enzyme on various matrix proteins. Acrosin degraded proteolytically fibronectin, type IV collagen and heat denatured type I collagen, whereas neither native type I collagen nor laminin were cleaved by the enzyme. The specific activity of acrosin with type IV collagen as substrate (66.6 g/h/g) was 125-fold higher than that of known type IV collagenase or stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase.     

Angiogenesis is associated with collagenous protein synthesis and degradation in the chick chorioallantoic membrane.

Extracts of the chick embryo chorioallantoic membrane (CAM) obtained from 7-20 day old embryos, contained enzyme activity that could degrade type IV collagen. Peak enzyme activity was observed on days 8-10 of embryogenesis, which coincides with the stage of maximum angiogenesis. This activity decreased to lowest values at days 13-15 and increased thereafter up to day 20. Maximum rate of collagen biosynthesis in CAM was observed between days 7 and 10, with a drastic decrease at day 12, when vascular density has reached a plateau. The type IV collagen-degrading activity of CAM was of the metalloprotease type, since it was inhibited by 1,10-phenanthroline and EDTA but was also partially inhibited by serine and thiol protease inhibitors.     

Purification and characterization of a murine basement membrane collagen-degrading enzyme secreted by metastatic tumor cells

A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and type IV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m. The apparent molecular weight of the enzyme was 160,000 but about 70,000 when Triton X-100 was added to the column buffer. The purified enzyme protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptide chains of about 68,000 and 62,000 daltons. The enzyme activity could be increased by preincubation with trypsin and it is possible that the two chains represent latent and active enzyme forms. The enzyme activity was not reduced in the presence of dithiothreitol, it had a pH optimum of 7.6 and was inhibited by EDTA but not N-ethylmaleimide, phenylmethylsulfonyl fluoride, or Trasylol. The inhibition with EDTA was reversible. The pro-alpha 1(IV) and pro-alpha 2(IV) chains of the type IV procollagen substrate were both degraded at a similar rate to form two pairs of degradation fragments corresponding in molecular weights to about 70 and 30% of the original size chains. The presence of Triton X-100 increased slightly the activity of the enzyme and diminished the reduction of its activity upon freezing, indicating that the enzyme is a hydrophobic protein.     

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.     

Ras oncogene mediated induction of a 92 kDa metalloproteinase; strong correlation with the malignant phenotype

We have previously demonstrated that activated ras oncogenes can induce the metastatic phenotype and type IV collagenolytic activity in NIH/3T3 cells (Thorgeirsson et al. Mol. Cell. Biol. 5:259-262, 1985). The present study demonstrates ras-mediated induction of a 92 kDa metalloproteinase, which degrades gelatin and type IV collagen. Association of the 92 kDa proteinase expression with the malignant phenotype was also observed in human tumor cell lines. Our data indicate that the 92 kDa gelatin-collagen IV degrading metalloproteinase is an important participant in the proteolytic process involving tumor cell invasion and metastasis.     

Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen

Two major gelatinolytic metalloproteinases (gelatinases) of 65 kDa and 92 kDa were purified from a tumor cell line. Analysis of collagen degradation showed that native full-length Engelbreth-Holm-Swarm (EHS) type IV collagen was not cleaved by the purified gelatinases under conditions where native pepsin-extracted human placental type IV and V collagen and heat-denatured collagens were markedly degraded. However, EHS type IV collagen degradation was noted at 37 degrees C, i.e., under conditions that would favor denaturation of the collagen molecule in solution. The pattern of degradation of human placental type IV and V collagen appeared similar for both gelatinases. Zymogram analysis of gelatinase activity in the absence of sodium dodecyl sulfate (SDS) (to eliminate possible SDS-mediated denaturation of type IV collagen) confirmed the inability of 65 and 92-kDa gelatinases to degrade native full-length EHS type IV collagen. Under the same conditions and in SDS-polyacrylamide gel electrophoresis zymograms the gelatinases degraded pepsin-predigested EHS type IV collagen and pepsin-extracted human placental type IV collagen. These data suggest that the 65- and 92-kDa tumor cell gelatinases are not true type IV collagenases. Their ability to degrade pepsin-solubilized, or denatured, type IV collagen suggests a specificity for telopeptide precleaved or conformationally altered forms of this molecule.     

Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.     

Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

The appearance of a high molecular weight gelatinolytic enzyme (230 kDa) correlated with cartilage collagen loss in chick embryonic tibias cultured with lipopolysaccharide. This 230 kDa enzyme was purified and its activity was measured on synthetic and natural substrates. The enzyme was activated by aminophenylmercuric acetate and inhibited by ethylenediaminetetraacetic acid, phenanthroline, marimastat or tissue inhibitors of metalloproteinases. Amino acid sequences of peptides derived from the purified enzyme showed identity with avian MMP-9. Digestion of the intact enzyme with chondroitinase decreased the size of the molecule to 80 kDa on SDS-PAGE. When chick embryonic tibia cultures were radiolabeled with (35)S-sulfate, the radiolabel co-purified with the 230 kDa gelatinase. Chondroitinase treated 230 kDa gelatinase also reacted with specific anti-chondroitin sulfate antibodies and FACE analysis revealed a predominance of chondroitin-4-sulfate. These results demonstrate this avian matrix metalloproteinase contained glycosaminoglycan chains. To our knowledge, this is the first report of a matrix metalloproteinase in a proteoglycan form.     

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.     

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”.     

Matrix metalloproteinase-like activity from hemocytes of the eastern oyster, Crassostrea virginica

Investigation of oyster blood cell lysate revealed one prominent band of proteolytic activity when analyzed using gelatin and collagen impregnated polyacrylamide gel electrophoresis. The proteolytic activity was inhibited by 1,10 phenanthroline and EDTA, but not by other proteinase inhibitors. Maximal activity was shown at pH 8.2 and the molecular weight of the protein responsible for the activity was estimated to be 68 kDa. Proteolytic activity was also measured by fluorescence assays containing hemocyte lysate and fluorescein-labeled gelatin, type I or type IV collagen. Characteristics of this proteolytic activity suggest that an invertebrate matrix metalloproteinase is responsible.     

Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components

A metalloproteinase, 'proteoglycanase', that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the 'weak region' of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.     

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.     

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo r?le of gelatinases is discussed.     

Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture.

Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al. Biol Reprod 1990; 43:946-955, 956-964). We have now investigated the regulation of testicular cell type IV collagenase and other metalloproteinases in vitro. Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels. However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mRNA levels. Zymographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-kDa type IV collagenase protein levels. However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloproteinases (83 kDa and 110 kDa and in an activation of the 72-kDa rat type IV collagenase. Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation. Immunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells. Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern. Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 kDa, 83 kDa, and 110 kDa and a triton-insoluble gelatinase of 225 kDa. These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.     

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.     

Partial purification and characterization of a neutral proteinase with collagen telopeptidase activity produced by human gingival fibroblasts

A neutral proteinase was purified 1930-fold from medium conditioned by the culture of human gingival fibroblasts that had been stimulated to secrete enzymes by concanavalin A. This enzyme had an apparent molecular weight of 35,000 (gel chromatography) and apparent isoelectric point of 4.3 (chromatofocusing). It was inhibited by chelating agents, serum, and nonactivated conditioned fibroblast medium, but not by phenylmethylsulphonyl fluoride or N-ethylmaleimide. This proteinase removes the C-telopeptide from the alpha 1 chain of type I collagen, an activity which could be important in the degradation of collagen in the extracellular matrix. It was also found to digest fibronectin but had no effect on proteodermatan sulphate under the conditions used. It appears to be unrelated to previously described fibroblast extracellular proteinases and we, therefore, tentatively propose the name fibroblast metalloproteinase IV.     

Purification and properties of a new type of protease produced by Microbacterium liquefaciens

A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the alpha-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.