Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stromelysin/Transin is a member of the matrix metalloprotease gene family. This metalloprotease is synthesized as a preproenzyme with a predicted size of 53,977 Da including a 17 amino acid signal peptide. Prostromelysin is secreted from normal and transformed cells in two forms with apparent molecular masses on NaDodSO4 gels of 60 and 58-kDa. The minor 60-kDa species contains N-linked oligosaccharide(s). Stromelysin consists of three domains the amino terminal propeptide(s) domain contains the tribasic amino acid sequence RRK which is important in the proteolytic activation of this zymogen by trypsin-like serine proteases. The second domain consists of the catalytic domain which contains the zinc binding site. The carboxyl-terminal hemopexin domain has no known function and can be removed without a loss of enzymatic activity. Stromelysin has a broad range of substrate specificity including proteoglycans, casein, fibronectin, laminin, native type IV and IX collagen and gelatin but not type I collagen. In the presence of trypsin or plasmin, catalytic amounts of this enzyme can also fully activate interstitial fibroblast collagenase. We have developed a panel of monoclonal antibodies against stromelysin which will be useful for the tissue localization of the various species of this enzyme in tissues. In addition, we have demonstrated that either human rIL-1 (alpha) or rTNF (alpha) can stimulate the expression of this enzyme in cultured bovine articular cartilage at least 10-fold. Based on western blot analysis, the zymogen form of the enzyme was the major enzyme species detected in either the media or cartilage matrix compartments of cytokine treated cultures.
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PMID:Primary structure and function of stromelysin/transin in cartilage matrix turnover. 148 63

The sequence of hemopexin consists almost entirely of two homologous domains joined by a short hinge region; the domain structure, with its own characteristic features, is derived from four short tandem repeats. Each repeat contains several alternating clusters of hydrophobic and hydrophilic residues but also has some individual features as a consequence of its position in the domain. Here we present evidence for the presence of a single hemopexin domain in an interstitial collagenase and in a collagenase homolog, as well as of two copies of the domain in vitronectin. The functions of all of these proteins involve binding to various proteins and smaller molecules. We suggest that the presence of this domain may facilitate these binding activities. Our analysis also suggests a tentative identification of substrate-binding and catalytic domains in the collagenase and its homolog.
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PMID:A domain structure common to hemopexin, vitronectin, interstitial collagenase, and a collagenase homolog. 245 21

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.
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PMID:Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members. 255 50

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.
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PMID:Structure-function relationships in the collagenase family member transin. 284 36

Primary cultures of hepatocytes isolated by collagenase perfusion of adult rats were transformed by infection with adenovirus type 5 or transfection with adenovirus DNA. Total virion DNA or recombinant plasmid DNA containing the adenovirus E1A and E1B genes transformed hepatocytes at comparable frequencies. No foci of replicating hepatocytes were detected after transfection with a plasmid containing the E1A gene alone. The frequency of transformation by the adenovirus E1A and E1B genes was dependent on the composition of the culture medium. Transformation occurred at a low frequency when the transfected hepatocytes were maintained in a chemically defined medium (CDM), but the frequency was enhanced 8- to 10-fold when the cells were maintained in (i) serum-supplemented medium or (ii) CDM supplemented with epidermal growth factor. Cell lines derived from the adenovirus-transformed colonies of hepatocytes expressed adenovirus E1A and E1B RNAs. When hepatocytes were maintained in CDM supplemented with dimethyl sulfoxide and transfected with plasmids containing the E1A and E1B genes, it was possible to derive cell lines that retained the ability to express several liver-specific genes, including albumin, transferrin, hemopexin, and the third component of complement. The amount of albumin secreted per cell varied from 1 to 5 pg per cell per 24 h, and in one cell line it was below detectable levels by passage 9. Adenovirus-transformed hepatocytes were not tumorigenic when inoculated subcutaneously into neonatal syngeneic rats. We conclude that the adenovirus E1A and E1B genes are capable of transforming adult rat hepatocytes, a differentiated epithelial cell type.
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PMID:Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA. 366 53

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.
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PMID:Recombinant porcine collagenase: purification and autolysis. 784 Jun 5

In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 x 10(-10) M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher M(r) complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 x 10(-9) M for the truncated variant's interaction with TIMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 x 10(-9) M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.
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PMID:Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases. C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin. 817 79

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.
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PMID:Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. 821 28

The coding region for human neutrophil short form procollagenase lacking the hemopexin like domain coding region was amplified by polymerase chain reaction. Recombinant short form procollagenase was expressed in E. coli and purified in a three step procedure. Renaturation of this proenzyme was carried out by an effective new method using Q-Sepharose chromatography. Treatment of short form procollagenase with mercurials resulted in active short form collagenase M(r) 21,000 and an intermediate product of M(r) 23,000. These two products were separated by hydroxamate affinity chromatography. The active, short form collagenase M(r) 21,000 is stable. Despite full proteolytic activity, it lacks type I collagen substrate specificity and forms the basis for crystallisation experiments.
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PMID:The recombinant catalytic domain of human neutrophil collagenase lacks type I collagen substrate specificity. 846 Sep 92

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
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PMID:Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme. 853 33


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