Gelatinase Inhibitor Research Articles

The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Expression of Interstitial Collagenase, 92-kDa Gelatinase, and Tissue Inhibitor of Metalloproteinases-1 in Granuloma Annulare and Necrobiosis Lipoidica Diabeticorum

Granuloma annular (GA) and necrobiosis lipoidica diabeticorum (NLD) are disorders characterized by granulomatous inflammation and degenerative changes in collagen and elastic fibers. Because these disorders have often been described as being associated with altered extracellular matrix deposition, we studied the in situ expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases (TIMP)-1. Twelve lesions each of GA and NLD of different histopathologic types and durations were examined. Interstitial collagenase mRNA was seen in histiocyte-like cells in one-third of the cases of both diseases, typically in younger lesions. In GA, collagenase mRNA was only detected in lesions of the palisading type. Signal for 92-kDa gelatinase mRNA was observed in eosinophils, which were present in low numbers in five of 12 GA and three of 12 NLD samples. The signal for this enzyme and the presence of eosinophils did not correlate with the age of lesion. TIMP-1 mRNA was consistently expressed by histiocyte-like cells in both disorders. In GA, TIMP-1 mRNA was detected at the outer edge of the palisading granulomas, but in NLD, inhibitor expression was seen in the perivascular and periadnexal accumulation of inflammatory cells. Our data indicate that collagenase and TIMP are expressed early in these disorders and that these proteins may contribute to stromal remodeling associated with necrobiotic lesions. Our results further indicate that the localization of TIMP-1 production may provide a distinction between the two disorders, whereas metalloproteinase expression is not sufficiently specific to aid in the differential diagnosis of GA and NLD.

Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme.

The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.

Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and Stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH 3CH 2CH 2( R,S)CH(COOH)-NHLeuPheAlaNH 2, competitively inhibited cleavage of dinitrophenylProLeuGlyLeuTrpAla d ArgNH 2 at the GlyLeu bond by MMP-1 and MMP-2 ( K I = 30 and 40 μ m, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 m NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TC A and TC B fragments as well as slower hydrolysis of the α2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195–207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent M r of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly 6Phe 7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.

Effect of immunosuppressive drugs on the release of metalloproteinases from human polymorphonuclear leukocytes

The concentration of the metalloproteinases type I collagenase and gelatinase was measured in isolated polymorphonuclear leukocytes (PMNLs) of renal transplant recipients treated either with cyclosporin A (CyA) and prednisolone (Pr) (n = 8) or azathioprine (Aza) and Pr (n = 8), and of healthy subjects (n = 12). PMNLs of CyA- and Aza-treated transplant patients displayed markedly higher gelatinase content (2427 +/- 489 and 3284 +/- 357 ng/10(7) cells) than PMNLs of controls (528 +/- 83 ng/10(7) cells). There was also a higher content of type I collagenase in PMNLs (3374 +/- 292 ng/10(7) cells) of Aza-treated patients and significantly elevated levels in PMNLs of patients receiving CyA (3625 +/- 229 ng/10(7) cells) compared with healthy subjects (2878 +/- 151 ng/10(7) cells). In contrast, neutrophil lactoferrin content was lower in transplant patients. Thus, immunosuppressive drugs may reduce the release of leukocyte proteinases, which are known for their deleterious role in proteolytic tissue and matrix breakdown. In vitro, the effects of different immunosuppressive drugs on the release of lactoferrin, collagenase and gelatinase were investigated on FMLPNTL-stimulated PMNLs isolated from healthy subjects. CyA but not Aza or Pr caused inhibition of gelatinase, collagenase and lactoferrin release.

Effect of immunosuppressive drugs on the release of metalloproteinases from human polymorphonuclear leukocytes

Abstract. The concentration of the metalloproteinases type I collagenase and gelatinase was measured in isolated polymorphonuclear leukocytes (PMNLs) of renal transplant recipients treated either with cyclosporin A (CyA) and prednisolone (Pr) (n= 8) or azathioprine (Aza) and Pr (n= 8), and of healthy subjects (n= 12). PMNLs of CyA-and Aza-treated transplant patients displayed markedly higher gelatinase content (2427 ± 489 and 3284 ± 357 ng/107cells) than PMNLs of controls (528 ± 83 ng/107cells). There was also a higher content of type I collagenase in PMNLs (33741292 ng/107cells) of Aza-treated patients and significantly elevated levels in PMNLs of patients receiving CyA (3625 ± 229 ng/107cells) compared with healthy subjects (2878 ± 151 ng/107cells). In contrast, neutrophil lactoferrin content was lower in transplant patients. Thus, immunosuppressive drugs may reduce the release of leukocyte proteinases, which are known for their deleterious role in proteolytic tissue and matrix breakdown. In vitro, the effects of different immunosuppressive drugs on the release of lactoferrin, collagenase and gelatinase were investigated on FMLPNTL-stimulated PMNLs isolated from healthy subjects. CyA but not Aza or Pr caused inhibition of gelatinase, collagenase and lactoferrin release.

Tissue inhibitor of metalloproteases (TIMP) is matrix associated in aortic tissue: Report of a radioimmunoassay

Tissue inhibitor of metalloproteinases (TIMP) is the major inhibitor of collagenase, gelatinase, proteoglycanase, stromelysin, and metalloelastases. An imbalance between proteases and inhibitors has been implicated in numerous disease processes including tumor invasion, rheumatoid arthritis, emphysema, and aortic aneurysm disease. The purpose of this investigation was to develop a polyclonal antibody to recombinant TIMP and establish an immunoassay to measure immunoreactive protein in normal and diseased tissues. A polyclonal antibody was produced in rabbit against recombinant human TIMP which was characterized and used to establish a radioimmunoassay. The assay was used to measure immunoreactive protein in fibroblast conditioned medium, human serum, and aortic extracts. There was more immunoreactive TIMP in matrix associated urea extracts than soluble salt extracts from human aorta, suggesting that TIMP is matrix associated. The sensitivity of the assay enables the specific measurement of this inhibitor in serum, fibroblast culture medium, and tissue extracts.

Human osteoblasts in vitro secrete tissue inhibitor of metalloproteinases and gelatinase but not interstitial collagenase as major cellular products.

Human osteoblast cultures (hOB) were examined for the production of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP), and gelatinolytic enzymes. Cells were isolated by bacterial collagenase digestion of trabecular bone (vertebra, rib, tibia, and femur) from 11 subjects (neonatal to adult). Confluent cultures were exposed to phorbol 12-myristate 13-acetate, PTH, PGE2, epidermal growth factor, 1,25(OH)2 vitamin D3, recombinant human IL-1 beta, and dexamethasone. Collagenase and TIMP were assayed immunologically and also by measurements of functional activity. Collagenase was not secreted in significant quantities by human bone cells under any tested condition. Furthermore, collagenase mRNA could not be detected in hOB. However, hOB spontaneously secreted large amounts of TIMP for at least 72 h in culture. hOB TIMP was found to be identical to human fibroblast TIMP by double immunodiffusion, metabolic labeling and immunoprecipitation, Northern blot analysis, and stoichiometry of collagenase inhibition. SDS-substrate gel electrophoresis of hOB-conditioned media revealed a prominent band of gelatinolytic activity at 68 kD, and specific polyclonal antisera established its identity with the major gelatinolytic protease of human fibroblasts. Abundant secretion of gelatinolytic, but not collagenolytic, enzymes by hOB may indicate that human osteoblasts do not initiate and direct the cleavage of osteoid collagen on the bone surface, but may participate in the preparation of the bone surface for osteoclast attachment by removal of denatured collagen peptides. The constitutive secretion of TIMP may function to regulate metalloproteinase activity.

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).

Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium.

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serum-less conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.