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L J Windsor

Publications and source records attributed to L J Windsor.

At least 19 recordsLinked to original sources
PubMed2007

Heterogeneity in the collagen-degrading ability of Porphyromonas gingivalis-stimulated human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The purpose of this study was to characterize the heterogeneity of the collagen-degrading ability of different human gingival fibroblast cell lines treated with Porphyromonas gingivalis supernatant. MATERIAL AND METHODS: Seven human gingival fibroblast cell lines were analyzed for their ability to cleave Type I collagen in the presence and absence of culture supernatant from P. gingivalis ATCC 33277 (10% v/v). The matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) produced by these human gingival fibroblasts were monitored at the protein level by zymography and/or western blot analyses, as well as at the mRNA level by reverse transcription-polymerase chain reaction. RESULTS: The collagen-degrading ability of the human gingival fibroblasts increased in four cell lines (aggressive) and was only slightly altered in the other three cell lines (nonaggressive) in the presence of P. gingivalis supernatant. MMP-1, MMP-2, and MMP-3 more readily underwent activation while the TIMP-1 level was decreased in the conditioned media from a P. gingivalis-treated human gingival fibroblast aggressive cell line. None of these was altered in a nonaggressive cell line. The mRNA levels of the MMPs and TIMPs were only slightly different between these two cell lines. CONCLUSION: Heterogeneity exists in human gingival fibroblasts in regard to their collagenolytic activity in the presence of P. gingivalis.

Cell Line↗
PubMed2006

Temporomandibular joint synovial fibroblasts mediate serine proteinase dependent Type I collagen degradation.

Degenerative temporomandibular joint (TMJ) disorders are characterized by the excessive turnover of collagen. In addition to the matrix metalloproteinase (MMP) and cathepsin mediated collagen degradation pathways, a serine proteinase dependent pathway has recently been identified in TMJ fibroblasts. This study focused on further characterizing this serine proteinase pathway utilizing a media-mediated collagen degradation assay and zymography. The conditioned media from cell-mediated collagen degradation assays were incubated with Type I collagen at pH 7.5 with or without a MMP inhibitor (1,10-phenanthroline), serine proteinase inhibitors (alpha1-antitrypsin and soybean trypsin inhibitor, STI), or cysteine proteinase inhibitors. The data showed that 1,10-phenanthroline and STI reduced the collagen cleavage by 12.33% and 47.78%, respectively. The cysteine proteinase inhibitors had no effect. The combination of alpha1-antitrypsin and 1,10-phenanthroline inhibited the cleavage by 79.22%, while STI and 1,10-phenanthroline together blocked the cleavage by 85.44%. Zymography identified a proteinase at approximate 22.5 kDa, which was more effectively blocked by serine proteinase inhibitors than by MMP or cysteine proteinase inhibitors. Reverse transcript-PCR and real-time PCR results demonstrated that TMJ cells did not express trypsinogen-2 mRNA, a collagen cleaving serine proteinase. This study demonstrated that TMJ fibroblasts can predominantly utilize a serine proteinase to mediate collagen degradation, which is not trypsinogen-2.

Cell Line, Tumor↗
PubMed2004

Novel nonmatrix-metalloproteinase-mediated collagen degradation.

Matrix metalloproteinases (MMPs) and their inhibitors have long been believed to play a major role in the collagen loss seen in destructive temporomandibular joint (TMJ) disorders. This project was originally designed to examine the expression of MMPs and the tissue inhibitors of MMPs (TIMPs) by diseased human TMJ synovial fibroblasts and to determine their ability to degrade Type I collagen. Reverse transcriptase-PCR indicated that these TMJ synovial fibroblasts expressed mRNA for multiple MMPs and TIMPs. The collagen degradation assay showed that these TMJ synovial fibroblasts at passage 3 to 8 were capable of digesting the collagen underneath them on collagen-coated plates. This degradation was inhibited by GM6001, a synthetic MMP inhibitor. During passage 8 to 13, these TMJ fibroblasts were able to digest all the collagen in the wells. This degradation was completely inhibited by combining GM6001 and soybean trypsin inhibitor (STI), a serine proteinase inhibitor. The collagen cleavage activity of collected conditioned media was dramatically inhibited by STI but not by 1,10-phenanthroline, an MMP inhibitor. The data suggest that these TMJ cells utilize a MMP-dependent pathway and a novel MMP-independent pathway to digest Type I collagen.

Cells, Cultured↗
PubMed2003

Odontoblast morphology and dental repair.

OBJECTIVES: To investigate the changes in morphology and activity of pulp odontoblasts in response to cavity restoration variables and patient factors. METHODS: Class V non exposed cavities were prepared in the intact 1st or 2nd premolar teeth of 27 patients, aged between 9 and 17 years-old. Following tooth extraction, the area of reactionary dentine and the area of the odontoblasts were measured using computerised histomorphometry. RESULTS: The cytoplasm to nucleus ratio of the odontoblasts was found to increase beneath cut dentinal tubules, following the secretion of reactionary dentine. However, none of the patient or preparation variables were found to be correlated with changes in the odontoblast cytoplasm to nucleus ratio. CONCLUSIONS: Morphological changes in human odontoblasts is directly related to their capacity to repair dentine injuries and provide pulp protection. Changes in odontoblast morphology reflect secretory activity.

Adolescent↗
PubMed2003

Remaining dentine thickness and human pulp responses.

AIM: To evaluate pulp responses as a function of remaining dentine thickness (RDT) of 98 class V cavity preparations in 49 teeth of 31 patients aged 10-16 years. METHODOLOGY: Shallow cavities were restored with amalgam, deeper cavities or pulp exposures were restored with amalgam lined with calcium hydroxide or with zinc oxide eugenol. Teeth were extracted after 3-89 days for orthodontic reasons. Following processing for light microscope analysis, the number of odontoblasts, pulp inflammation, and repair was recorded. RESULTS: In comparison with independent odontoblasts, the numbers of odontoblasts were reduced by 13.6% beneath a RDT of 2.5-0.5 mm, 33.7% beneath a RDT of 0.5-0.01 mm and 99.0% beneath pulp-exposed cavities. Reparative dentine was observed following pulp exposure and reactionary dentine was observed with a mean RDT of 0.77 mm (2.5-0.01 mm). Reactionary dentine secretion was influenced by RDT and restorative materials. Pulp inflammation was not influenced by RDT in the present study. CONCLUSIONS: Cavity RDT mediates a powerful influence on underlying pulp tissue vitality but it has little effect on reactionary dentine secretion and inflammatory activity. Gross tissue injury explains the poor pulp capping prognosis following exposure and underlies the need to avoid this type of injury. Following restoration, a RDT of 0.5 mm or greater is necessary to avoid evidence of pulp injury.

Adolescent↗
PubMed2003

Histomorphometric analysis of odontoblast-like cell numbers and dentine bridge secretory activity following pulp exposure.

AIM: The purpose of this study was to collect quantitative information about the numbers and dentine bridge secretory activity of odontoblast-like cells following dental pulp exposure. METHODOLOGY: The numbers and secretory activity of odontoblast-like cells were measured histomorphometrically between 7 days and 2 years in 161 pulp-exposed nonhuman primate teeth. The area of dentine bridges and the dimensions of cavity preparations were measured. The density of odontoblast-like cells and subjacent reorganizing tissue cells were measured beneath dentine bridge formation. The presence of operative dentine debris and tunnel defects in bridges was noted. Pulp inflammation was categorized according to ISO standards. Bacteria were detected using McKay's stain. RESULTS: The area of dentine bridges was mediated by the density and secretory activity of odontoblast-like cells over time. The cell density of subjacent reorganizing tissue was found to be strongly associated with that of odontoblast-like cells. Bacterial microleakage was found to impede dentine bridge secretion by odontoblast-like cells. CONCLUSIONS: Pulp reparative activity occurs naturally beneath capping materials in the absence of bacterial microleakage. The outcome of pulp-capping treatments could be beneficially influenced by concentrating attention on limiting the width of pulp exposure, minimizing pulp injury by limiting the creation of operative debris and placing materials which prevent bacterial microleakage.

Analysis of Variance↗
PubMed2001

Membrane type I-matrix metalloproteinase-mediated degradation of type I collagen by oral squamous cell carcinoma cells.

Oral squamous cell carcinomas are highly invasive lesions that destroy adjacent tissues and invade bone and muscle, which is most likely the result of matrix metalloproteinase (MMP) activity. We examined three cell lines derived from squamous cell carcinoma of the tongue for their intrinsic capacities to degrade interstitial collagen with the goal of identifying the matrix-degrading enzymes. SCC-25 and SCC-15 cells degrade reconstituted fibrillar type I collagen in the absence of exogenous growth factors or cytokines when seeded as a colony on dried films. Degradation is confined to the subjacent matrix, is enhanced 2-3-fold by phorbol ester, and is strictly MMP-dependent, as it is blocked by BB-94 and tissue inhibitor of metalloproteinases-2 but not by inhibitors of serine and cysteine proteinases. Both cell lines express active (M(r) 57,000) membrane type I-MMP (MT1-MMP) on their surfaces, as detected by surface biotinylation and immunoprecipitation. Concomitantly, both cell lines activate endogenous MMP-2 when cultured on type I collagen films, as assessed by zymography. Phorbol ester treatment enhances collagen-induced MMP-2 activation, which is accompanied by the appearance of a surface-labeled M(r) 43,000 form of MT1-MMP. Treatment of cells with a synthetic furin inhibitor, which inhibits processing of the MT1-MMP zymogen, blocks collagen degradation. In contrast, CAL 27 cells do not degrade collagen under either basal or phorbol 12-myristate 13-acetate-stimulated conditions. Although proMT1-MMP (M(r) 63,000/65,000) is detectable in these cells by immunoblot analysis, they express greatly reduced levels of active MT1-MMP on their surfaces relative to SCC-25 and SCC-15 cells. Correspondingly, CAL 27 cells cultured on collagen express neither latent nor active gelatinases. Immunoblots of lysates and conditioned media revealed the constitutive expression of proMMP-1 and proMMP-13 in all three cell lines. We conclude that in the absence of exogenous growth factors or accessory stromal cells, degradation of interstitial collagen by oral squamous cell carcinoma cells requires a threshold level of active MT1-MMP on cell surfaces.

Animals↗
PubMed2001

Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: a possible mechanism of basement membrane and integrin alterations.

We have previously described decreased immunostaining of nidogen-1/entactin; laminin chains alpha1, alpha5, beta1,gamma1; and epithelial integrin alpha3beta1 in human diabetic retinopathy (DR) corneas. Here, using 142 human corneas, we tested whether these alterations might be caused by decreased gene expression levels or increased degradation. By semiquantitative reverse transcription-polymerase chain reaction, gene expression levels of the alpha1, alpha5, and beta1 laminin chains; nidogen-1/entactin; integrin alpha3 and beta1 chains in diabetic and DR corneal epithelium were similar to normal. Thus, the observed basement membrane and integrin changes were unlikely to occur because of a decreased synthesis. mRNA levels of matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly elevated in DR corneal epithelium and stroma, and of MMP-3/stromelysin-1, in DR corneal stroma. No such elevation was seen in keratoconus corneas. These data were confirmed by immunostaining, zymography, and Western blotting. mRNA levels of five other proteinases and of three tissue inhibitors of MMPs were similar to normal in diabetic and DR corneal epithelium and stroma. The data suggest that alterations of laminins, nidogen-1/entactin, and epithelial integrin in DR corneas may occur because of an increased proteolytic degradation. MMP-10 overexpressed in the diabetic corneal epithelium seems to be the major contributor to the observed changes in DR corneas. Such alterations may bring about epithelial adhesive abnormalities clinically seen in diabetic corneas.

Adult↗
PubMed2001

TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function.

PURPOSE: To determine transforming growth factor (TGF) beta effects on matrix metalloproteinases (MMPs) as a potential cause of the blood-retinal barrier breakdown at the onset of angiogenesis. Previously, glial cells were shown to play a role in the angiogenesis process and to express the angiogenic regulating factor TGF-beta, which becomes active under hypoxia conditions. Here, the authors demonstrate that retinal endothelial cells express MMP-9 when treated with TGF-beta or cocultured with glial cells and that both TGF-beta and MMP-9 increase endothelial cell permeability. METHODS: Primary cultures of bovine retinal endothelial (BRE) cells grown on porous membranes were treated with TGF-beta or purified MMP-9, and permeability changes were assayed. The amount and distribution of the tight junction protein occludin also was analyzed by immunocytochemistry and Western blotting. Cell extracts or conditioned media from TGF-beta-treated BRE cells and from glial cell-BRE cocultures were analyzed for MMP-9 content by substrate gel electrophoresis (zymography) or Western blotting. RESULTS: Both TGF-beta and MMP-9 increased the permeability of BRE monolayers and reduced the levels of the junction protein occludin. The effect of MMP-9 on permeability was rapid, but the TGF-beta-induced permeability required longer incubation and was blocked by anti-TGF-beta and anti-MMP-9 antibodies as well as by TGF-beta latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE cultures, was dramatically increased by TGF-beta as well as by coculturing with either astrocytes or Müller glial cells. Anti-TGF-beta antibody blocked the TGF-beta effect, but not the coculture effect on MMP-9 production. CONCLUSIONS: These data indicate a direct correlation between TGF-beta-induced MMP-9 activity and increased endothelial cell permeability. Moreover, endothelial cell production of MMP-9 is regulated by glial cells through expression of TGF-beta or by direct cell-to-cell contact. During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retinal barrier by stimulating endothelial cell MMP-9 production.

Animals↗
PubMed2000

Induction of collagenase-3 (MMP-13) in rheumatoid arthritis synovial fibroblasts.

There is a growing body of evidence that implicates matrix metalloproteinases (MMPs) as major players in numerous diseased conditions. The articular cartilage degradation that is characteristic of rheumatoid arthritis (RA) is believed to be mediated by the collagenase subfamily of matrix metalloproteinases. The preference of collagenase-3 (CL-3) for collagen type II makes it a likely candidate in the turnover of articular cartilage and a potential target for drug development. In this study, RA synovial membrane tissue was shown to express CL-3 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunohistochemistry. Fibroblasts isolated and cultured from RA synovial membrane tissue were induced to express CL-3 mRNA. CL-3 mRNA was detected after PMA treatment in 16 of the 18 RA synovial membrane fibroblast cell lines established for this study. These fibroblasts also expressed mRNA for collagenase-1 (CL-1, MMP-1), membrane type-1 matrix metalloproteinase, gelatinase A, gelatinase B, stromelysin-1, stromelysin-2, TIMP-1, and TIMP-2. They were further shown to express CL-1 mRNA constitutively and CL-3 mRNA only after stimulation with PMA, IL-1, TGF-beta1, TNF-alpha, or IL-6 with IL-6sR. These fibroblasts also expressed after induction both CL-1 and CL-3 at the protein level as determined by Western blot analyses and immunofluorescence.

Antibodies↗
PubMed2000

Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1.

Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) family of enzymes. The active site glutamic acid of the MMPs is conserved throughout the family and plays a pivotal role in the catalytic mechanism. The structural and functional consequences of a glutamate to glutamine substitution in the active site of stromelysin-1 were investigated in this study. In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activated by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue inhibitor of metalloproteinases, after cleavage of the propeptide with trypsin. A second construct containing the glutamine substitution but lacking the propeptide was also inactive in the proteolysis assays and capable of TIMP-1 binding. X-ray structures of the wild-type and mutant proteins complexed with the propeptide-based inhibitor Ro-26-2812 were solved and in both structures the inhibitor binds in an orientation the reverse of that of the propeptide in the pro-form of the enzyme. The inhibitor makes no specific interactions with the active site glutamate and a comparison of the wild-type and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.

Amino Acid Substitution↗
PubMed1999

Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked by APMA ((4-Aminophenyl)mercuric acetate).

Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matrix metalloproteinases (MMPs). The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological conditions. SL-1 like the other MMPs can be activated in vitro by the stepwise removal of the propeptide that contains a single unpaired cysteine which coordinates the active site zinc. Other residues in the propeptide also play a role in maintaining the latency of the enzymes. Deletion mutants and single-site amino acid replacements within the propeptide of a carboxyl-terminally truncated stromelysin-1 (mini-SL-1) were constructed and expressed in Escherichia coli to further examine what amino acids within the propeptide of SL-1 are important for maintaining latency. While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower Mr in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E. coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide. Mutant Delta63 as well as other mutants in which most of the propeptide had been deleted no longer responded to exposure to the organomercurial APMA by accelerated autolytic processing. Rather, APMA inhibited the autolytic processing in these mutants, further confirming the complexity of the action of this organomercurial in the activation of pro-MMPs.

Amino Acid Sequence↗
PubMed1999

92K-GL (MMP-9) and 72K-GL (MMP-2) are produced in vivo by human oral squamous cell carcinomas and can enhance FIB-CL (MMP-1) activity in vitro.

Previous studies have shown a correlation between the production of certain matrix metalloproteinases (MMPs), especially the gelatinases, by malignant tumors and the progression of these cancers as they invade and metastasize through the extracellular matrix and basement membranes. However, very few of these studies examined this relationship in human oral cancer in vivo, and none addressed the issue of how combinations of the MMPs may further enhance tumor progression. To determine which MMPs are produced in vivo by human oral cancers, we used specific anti-human-MMP antibodies and immunocytochemistry (ICC) methods to examine oral cancer tissue specimens from 20 surgery patients. The ICC data indicated that 72-kDa (72K-GL) and 92-kDa gelatinases (92K-GL) were produced in vivo by discreet clusters of tumor cells and by stromal fibroblasts, vascular endothelial cells (72K-GL), and PMNs (92K-GL). Some stromal fibroblasts near the tumors also appeared to produce fibroblast-type collagenase (FIB-CL), a finding confirmed by Western blot analysis of media conditioned by oral tumor explant cultures. ICC results indicated that 5 of the 20 tumors coincidentally produced all three MMPs. To examine how the two gelatinases and FIB-CL may interact in vitro to degrade fibrillar type I collagen, a major structural component of the extracellular matrix, we used a modified FIB-CL activity assay. Combinations of the gelatinases and FIB-CL were incubated with a 3H-collagen substrate, with the results compared with the combination of stromelysin-1 (SL-1, a superactivator of FIB-CL) and FIB-CL. 92K-GL caused a nine-fold increase in collagenase activity, equivalent to SL-1, while 72K-GL produced a four-fold increase. These results indicate that human oral cancers produce 92K-GL, 72K-GL, and FIB-CL in vivo and that the gelatinases and FIB-CL cooperate to enhance collagen degradation greatly in vitro.

Antibodies↗
PubMed1998

Transcriptional suppression of matrix metalloproteinase-2 gene expression in human astroglioma cells by TNF-alpha and IFN-gamma.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that function in the turnover of extracellular matrix components during development. In addition, MMPs also contribute to pathological conditions associated with inflammation, angiogenesis, and tumor invasion. A 72-kDa type IV collagenase, also referred to as gelatinase A or MMP-2, has been proposed to potentiate the invasion and metastasis of malignant tumors. In particular, MMP-2 activity has been shown to constitute an important component of human astroglioma invasion. We investigated the influence of various cytokines, both proinflammatory and immunosuppressive, on MMP-2 gene expression in two human astroglioma cell lines (U251-MG and CRT). Our results indicate that the cell lines constitutively express high levels of MMP-2 mRNA, protein, and bioactivity as assessed by ribonuclease protection assay, immunoblotting, and zymography assays, respectively. The proinflammatory cytokines TNF-alpha and IFN-gamma individually can inhibit constitutive MMP-2 expression, and function in an additive manner for near-complete inhibition of MMP-2 expression. Inhibition of MMP-2 mRNA levels by TNF-alpha and IFN-gamma is not due to destabilization of the MMP-2 message; rather, inhibition is mediated at the transcriptional level. Furthermore, TNF-alpha/IFN-gamma inhibition of MMP-2 expression results in decreased invasiveness of the human astroglioma cells through an extracellular matrix. These results raise the possibility that TNF-alpha and IFN-gamma may have beneficial effects in attenuating astroglioma invasive properties.

Astrocytes↗
PubMed1998

Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and Mr 72000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2-4-fold lower than that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose-response curve suggested that the interaction with the Mr 72000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and Mr 72000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.

Amino Acid Sequence↗
PubMed1998

Induction of matrix metalloproteinases and a collagen-degrading phenotype in fibroblasts and epithelial cells by secreted Porphyromonas gingivalis proteinase.

Periodontitis is characterized by advancement of a narrow band of epithelium (1-10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiol-proteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.

Adhesins, Bacterial↗
PubMed1997

Replacement of conserved cysteines in human tissue inhibitor of metalloproteinases-1.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP-1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity against Mr 72,000 gelatinase (Mr 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC --> CQQC retained their ability to inhibit FIB-CL in a manner similar to wild-type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site "parent" mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.

Amino Acid Sequence↗