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L J Windsor

Publications and source records attributed to L J Windsor.

At least 37 records · Page 2Linked to original sources
PubMed1997

Catalytic domain comparisons of human fibroblast-type collagenase, stromelysin-1, and matrilysin.

The propeptide plus the catalytic domain of human fibroblast-type collagenase, stromelysin-1, and matrilysin were expressed in Escherichia coli to directly compare the properties of all three catalytic domains utilizing the same assays. Truncated fibroblast-type collagenase (mini-CL), truncated stromelysin-1 (mini-SL-1), and matrilysin, like their native counterparts, could be activated by organomercurials, trypsin, or SDS. The mini-CL and mini-SL-1 displayed catalytic properties similar to their native counterparts, except that the mini-CL could not cleave native type I collagen. The k(cat)/Km for matrilysin (355 microM(-1) h(-1)) on the synthetic Mca-peptide was much higher than that for mini-CL (69 microM(-1) h(-1)) or mini-SL-1 (23.6 microM(-1) h(-1)). Mini-SL-1 and matrilysin, but not mini-CL, were capable of superactivating collagenase thus increasing the rate of collagen cleavage. Mini-CL and mini-SL-1, but not matrilysin, were able to form SDS-stable complexes with TIMP-1 when co-incubated with an organomercurial and TIMP-1. The second-order rate constant (k(on)) for TIMP-1 inhibition of mini-CL and mini-SL-1 were similar, 0.635 x 10(5) M(-1) s(-1) and 1.52 x 10(5) M(-1) s(-1), respectively. The k(on) for TIMP-1 inhibition of matrilysin was lower (0.130 x 10(5) M(-1) s(-1)) supporting the observation that no SDS stable complexes were detected. This study demonstrates that these catalytic domains are distinct and play a major role in the specificity of these enzymes in regard to rate of catalysis, TIMP-1 binding, and superactivation of collagenase.

Catalysis↗
PubMed1997

Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis.

A critical outcome of periodontal disease is degradation of the collagenous periodontal ligament that connects teeth to bone in the dental arch. Periodontal diseases occur in response to bacterial colonization of the teeth, but their molecular pathogenesis is still speculative. One family of enzymes, known as the matrix metalloproteinases (MMPs), has been implicated in the degradation of the periodontal ligament. MMPs, which are also suspected to play a role in many other physiologic and pathologic remodeling processes, can be secreted by epithelial cells surrounding the teeth and are found in relative abundance in tissues and fluids near periodontally diseased sites. Since most MMPs are secreted as inactive zymogens which may be activated by limited proteolysis, it has been suggested that proteinases expressed by the infecting periodontal pathogens might activate latent host MMPs to initiate or accelerate degradation of the collegenous periodontal ligament. The aim of this work was to examine interactions between purified host MMPs and bacterial proteinase. In this article, we demonstrate that a proteinase isolated from the periodontopathogen Porphyromonas gingivalis can activate MMP-1, MMP-3, and MMP-9 and can catalyze the superactivation of MMP-1 by MMP-3. Activation of these MMPs is demonstrated to result from initial hydrolysis within their propeptide. Also, for MMP-1 and MMP-9, the P. gingivalis proteinase cleaves the MMP propeptide following a lysine residue at a previously unreported site which, for both MMPs, is one residue NH2-terminal to the known autocatalytic cleavage site. These data describe a mode of virulence for the periodontopathogen Porphyromonas gingivalis that involves activation of host-degradative enzymes.

Amino Acid Sequence↗
PubMed1996

The mechanism of inhibition of collagenase by TIMP-1.

Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight-binding inhibitor of fibroblast-type collagenase. Time-course data from inhibition experiments were analyzed by graphic analysis, by nonlinear regression of the analytic integrals of the rate equations and by nonlinear regression with numeric integration of the rate equations. With the same assumptions, approximations and data, all three methods of analysis produced the same model preferences and values for the kinetic parameters. The time-course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncompetitive two-step mechanism, in which an inactive, rapidly formed, reversible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 comparable to that of collagenase, it is apparent that free TIMP-1 also functions in the breakdown of the tight complex. The rapidly formed complex has a dissociation constant of 8 nM and reacts to the tight complex with a first-order rate constant of 0.003 s-1. The back reaction of the tight complex to the rapid complex has a second-order rate constant of 5 x 10(4) M-1 s-1. The resulting global dissociation constant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase concentration. Collagenase without the carboxyl-terminal domain (mini-collagenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 10(4) mol-1 s-1 and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s-1. The apparent global dissociation constant for the tight complex (2.7 nM) is higher than that for the fibroblast-type collagenase. Participation of TIMP-1 in the dissociation is not demonstrable. Therefore, the carboxyl-terminal domain of fibroblast-type collagenase is important for the initial, rapid binding of TIMP-1 and the initial complex contributes to the overall binding.

Collagenases↗
PubMed1996

APMA (4-aminophenylmercuric acetate) activation of stromelysin-1 involves protein interactions in addition to those with cysteine-75 in the propeptide.

Matrix metalloproteinases (MMPs) can be activated in vitro by multiple mechanisms such as treatment with proteases, organomercurials, oxidants, and detergents. The proposed cysteine switch model for activation suggests that these multiple methods for activation cause the dissociation of the single cysteine residue in the propeptide from the active site zinc. In particular, it has been suggested that organomercurials such as 4-aminophenylmercuric acetate (APMA) work by directly reacting with the sulfhydryl group of this cysteine residue, resulting in its displacement from the active site. However, recent data by Chen et al. [(1993) Biochemistry 32, 10289-10295] demonstrated that modification of this cysteine residue in the propeptide of stromelysin-1 by sulfhydryl reagents did not result in an active enzyme as predicted. To investigate the roles that this cysteine residue and the propeptide salt bridge (R74 to D79) might play in the APMA-induced activation of stromelysin-1, we have changed these residues by site-directed mutagenesis. Wild-type stromelysin-1 and the mutants were all expressed at detectable levels using a recombinant vaccinia virus system and determined to be catalytically competent by zymography. The wild-type stromelysin-1 and the cysteine mutants (C75S and C75H) underwent APMA-induced activation as determined by the characteristic reduction in molecular weight associated with activation and by their ability to cleave casein only when activated. On the other hand, mutants R74K, D79A, and C75H/D79A did not undergo APMA-induced activation. These results demonstrate that APMA-induced activation of stromelysin-1 involves protein interactions in addition to those with cysteine-75 in the propeptide and also suggest that the R74 to D79 salt bridge may play a role.

Amino Acid Sequence↗
PubMed1996

Activation of human progelatinase A by collagenase and matrilysin: activation of procollagenase by matrilysin.

Proteolytic and nonproteolytic methods were used to investigate the mechanism(s) by which human fibroblast progelatinase A and fibroblast-type procollagenase can be activated. Both collagenase and matrilysin were able to activate progelatinase A, resulting in an amino terminus in gelatinase A of Tyr81. The cleavage occurred distal to Cys73 within the sequence of PRCGNPDVAN80-Y81NFFPRKP. While several nonproteolytic reagents were tested, only the heavy metal Hg(II) and p-chloromercuribenzoate (PCMB) were able to induce activation of progelatinase A and resulted in the conversion of the latent 72-kDa gelatinase A to an active form of about 64.5 kDa. Matrilysin was also able to activate procollagenase and resulted in an amino terminus in collagenase of Phe81. These results suggest that fibroblast-type collagenase and matrilysin may be physiologically relevant activators of progelatinase A; the maintenance of latency and the process of activation for progelatinase A may occur through the cysteine-switch mechanism, and the proteolytic activation of procollagenase by matrilysin resulted in the same amino terminus as produced by stromelysin-1.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of residues in and around the active site of human fibroblast-type collagenase.

Mutants in and around the catalytic zinc-binding site of human fibroblast-type collagenase have been expressed in Escherichia coli. Replacement of each of the three zinc ligands, His-199, His-203, and His-209, in the active site sequence: VAAHEXGHXXGXXH, not only destroyed catalytic activity but also led to improper folding of the polypeptide, suggesting that this sequence also serves as a structural zinc-binding site. By comparison, mutation of His-194 immediately preceding this sequence had no measurable effect on catalytic activity or on folding. Replacement of Glu-200 in the active site yielded enzymes that either were completely inactive (E200Q) or had greatly diminished (E200D) catalytic activity. Both Glu-200 mutants, however, were fully capable of forming complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1) after reaction with organomercurials. Formation of complexes with TIMP-1 appear to require a properly folded, but not necessarily catalytically competent, active site. By contrast, complexes with alpha 2-macroglobulin form only with mutants with a catalytically competent active site. Two mutants identified in this study (E200Q and D212E) appeared to be properly folded but unable to generate any catalytic activity when exposed to either p-aminophenylmercuric acetate, trypsin, or SDS.

Amino Acid Sequence↗
PubMed1994

Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases).

To define domains of tissue inhibitor of metalloproteinases (TIMP-1) that are important to its ability to inhibit fibroblast-type collagenase (FIB-CL), two different approaches were used: (i) competition with synthetic peptides modeled after the human TIMP-1 sequence and (ii) localization of epitopes of blocking antibodies. TIMP-1 consists of six loops, held in place by six disulfide bonds arranged in three knotlike structures. Several long peptides (n = 20-34), together covering three-fourths of the human TIMP-1 sequence, were able to block inhibition of human FIB-CL by TIMP-1. While most of these peptides were modeled after sequences in the NH2-terminal domain of the molecule (loops 1, 2, and 3), they also included two-thirds of the residues of the COOH-terminal domain including loops 4 and 5 and the COOH-terminal tail but not loop 6. Refinement by competition with shorter peptides (7-10 residues) showed that the region surrounding the second "disulfide knot" (Cys13-Cys124, Cys127-Cys174) plays a major role in the inhibition of FIB-CL. This region consists of two strands, residues 10-25 and 121-129, connected through Cys13-Cys124. Peptides from this region also directly inhibited FIB-CL in the absence of TIMP-1. Additional competing peptides included T2-11 of the NH2-terminal domain and T34-42, a highly conserved region in the middle of loop 1. Among a series of monoclonal and polyclonal antibodies (mAbs and pAbs) to TIMP-1, we identified two, one mAb and one pAb, that neutralized the activity of TIMP-1 against FIB-CL. Both recognized epitopes in loop 3. The epitope for the mAb was located in the sequence that marks the transition between loops 3 and 4, GCEEC127, a region also identified as important by peptide competition experiments. By contrast, the epitope for a nonblocking mAb was located in a short 9-residue segment of loop 4, and a nonblocking pAb recognized epitopes in loop 1, loop 6, and the COOH-terminal tail. Our findings suggest that the FIB-CL-TIMP-1 complex possesses multiple contact sites that involve several different subdomains of the inhibitor.

Amino Acid Sequence↗
PubMed1993

Cell type-specific regulation of SL-1 and SL-2 genes. Induction of the SL-2 gene but not the SL-1 gene by human keratinocytes in response to cytokines and phorbolesters.

The stromelysin-2 (SL-2) gene is transcriptionally active in normal human keratinocytes and encodes a secreted, catalytically competent but latent matrix metalloproteinase. Phorbolester induction resulted in the emergence of SL-2 (but not SL-1 transcripts), whereas the opposite was true for human mucosal fibroblasts. Expression of keratinocyte SL-2 was also induced by the two keratinocyte growth factors, transforming growth factor-alpha and epidermal growth factor, by the proinflammatory cytokine, tumor necrosis factor-alpha, but, somewhat surprisingly, not by interleukin-1 beta. The latent SL-2 proenzyme was isolated from 12-O-tetradecanoylphorbol-13-acetate-induced keratinocytes by immunoaffinity chromatography using a cross-reactive antibody raised against human SL-1. This procedure led to the recovery of a single M(r) 54,000 molecular species at a level of approximately 0.2 microgram/ml of culture medium. Amino-terminal sequencing identified the protein as SL-2 and verified the predicted signal sequence cleavage site. Conformational activation of latent SL-2 precursor by SDS gave rise to a full-length, uncleaved (M(r) 54,000) active form and at the same time exposed a cryptic thiol group. By contrast, organomercurial activation resulted in autolytic truncation of the molecule with loss of M(r) approximately 10,000 propeptide. SL-2 shared with (human fibroblast) SL-1 the ability to cleave casein, to "superactivate" fibroblast type procollagenase, and to form apparently binary, SDS-resistant complexes with tissue inhibitor of metalloproteinases-1.

Amino Acid Sequence↗
PubMed1993

Matrix metalloproteinases: a review.

Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn(++)-endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in specific developmental or pathological conditions, including human periodontal diseases.

Amino Acid Sequence↗
PubMed1992

The latency of the human fibroblast collagenase precursor depends on an internal cysteine residue.

The proenzyme form of human fibroblast collagenase has been expressed in E. coli from its cDNA clone and has been shown to be functionally identical to the human enzyme. Mutants at one of three cysteine residues were constructed by site-directed mutagenesis of the cDNA and their relative activities compared to the wild type enzyme. A cysteine contained in the propeptide domain of procollagenase and other matrix metalloproteinases was shown to be essential for maintaining the proenzyme in an inactive state. A model to explain the importance of this highly conserved cysteine to the maintenance of latency is discussed.

Amino Acid Sequence↗
PubMed1992

Degradation of collagen fibrils by live cells: role of expression and activation of procollagenase.

We have examined the conditions for dissolution by live cells of an extracellular matrix composed of reconstituted type I collagen fibrils, using three different cell types which express varying constitutive or inducible levels of procollagenase and collagenase inhibitor. The two major conclusions from these studies were that (i) expression of collagenase is a necessary but not sufficient requirement for dissolution of the collagen fibrils and that (ii) activation of procollagenase is a rate-limiting step. Cells which secreted high levels of procollagenase dissolved collagen fibrils only to the extent that they were able to activate the enzyme. Cells which also expressed inhibitor failed to activate procollagenase in the culture medium and did not dissolve the collagen fibrils unless procollagenase-activation was assisted by exogenous proteinase activity. Cells that did not express inhibitor ultimately did activate procollagenase but the process was slow and incomplete. Introduction of exogenous proteinase activity either in the form of plasminogen, plasmin, or trypsin stimulated collagen breakdown by several fold. Analysis of the culture medium sampled from such cultures showed that the stimulating effect of exogenous proteinases could be ascribed to three separate, but synergistic events: elevated expression of procollagenase, conversion of procollagenase to active form and inactivation of collagenase inhibitor. Two lines of evidence suggested that the dissolution of collagen fibrils in these cultures was mediated by a collagenase-dependent pathway: (i) the rate of dissolution closely mirrored the level of expression of collagenase and (ii) the process was blocked by inhibitory collagenase-specific antibodies.

Adult↗
PubMed1991

An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase.

The cDNA that encodes the proenzyme form of human fibroblast collagenase has been expressed in Escherichia coli. It has been shown by a number of criteria to be functionally identical with the enzyme isolated from human sources. Mutations of each of three cysteine residues found in procollagenase were constructed by site-directed mutagenesis of the cDNA. The relative activities of these mutants were compared to the wild-type enzyme. All of the mutants retained proteolytic activity, but not necessarily on collagen. Mutations that interfere with the formation of the sulfhydryl bridge in the carboxy-terminal domain in some cases increased and in other cases decreased the rate of casein cleavage. On the basis of extensive autolysis within E. coli of a mutant with a replacement of cysteine-73, the procollagenase molecule produced appeared to be either spontaneously active or perhaps more susceptible to autolytic activation, despite the continued presence of the propeptide. Experiments designed to capture the active forms of the mutant by use of the irreversible inhibitor alpha 2-macroglobulin showed that some degree of latency still persisted in the autolytic mutant. These findings suggest that the cysteine at position 73 is important for maintaining the proenzyme in an inactive state but that the maintenance of latency in MMPs may be a complex process, involving a number of interactions between the propeptide domain and the remainder of the collagenase molecule.

Base Sequence↗
PubMed1990

Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site.

The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the alpha 1(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Comparison of pulp responses following restoration of exposed and non-exposed cavities.

OBJECTIVES: The purpose of this study is to compare and contrast differences of pulp responses between non-exposed and exposed cavity preparations in terms of inflammation, frequency of bacterial microleakage, odontoblast and odontoblastoid cell numbers, and tertiary dentine formation. METHODS: Class V non-exposed cavities (n=161) and exposed cavities (n=161 teeth) were prepared in non-human primate teeth. Cavities were restored with calcium hydroxide [Ca(OH)(2)], resin modified glass ionomer, or resin composite. Following extraction (7-730 days), bacteria were detected with McKays stain and pulp reactions were categorized according to ISO guidelines. Teeth were analyzed histomorphometrically and statistically using analysis of variance tests. RESULTS: Exposed cavities in comparison with non-exposed cavities were found to have more severe inflammation (p=0.0001), greater quantities of tertiary dentine (p=0.0001), and an increased frequency of bacterial microleakage (p=0.0034). The density of odontoblastoid cells beneath pulp exposed tertiary dentine was found to be 47.8% of odontoblast cell density beneath non-exposed dentine (p=0.0001). CONCLUSIONS: The restoration of exposed cavity preparations is associated with more traumatic pulp injury and repair responses. Consequently, efforts should be made to minimize iatrogenic dentine removal during cavity preparation and the creation of pulp exposures whenever possible.

Analysis of Variance↗
PubMedDate not supplied

Hierarchy of variables correlated to odontoblast-like cell numbers following pulp capping.

OBJECTIVES: Following tooth pulp exposure, pulpal repair is accomplished by dentine bridge secretion by odontoblast-like cells. However, little information is available about the hierarchy of variables, which influence odontoblast-like cell numbers. The purpose of this study was to examine correlations between pulp capping events and odontoblast-like cell numbers. METHODS: Two hundred and fifty standardised pulp exposed cavities were prepared in non-human primate teeth according to ISO usage guidelines. Exposed pulps were capped with Calcium hydroxide [Ca(OH)(2)], and multi-step and self-etching primer composite resins. Teeth were collected from 3 to 60-days to observe pulp reactions. Statistical analysis was evaluated using analysis of variance. RESULTS: The hierarchy of variables correlated to odontoblast-like cells were the dentine bridge area (P = 0.0001), time since pulp exposure (P = 0.0001), odontoblast numbers opposite the exposure site (P = 0.0002), and pulp capping materials (P = 0.0313). Other pulp capping variables were found to be less likely to be correlated with odontoblast-like cell numbers. CONCLUSIONS: The area of dentine bridge formation is directly related to the numbers of odontoblast-like cells, cell activity is time dependent, and the cell numbers are much lower than original odontoblast cells. The time-lag between the appearance of odontoblast-like cells at the site of pulp exposure, and the limited numbers of these cells, explain why pulpal repair is difficult to achieve successfully following pulp exposure.

Animals↗