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Biomedical subjects

C M Overall

Publications and source records attributed to C M Overall.

At least 19 recordsLinked to original sources
PubMed2006

Towards third generation matrix metalloproteinase inhibitors for cancer therapy.

The failure of matrix metalloproteinase (MMP) inhibitor drug clinical trials in cancer was partly due to the inadvertent inhibition of MMP antitargets that counterbalanced the benefits of MMP target inhibition. We explore how MMP inhibitor drugs might be developed to achieve potent selectivity for validated MMP targets yet therapeutically spare MMP antitargets that are critical in host protection.

Amino Acid Sequence↗
PubMed2001

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway.

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.

Animals↗
PubMed2001

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1.

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.

Amino Acid Sequence↗
PubMed2001

Tissue inhibitor of metalloproteinases-4 inhibits but does not support the activation of gelatinase A via efficient inhibition of membrane type 1-matrix metalloproteinase.

The tissue inhibitors of metalloproteinases 1-4 (TIMPs) have discrete regulatory roles in the activation of matrix metalloproteinase (MMP)-2 (gelatinase A), an important basement membrane-degrading MMP pivotal to tumor metastasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of progelatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a free MT1-MMP molecule for activation. To investigate the role of TIMP-4 in the activation process, we developed a new procedure for the expression and purification of recombinant human TIMP-4 from baby hamster kidney cells. The recombinant TIMP-4 was a potent inhibitor of gelatinase A (apparent K(i) [Ki(app.)] < or = 9 pM; k(on) (association rate constant), 4.57 +/- 0.13 x 10(6) M(-1)s(-1)) and was less dependent upon hemopexin C domain interactions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP (Ki(app.) < or = 100 pM; k(on), 3.49 +/- 0.34 x 10(6) M(-1)s(-1)) and blocked the concanavalin A-induced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 -/- fibroblasts or unstimulated MT1-MMP-transfected Timp2 -/- cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3-5 nM TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2 dependency of MT1-MMP-induced activation of gelatinase A and the fact that TIMP-4 cannot support activation. The dominance of TIMP-2 in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A in inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from the hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by efficient inhibition of MT1-MMP-mediated activation and by inhibiting the activated enzyme and, thus, is a tumor resistance factor in the peritumor stroma.

Amino Acid Sequence↗
PubMed2001

Functional interplay between type I collagen and cell surface matrix metalloproteinase activity.

Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves beta(1) integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both alpha(2)beta(1) and alpha(3)beta(1) integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of alpha(3)beta(1) strongly promotes activation of pro-MMP-2, whereas alpha(2)beta(1) integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.

Cell Adhesion↗
PubMed2001

Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils.

Leukolysin, originally isolated from human leukocytes, is the sixth member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily with a potential glycosylphosphatidylinositol (GPI) anchor. To understand its biological functions, we screened subpopulations of leukocytes and localized the expression of leukolysin at the mRNA level to neutrophils. Polyclonal and mono-specific antisera raised against a synthetic peptide from its hinge region recognized a major protein species at 56 kDa and several minor forms between 38 and 45 kDa in neutrophil lysates. In resting neutrophils, leukolysin is distributed among specific granules ( approximately 10%), gelatinase granules ( approximately 40%), secretory vesicles ( approximately 30%), and the plasma membrane ( approximately 20%), a pattern distinct from that of neutrophil MMP-8 and MMP-9. Consistent with its membrane localization and its reported GPI anchor, leukolysin partitions into the detergent phase of Triton X-114 and can be released from intact resting neutrophils by glycosylphosphatidylinositol-specific phospholipase C. Phorbol myristate acetate stimulates neutrophils to discharge 100% of leukolysin from specific and gelatinase granules and approximately 50% from the secretory vesicles and plasma membrane, suggesting that leukolysin can be mobilized by physiological signals to the extracellular milieu as a soluble enzyme. Indeed, interleukin 8, a neutrophil chemoattractant, triggered a release of approximately 85% of cellular leukolysins by a process resistant to a mixture of proteinase inhibitors, including aprotinin, BB-94, pepstatin, and E64. Finally, purified recombinant leukolysin can degrade components of the extracellular matrix. These results not only establish leukolysin as the first neutrophil-specific MT-MMP but also implicate it as a cytokine/chemokine-regulated effector during innate immune responses or tissue injury.

Amino Acid Sequence↗
PubMed2000

Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.

Animals↗
PubMed2000

Domain interactions in the gelatinase A.TIMP-2.MT1-MMP activation complex. The ectodomain of the 44-kDa form of membrane type-1 matrix metalloproteinase does not modulate gelatinase A activation.

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

Animals↗
PubMed2000

Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3.

Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.

Animals↗
PubMed2000

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.

Acarbose↗
PubMed2000

The matrix metalloproteinase gelatinase A in human dentine.

A dentine protein extraction protocol was modified in order to identify matrix metalloproteinase gelatinolytic activities in the non-mineralized and mineralized phases of human dentine. Dentine proteins from 24 individual permanent molars from patients aged 15-73 years were sequentially extracted, first with guanidinium chloride (G1 extract), then EDTA (E extract), and after this demineralization step, again by guanidinium chloride (G2 extract) to dissociate collagen-associated proteins. Extracts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the gels were processed by Western blotting and zymography to detect gelatinolytic activities. Active and latent forms of gelatinase A were identified in the non-mineralized dentine fraction (G1 extract) of 58% of the teeth. Other gelatinolytic species were also detected by zymography with apparent M(r) of 92, 54 and 30 kDa. Although gelatinase A was detected in the G1 extracts of teeth from all ages, indicating more recent synthesis and remodelling of the predentine, gelatinase A was never detected in any E extract or in the G2 extracts of patients older than 41 years. The presence of the active form of gelatinase A in mineralized human dentine implicates this enzyme in dentine mineralization.

Adolescent↗
PubMed2000

Use of a fluorogenic septapeptide matrix metalloproteinase assay to assess responses to periodontal treatment.

BACKGROUND: Quantification of gingival crevicular fluid matrix metalloproteinase activity may provide improved assessment of periodontal disease status and response to treatment. A fluorogenic matrix metalloproteinase substrate assay (FSA) has been developed using a methoxycoumarin-containing septapeptide analog of the alpha2(I) collagen cleavage site. This substrate exhibits increased fluorescence following cleavage by many matrix metalloproteinases, and the enzyme activity can be readily estimated with a fluorimeter. Here we compared this assay with classical methods of periodontal assessment including bleeding on probing, crevicular fluid flow, and probing depth to assess its utility as an indicator of changes in periodontal status and treatment response. METHODS: Complete measurements of probing depth were obtained for Ramfjord teeth on subjects who had been previously treated for periodontitis. Subjects were subsequently divided into groups based on existing periodontal disease severity: gingivitis (n = 21), stable periodontitis (n = 41), and severe periodontitis (n = 50). Crevicular fluid volume, bleeding on probing, and FSA were measured at each Ramfjord tooth or substitute. After baseline measurements, subjects received subgingival scaling and prophylaxis; 3 months later, they were reassessed. RESULTS: FSA measurements were positively associated with severity of disease at baseline. After treatment there were substantial reductions of FSA in gingivitis (approximately 51%; P <0.01) and severe periodontitis (approximately 45%; P <0.001), but not in stable periodontitis (13%; P >0.2). All groups showed a positive association between FSA measurements and higher bleeding scores at individual sites. FSA measurements were also positively associated with crevicular fluid flow at baseline, but after treatment there was a approximately 67% decrease (P <0.01) in the highest crevicular fluid flow class. There were significant reductions of FSA at follow-up for sites with probing depths between 0 to 3 mm (23%; P <0.05) and 4 to 6 mm (31%; P <0.05). However, the largest reduction was for sites with probing depth between 7 to 9 mm (49%; P <0.001). CONCLUSIONS: These results indicate that monitoring patients by measurement of matrix metalloproteinase levels in gingival crevicular fluid with the quenched fluorescent substrate assay provides estimates of inflammatory status, periodontal destruction, and response to treatment, especially in more severe periodontitis lesions.

Adult↗
PubMed2000

Gelatinase A in human dentin as a new biochemical marker for age estimation.

Dentin proteins from 24-individual permanent molars from patients aged 15 to 73 years were sequentially extracted, with guanidinium chloride (G1-extract) and then with EDTA; after demineralization, the material was again extracted with guanidinium chloride (G2-extract). Extracts were analyzed by SDS-PAGE electrophoresis and the gels were processed for zymography to detect gelatinolytic activities. The patterns of gelatinase A distribution differed in the different dentin protein fractions, and the changes varied with age. Significant differences were detected in gelatinase A in G2-extracts between individual younger than 20 years old and the rest of the sample (chi2exp = 19.429; 1 d.f.; p < or = 0.001). The incidence of true and false positives and negatives, and sensitivity and specificity for the presence of gelatinase A in dentin extracts, were calculated. Determination of gelatinase A in human dentin may be a useful marker to estimate age, especially when other morphological methods are of limited usefulness.

Adolescent↗
PubMed1999

Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin carboxyl domain of human gelatinase A by site-directed mutagenesis. The hierarchical role in binding TIMP-2 of the unique cationic clusters of hemopexin modules III and IV.

Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617 reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain.TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (Kd 3.0 x 10(-8) M), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2. C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site.

Amino Acid Sequence↗
PubMed1999

Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.

Amino Acid Sequence↗
PubMed1999

Activation of neutrophil collagenase in periodontitis.

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.

Case-Control Studies↗