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Harald Tschesche - One of the best experts on this subject based on the ideXlab platform.
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126 Neutrophil Collagenase
Handbook of Proteolytic Enzymes (Second Edition)#R##N#Aspartic and Metallo Peptidases, 2004Co-Authors: Harald TschescheAbstract:Publisher Summary This chapter examines structural chemistry and biological properties of Neutrophil Collagenase. Neutrophil Collagenase is stored intracellularly as a latent proenzyme in the specific granules of polymorphonuclear leukocytes. Activation of the proCollagenase occurs in the extracellular space after secretion. The active enzyme is capable of cleaving types I, II and III triple-helical collagen into the characteristic one-quarter and three-quarter fragments. Human Neutrophil preproCollagenase consists of a signal peptide, a propeptide containing a sequence motif responsible for maintaining the enzyme in the latent form, a catalytic domain and a hemopexin-like domain responsible for binding native collagen. The gene for human Neutrophil Collagenase is located on the long arm of chromosome 11 and mapped to Ilq21-q22 by in situ hybridization. The cDNA sequences show that Neutrophil Collagenase and interstitial Collagenase are coded by two different genes. The three highly homologous human Collagenases which include Neutrophil Collagenase, fibroblast Collagenase and Collagenase 3 share more than 50% sequence identity. It is found that all three enzymes cleave type I, II and III triple-helical collagen into the characteristic one-quarter and three-quarter fragments, but with different substrate specificity.
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modulation of the catalytic activity of Neutrophil Collagenase mmp 8 on bovine collagen i role of the activation cleavage and of the hemopexin like domain
Journal of Biological Chemistry, 2002Co-Authors: Magda Gioia, Giovanni Francesco Fasciglione, Oliver Diekmann, Vincenzo Politi, Harald Tschesche, Stefano Marini, Michael Pieper, Giampiero De Sanctis, Silvana Dalessio, Massimo ColettaAbstract:The cleavage of bovine collagen I by Neutrophil Collagenase MMP-8 has been followed at pH 7.4, 37 °C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990)Biochemistry 29, 10628–10634; Knauper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Blaser, J., and Tschesche, H. (1993) Biochem. J. 291, 847–854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657–18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met80 of the proenzyme (MetMMP-8) and the former Phe79 of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much fasterk cat/K m , fully attributable to a lower K m , suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227–233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
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Modulation of the catalytic activity of Neutrophil Collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain.
Journal of Biological Chemistry, 2002Co-Authors: Magda Gioia, Giovanni Francesco Fasciglione, Silvana D'alessio, Oliver Diekmann, Vincenzo Politi, Michael P Pieper, Harald Tschesche, Stefano Marini, Massimo ColettaAbstract:The cleavage of bovine collagen I by Neutrophil Collagenase MMP-8 has been followed at pH 7.4, 37 degreesC. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Knauper, V., Osthues, A., DeClerk, Y. A., Langley, K. A, Blaser, J., and Tschesche, H. (1993) Biochem. J. 291, 847854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80)of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K-m, fully attributable to a lower K-m, suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
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computational study of the catalytic domain of human Neutrophil Collagenase specific role of the s3 and s 3 subsites in the interaction with a phosphonate inhibitor
Journal of Computer-aided Molecular Design, 2002Co-Authors: Massimiliano Aschi, Harald Tschesche, Michael Pieper, Enrico Gavuzzo, Giorgio Pochetti, Carlo Gallina, Danilo Roccatano, Alfredo Di Nola, Fernando MazzaAbstract:Human Neutrophil Collagenase (HNC, MMP-8) is one of the target enzymes for drug treatment of pathologic extracellular matrix degradation. Peptidomimetic inhibitors bind in the S'-side of the enzyme active site occupying the S-1(') primary specificity pocket by their large hydrophobic side-chains. The crystal structure of the complex between the catalytic domain of MMP-8 and Pro-Leu-l-Trp(P)(OH)(2) (PLTP) showed that this phosphonate inhibitor binds in the S side of the active site. This finding was unexpected since it represents the first example of accommodation of the bulky Trp indolyl chain in the S-1 rather than in the S-1(') subsite. Dynamical and structural factors favouring this uncommon mode of binding were therefore investigated. MD simulations performed on the uncomplexed enzyme show that its structure in aqueous solution is only slightly different from the crystal structure found in the complex with PLTP. ED analysis of the MD simulations, performed on PLTP alternatively interacting with the S- or S'-side of the active site, shows that the enzyme fluctuation increases in both cases. The main contribution to the overall enzyme fluctuation is given by the loop 164-173. The fluctuation of this loop is spread over more degrees of freedom when PLTP interacts with the S-side. This dynamical factor can enhance the preference of PLTP for the S subsites of MMP-8. MD simulations also show that ligation of PLTP in the S subsites is further favoured by better zinc chelation, a cation-pi interaction at the S-3 subsite and unstrained binding conformations. The role of the S-3, S-3(') and S-1(') subsites in determining the inhibitor binding is discussed.
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Two Crystal Structures of Human Neutrophil Collagenase, One Complexed with a Primed- and the Other with an Unprimed-Side Inhibitor: Implications for Drug Design†
Journal of medicinal chemistry, 2000Co-Authors: Enrico Gavuzzo, Silvana D'alessio, Michael P Pieper, Harald Tschesche, Giorgio Pochetti, Fernando Mazza, Carlo Gallina, Barbara Gorini, Paul A. TuckerAbstract:Two crystal structures of human Neutrophil Collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-l-TrpP(OH)2 occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S1 subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3,4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained d-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S1‘ subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation−aromatic interactions can stabilize the f...
Ghislain Opdenakker - One of the best experts on this subject based on the ideXlab platform.
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Research Article Inhibition of Neutrophil Collagenase/MMP-8 and Gelatinase B/MMP-9 and Protection against Endotoxin Shock
2016Co-Authors: Ghislain Opdenakker, Min WangAbstract:Copyright © 2014 Zheng Qiu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Endotoxin shock is a life-threatening disorder, associated with the rapid release of Neutrophil enzymes, including Neutrophil Collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9). After activation, these enzymes cleave extracellular matrix components and cytokines and thus may contribute to shock syndrome development. MMP inhibitors have been suggested as immunotherapy of endotoxin shock. However, little is known about the therapeutic time window of MMP inhibition. Here, a sublethal endotoxin shock mouse model was used to evaluate the effect of an MMP inhibiting peptide (P2) after intravenous or intraperitoneal injection and to study the time window between LPS and inhibitor injections. With the use of a specific ELISA the plasma P2 concentrations were monitored. Whereas we corroborated the treatment strategy of MMP targeting in endotoxin shock with a new inhibitor, we also demonstrated that the time window, within which effective MMP inhibition increased the survival rates, is rather limited. 1
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inhibition of Neutrophil Collagenase mmp 8 and gelatinase b mmp 9 and protection against endotoxin shock
Clinical & Developmental Immunology, 2014Co-Authors: Zheng Qiu, Ghislain Opdenakker, Jianghai Chen, Philippe E Van Den Steen, Min WangAbstract:Endotoxin shock is a life-threatening disorder, associated with the rapid release of Neutrophil enzymes, including Neutrophil Collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9). After activation, these enzymes cleave extracellular matrix components and cytokines and thus may contribute to shock syndrome development. MMP inhibitors have been suggested as immunotherapy of endotoxin shock. However, little is known about the therapeutic time window of MMP inhibition. Here, a sublethal endotoxin shock mouse model was used to evaluate the effect of an MMP inhibiting peptide (P2) after intravenous or intraperitoneal injection and to study the time window between LPS and inhibitor injections. With the use of a specific ELISA the plasma P2 concentrations were monitored. Whereas we corroborated the treatment strategy of MMP targeting in endotoxin shock with a new inhibitor, we also demonstrated that the time window, within which effective MMP inhibition increased the survival rates, is rather limited.
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Inhibition of Neutrophil Collagenase/MMP-8 and Gelatinase B/MMP-9 and Protection against Endotoxin Shock
Hindawi Limited, 2014Co-Authors: Zheng Qiu, Ghislain Opdenakker, Jianghai Chen, Philippe E Van Den Steen, Min WangAbstract:Endotoxin shock is a life-threatening disorder, associated with the rapid release of Neutrophil enzymes, including Neutrophil Collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9). After activation, these enzymes cleave extracellular matrix components and cytokines and thus may contribute to shock syndrome development. MMP inhibitors have been suggested as immunotherapy of endotoxin shock. However, little is known about the therapeutic time window of MMP inhibition. Here, a sublethal endotoxin shock mouse model was used to evaluate the effect of an MMP inhibiting peptide (P2) after intravenous or intraperitoneal injection and to study the time window between LPS and inhibitor injections. With the use of a specific ELISA the plasma P2 concentrations were monitored. Whereas we corroborated the treatment strategy of MMP targeting in endotoxin shock with a new inhibitor, we also demonstrated that the time window, within which effective MMP inhibition increased the survival rates, is rather limited
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definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase b matrix metalloproteinase 9 mmp 9 and tnf α converting enzyme tace adam 17
Journal of Enzyme Inhibition and Medicinal Chemistry, 2012Co-Authors: Zheng Qiu, Philippe E Van Den Steen, Min Wang, Ming Yan, Datao Liu, Ghislain OpdenakkerAbstract:Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn2+-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on Neutrophil Collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.
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targeting Neutrophil Collagenase matrix metalloproteinase 8 and gelatinase b matrix metalloproteinase 9 with a peptidomimetic inhibitor protects against endotoxin shock
Biochemical Pharmacology, 2005Co-Authors: Philippe E Van Den Steen, Chris Dillen, Ghislain OpdenakkerAbstract:Abstract Gram-negative sepsis, bacterial meningitis and endotoxin shock are life-threatening disorders, associated with the rapid release of Neutrophil enzymes. Neutrophil Collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9) are contained in granules, are quickly exocytosed upon granulocyte activation and efficiently cleave intact and denatured collagens, respectively. Genetic ablation of gelatinase B protects against endotoxin-induced mortality. Therefore, we designed and synthesized a peptidomimetic gelatinase B inhibitor Regasepin1, and compared the selectivity for the Collagenases MMP-1, MMP-8 and MMP-13. Regasepin1 was found to inhibit, almost to the same degree, the Neutrophil enzymes MMP-8 and MMP-9 and the monocytic tumor necrosis factor-α (TNF-α) converting enzyme (TACE/ADAM-17) in vitro. With the use of mass spectrometry analysis, the plasma half-life of inhibitor levels was determined after an intraperitoneal bolus injection in mice. Plasma peak levels of the inhibitor were reached at 50 min after intraperitoneal injection and the subsequent half-life in the circulation exceeded 40 min. Regasepin1 protected mice against lethal endotoxinemia by intraperitoneal and intravenous injection routes. This proofs the principle that early Neutrophil MMP inhibition followed by TACE blockade may become a treatment strategy of gram-negative sepsis, endotoxinemia and other life-threatening inflammatory reactions.
Vera Knäuper - One of the best experts on this subject based on the ideXlab platform.
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analysis of the contribution of the hinge region of human Neutrophil Collagenase hnc mmp 8 to stability and collagenolytic activity by alanine scanning mutagenesis
FEBS Letters, 1997Co-Authors: Vera Knäuper, Harald Tschesche, Andrew J P Docherty, Bryan D Smith, Gillian MurphyAbstract:Analysis of the hinge region of Neutrophil Collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1′ position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239–Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247→Ala (P1 of the minor cleavage site Pro247–Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of Neutrophil Collagenase is important for collagenolytic activity. The alteration of the Gly242–Leu–Ser–Ser–Asn–Pro–Ile–Gln–Pro247 sequence motif to Gly242–Ala–Ala–Ala–Ala–Pro–Ala–Ala–Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247–Thr–Gly–Pro–Ser–Thr–Pro–Lys–Pro258 to Pro247–Ala–Ala–Pro–Ala–Ala–Pro–Ala–Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme. © 1997 Federation of European Biochemical Societies.
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research communicationa one step sandwich enzyme immunoassay for human matrix metalloproteinase 8 Neutrophil Collagenase using monoclonal antibodies
Clinica Chimica Acta, 1996Co-Authors: Hirokazu Matsuki, Vera Knäuper, Noboru Fujimoto, Kazushi Iwata, Yasunori Okada, Taro HayakawaAbstract:A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 8 (MMP-8, Neutrophil Collagenase, EC 3.4.24.7) has been established with a pair of monoclonal antibodies prepared against the zymogen of MMP-8 purified from human Neutrophils. MMP-8 in samples simultaneously reacted with both solid-phase and peroxidaselabeled antibodies. Sensitivity of this EIA system was 0.34 μg/l (5.7 pg/assay) and linearity was obtained between 0.5 and 500 μg/l (8.3–8300 pg/assay). The EIA system recognized both precursor and active forms of MMP-8 but not MMP-8 complexed with tissue inhibitors of metalloproteinases. There was no difference in the MMP-8 levels between the plasma samples from patients with rheumatoid arthritis or osteoarthritis and those from healthy subjects (median 6.2 μg/l, range 1.5–28 μg/l). However, the level in synovial fluids from patients with rheumatoid arthritis (median 345 μg/l, range 84–2860 μg/l) was shown to be higher than that from osteoarthritic patients. MMP-8 levels in human whole saliva from patients with periodontal diseases (median 282 μg/l, range 0–1420 μg/l) were also significantly higher than those from clinically healthy subjects (median 25 μg/l, range 0–100 μg/l). Immunoreactivity analyses showed that MMP-8 species in normal human plasma exists as a precursor but not as a complex form with tissue inhibitor of metalloproteinases (TIMP)-1 or TIMP-2.
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activation of human Neutrophil proCollagenase by stromelysin 2
FEBS Journal, 1996Co-Authors: Vera Knäuper, Gillian Murphy, Harald TschescheAbstract:Neutrophil proCollagenase (MMP-8) was efficiently activated by incubation with active stromelysin 2 (MMP-10). A single-step activation mechanism involving the cleavage of the Gly78-Phe79 peptide bond at the end of the propeptide domain was observed. Determination of the collagenolytic activity revealed the generation of active Neutrophil Collagenase displaying high specific activity. When compared with the specific activity following mercurial activation, which generates active Collagenase by autoproteolytic cleavage of either Phe79-Met80 or Met80-Leu81 peptide bonds [Blaser, J., Knauper, V., Osthues, A., Reinke, H. & Tschesche, H. (1991) Eur J. Biochem. 202, 1223–1230], the specific activity of the stromelysin-2-activated enzyme was considerably higher. Thus, human Neutrophil proCollagenase was ‘superactivated’ by stromelysin 2, as was recently shown for the stromelysin-1-activated enzyme [Knauper, V., Wilhelm, S. M., Seperack, P. K., De Clerck, Y. A., Langley, K. E., Osthues, A. & Tschesche, H. (1993a) Biochem. J. 295, 581–586].
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Neutrophil Collagenase mmp 8 cleaves at the aggrecanase site e373 a374 in the interglobular domain of cartilage aggrecan
Biochemical Journal, 1994Co-Authors: Amanda J Fosang, Harald Tschesche, Clare Elizabeth Hughes, Peter J Neame, Gillian Murphy, Vera Knäuper, Bruce Caterson, Timothy E. HardinghamAbstract:Native and recombinant Neutrophil Collagenase (MMP-8) was shown to cleave at the E373-A374 'aggrecanase' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.
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Neutrophil Collagenase mmp 8 cleaves at the aggrecanase site e373 a374 in the interglobular domain of cartilage aggrecan
Biochemical Journal, 1994Co-Authors: Amanda J Fosang, Harald Tschesche, Clare Elizabeth Hughes, Peter J Neame, Gillian Murphy, Vera Knäuper, Bruce Caterson, Timothy E. HardinghamAbstract:Native and recombinant Neutrophil Collagenase (MMP-8) was shown to cleave at the E373-A374 'aggrecanase' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.
Timo Sorsa - One of the best experts on this subject based on the ideXlab platform.
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pilot study on oral health status as assessed by an active matrix metalloproteinase 8 chairside mouthrinse test in adolescents
Journal of Periodontology, 2016Co-Authors: Anna Maria Heikkinen, Solomon O Nwhator, Timo Sorsa, Nilminie Rathnayake, Paivi Mantyla, Paivi VatanenAbstract:Background: Matrix metalloproteinase (MMP)-8 is a major destructive Collagenase involved in periodontitis and can be regarded as a periodontitis biomarker. A Neutrophil Collagenase 2 (active MMP-8 [aMMP-8]) oral fluid immunoassay has recently been demonstrated to be a periodontitis risk indicator among adults. The aim of this study is to investigate whether a point-of-care mouthrinse test based on an aMMP-8 immunoassay could identify patients with oral inflammatory burden (periodontitis and caries) among adolescents with early pathologic findings.Methods: This cross-sectional study was carried out at the Kotka Health Center, Finland. First, the aMMP-8 chairside mouthrinse test was performed on enrolled individuals (adolescents aged 15 to 17 years, n = 47), and the results were read based on a color change within 5 minutes. Then, full-mouth clinical parameters of oral health were assessed, including periodontal, oral mucosal, and caries status.Results: The sensitivity and specificity of the test for bleedi...
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clinical correlates of a lateral flow immunoassay oral risk indicator
Journal of Periodontology, 2014Co-Authors: Solomon O Nwhator, P O Ayanbadejo, K A Umeizudike, O I Opeodu, G A Agbelusi, Joseph A Olamijulo, Modupe O Arowojolu, Timo Sorsa, B S Babajide, D O OpedunAbstract:Background: The aim of the present study is to investigate the clinical correlates of a novel lateral-flow immunoassay with bleeding on probing (BOP), oral hygiene, and periodontal probing depth. This report offers a simple, rapid, and highly sensitive tool that addresses two issues important to periodontists: 1) detecting active periodontitis, and 2) predicting chronic periodontitis. Methods: Seventy-six of 86 males requiring seminal fluid analysis as part of a separate study were serially recruited into the study. After basic dental and periodontal examination under natural light and with the use of the community periodontal index of treatment needs (CPITN) probe, debris and calculus indices were recorded per participant. Participants were subsequently grouped into ‘‘good,’’ ‘‘fair,’’ and ‘‘poor’’ oral hygiene categories based on a simplified oral hygiene index. BOP was assessed with the ball-ended tip of the probe, and periodontitis was assessed with pocket probing as well as a lateral flow of Neutrophil Collagenase-2 immunoassay, which measures levels of matrix metalloproteinase-8. Results: Neutrophil Collagenase-2 immunoassay was 96% sensitive for poor oral hygiene, 95% sensitive for chronic periodontitis (defined as at least two sites with periodontal pockets), and 82.6% sensitive for at least two sites with BOP. Conclusion: Neutrophil Collagenase-2 immunoassay had a high sensitivity for at least two sites with BOP and two sites with periodontal pockets but a lower relationship for single-site pockets and BOP. JP eriodontol 2014;85:188-194.
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human Neutrophil Collagenase mmp 8 in peri implant sulcus fluid and its inhibition by clodronate
Journal of Dental Research, 1997Co-Authors: Olli Teronen, Y T Konttinen, Tuula Ingman, Christian Lindqvist, Tuula Salo, Anneli Lauhio, Y Ding, S Santavirta, Timo SorsaAbstract:The exact molecular mechanisms of the loosening of a dental implant are not well-known. The characteristics of implant sulci are similar to those of periodontal sulci regarding gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF). Proteolytic enzymes, matrix metalloproteinases (MMPs), participate in peri-implant tissue remodeling. Clodronate is a well-tolerated bisphosphonate-group drug currently used in bone-resorption-related diseases in humans. The mechanisms of bisphosphonate action are not clarified. Collagenase activity in diseased PISF was significantly higher than in the clinically healthy group. Immunoblotting disclosed that diseased PISF contained increased immunoreactives MMP-8 compared with the healthy PISF. The residual latent Collagenase activity in the diseased PISF was activated by gold thioglucose and inhibited completely by 100 microM of doxycycline closely resembling pure Neutrophil Collagenase (MMP-8). The presence of MMP-8 in diseased but not in clinically healthy PISF may prove to be a useful biochemical indicator to monitor peri-implant health and disease. Pure human Neutrophil Collagenase (MMP-8) and the MMP-8 present in PISF and in the GCF of both loosening implants and periodontitis-affected teeth were efficiently inhibited in vitro by clodronate (50% inhibition [IC50] was achieved by 150 microM of clodronate), an osteoactive, antiresorptive bisphosphonate. Furthermore, the new finding suggests an extended and hitherto-undescribed potential for clodronate in preventing the loosening of both implants and teeth, based on a dual beneficial effect: prevention of both bone resorption/osteolysis and of soft tissue/dental ligament destruction. Potential new therapeutic indications based on the Collagenase-inhibiting effect of clodronate provide potential new therapeutic indications for a variety of diseased involving connective tissue breakdown, such as periodontal disease, arthritides, and tumor invasion.
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human Neutrophil Collagenase mmp 8 identified in bronchiectasis bal fluid correlates with severity of disease
Chest, 1995Co-Authors: Ruth Sepper, Y Ding, Y T Konttinen, Michiaki Takagi, Timo SorsaAbstract:Collagenases in bronchoalveolar lavage fluid (BALF) of patients with bronchiectasis and healthy subjects were characterized using specific functional and immunologic assays. The BAL fluid contained interstitial Collagenase and collagenolytic proteinases of bacterial origin. Collagenase activities, obtained after organomercurial activation, correlated with the severity of bronchiectasis. In severe cases, Collagenase activities were 3.5×10 −7 IU/L/48 h or 4.8×10 −6 IU/g/48 h (p −7 IU/L/48 h or 3.35×10 −6 IU/g/48 h (p −7 IU/L/48 h or 0.7×10 −6 IU/g/48 h (p −7 IU/L/48 h or 0.13×10 −6 IU/g/48 h. The cellular origin of interstitial Collagenase was assessed with doxycycline inhibition test utilizing the differential sensitivity of fibroblast-type Collagenase/MMP-1 (IC 50 =280 μ M) and Neutrophil-type Collagenase/MMP-8 (IC 50 =26 μ M) to the anticollagenolytic, nonantimicrobial doxycycline action. Interstitial Collagenase, contained in BALF, was totally inhibited by 100 μ M of doxycycline. It can therefore be concluded that most of mammalian Collagenase presented in inflamed fluid of bronchiectasis originated from Neutrophils. The molecular forms of Neutrophil-type Collagenase/MMP-8 were confirmed and analyzed by Western-blot, which showed evidence of the proteolytic conversion of the latent 85-kD MMP-8 proenzyme species into active 65-kD molecular weight species. These findings strongly suggest involvement of proteolytic activation pathway of proMMP-8, especially in severe and moderate forms of bronchiectasis. Furthermore, collagenolytic proteases of bacterial origins may also participate in tissue destruction of the lung.
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reduction of matrix metalloproteinase 8 Neutrophil Collagenase levels during long term doxycycline treatment of reactive arthritis
Antimicrobial Agents and Chemotherapy, 1994Co-Authors: Anneli Lauhio, Harald Tschesche, Y T Konttinen, L M Golub, Tuula Salo, Dan Nordstrom, Juhani Lahdevirta, Timo SorsaAbstract:The aim of this work was to determine whether human polymorphonuclear Neutrophilic interstitial Collagenase (matrix metalloproteinase 8 [MMP-8]) levels are reduced during long-term doxycycline treatment in humans with reactive arthritis. Serum MMP-8 levels were reduced (mean +/- standard error of the mean, 678.9 +/- 185.6 versus 491.2 +/- 144.8 ng of MMP-8 per ml), but not statistically significantly. However, the reduction of salivary MMP-8 levels was statistically significant (3,729 +/- 1,905.3 versus 1,866 +/- 780.0 ng of MMP-8 per ml, P < 0.05). This study demonstrated that a 2-month regimen of doxycycline can reduce MMP-8 levels in serum and especially in body fluids (i.e., saliva) containing inflammatory exudates and thus may contribute to reduced tissue destruction.
Massimo Coletta - One of the best experts on this subject based on the ideXlab platform.
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characterization of the mechanisms by which gelatinase a Neutrophil Collagenase and membrane type metalloproteinase mmp 14 recognize collagen i and enzymatically process the two α chains
Journal of Molecular Biology, 2007Co-Authors: Magda Gioia, Giovanni Francesco Fasciglione, Stefano Marini, Susanna Monaco, Anna Coletti, Andrea Modesti, Massimo ColettaAbstract:The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which Neutrophil Collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.
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modulation of the catalytic activity of Neutrophil Collagenase mmp 8 on bovine collagen i role of the activation cleavage and of the hemopexin like domain
Journal of Biological Chemistry, 2002Co-Authors: Magda Gioia, Giovanni Francesco Fasciglione, Oliver Diekmann, Vincenzo Politi, Harald Tschesche, Stefano Marini, Michael Pieper, Giampiero De Sanctis, Silvana Dalessio, Massimo ColettaAbstract:The cleavage of bovine collagen I by Neutrophil Collagenase MMP-8 has been followed at pH 7.4, 37 °C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990)Biochemistry 29, 10628–10634; Knauper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Blaser, J., and Tschesche, H. (1993) Biochem. J. 291, 847–854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657–18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met80 of the proenzyme (MetMMP-8) and the former Phe79 of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much fasterk cat/K m , fully attributable to a lower K m , suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227–233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
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Modulation of the catalytic activity of Neutrophil Collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain.
Journal of Biological Chemistry, 2002Co-Authors: Magda Gioia, Giovanni Francesco Fasciglione, Silvana D'alessio, Oliver Diekmann, Vincenzo Politi, Michael P Pieper, Harald Tschesche, Stefano Marini, Massimo ColettaAbstract:The cleavage of bovine collagen I by Neutrophil Collagenase MMP-8 has been followed at pH 7.4, 37 degreesC. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Knauper, V., Osthues, A., DeClerk, Y. A., Langley, K. A, Blaser, J., and Tschesche, H. (1993) Biochem. J. 291, 847854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80)of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K-m, fully attributable to a lower K-m, suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
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ph and temperature dependence of functional modulation in metalloproteinases a comparison between Neutrophil Collagenase and gelatinases a and b
Biophysical Journal, 2000Co-Authors: Giovanni Francesco Fasciglione, Vincenzo Politi, Stefano Marini, Silvana Dalessio, Massimo ColettaAbstract:Metalloproteases are metalloenzymes secreted in the extracellular fluid and involved in inflammatory pathologies or events, such as extracellular degradation. A Zn(2+) metal, present in the active site, is involved in the catalytic mechanism, and it is generally coordinated with histidyl and/or cysteinyl residues of the protein moiety. In this study we have investigated the effect of both pH (between pH 4.8 and 9.0) and temperature (between 15 degrees C and 37 degrees C) on the enzymatic functional properties of the Neutrophil interstitial Collagenase (MMP-8), gelatinases A (MMP-2) and B (MMP-9), using the same synthetic substrate, namely MCA-Pro-Leu-Gly approximately Leu-DPA-Ala-Arg-NH(2). A global analysis of the observed proton-linked behavior for k(cat)/K(m), k(cat), and K(m) indicates that in order to have a fully consistent description of the enzymatic action of these metalloproteases we have to imply at least three protonating groups, with differing features for the three enzymes investigated, which are involved in the modulation of substrate interaction and catalysis by the enzyme. This is the first investigation of this type on recombinant Collagenases and gelatinases of human origin. The functional behavior, although qualitatively similar, displays significant differences with respect to what was previously observed for stromelysin and porcine Collagenase and gelatinase (Stack, M. S., and R. D. Gray. 1990. Arch. Biochem. Biophys. 281:257-263; Harrison, R. K., B. Chang, L. Niedzwiecki, and R. L. Stein. 1992. Biochemistry. 31:10757-10762). The functional characterization of these enzymes can have some relevant physiological significance, since it may be related to the marked changes in the environmental pH that Collagenase and gelatinases may experience in vivo, moving from the intracellular environment to the extracellular matrix.
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cleavage of bovine collagen i by Neutrophil Collagenase mmp 8 effect of ph on the catalytic properties as compared to synthetic substrates
Journal of Biological Chemistry, 2000Co-Authors: Stefano Marini, Giovanni Francesco Fasciglione, Vincenzo Politi, Giampiero De Sanctis, Silvana Dalessio, Massimo ColettaAbstract:The enzymatic processing of bovine collagen I by Neutrophil Collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.