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John D. Sandy - One of the best experts on this subject based on the ideXlab platform.
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biochemical identification and immunolocalizaton of aggrecan adamts5 and inter alpha trypsin inhibitor in equine degenerative suspensory ligament desmitis
Journal of Orthopaedic Research, 2011Co-Authors: Anna Plaas, John D. Sandy, Daniel Schenkman, Haowen Liu, Michael A. Diaz, Robert P. Magnus, Paul W. Kopesky, Vincent M. Wang, Courtney Bolambretl, Jorge O. GalanteAbstract:We describe analysis of suspensory ligaments from horses with advanced degenerative suspensory ligament desmitis (DSLD) to identify the major proteoglycans (PGs), ADAMTS-aggrecanases and inter-alpha-trypsin inhibitor (IαI) components associated with ligament degeneration. Specific anatomical regions of suspensory ligaments from two normal horses and four diagnosed with DSLD were analyzed by Western blot and immunohistochemistry for the following: aggrecan, aggrecan fragments, decorin, ADAMTS4, ADAMTS5, and IαI components. When compared to normal, DSLD ligaments showed about a 15-fold increase (P < 0.0014) in aggrecan levels and markedly enhanced staining with Safranin O. The aggrecan was composed of two distinct high molecular weight core protein species. The largest species was found only in DSLD samples and it co-migrated with aggrecan synthesized by equine mesenchymal stem cells (MSC). Many of the DSLD samples also contained abnormally high concentrations of ADAMTS4, ADAMTS5, and IαI. Notably, the ADAMTS5 in DSLD samples, but not normals, was present largely as a high molecular weight complex. We conclude that ligament degeneration in DSLD is associated with matrix changes characteristic of an inflammatory nonhealing wound, specifically containing chondrogenic progenitor cells. Since aggrecan accumulation is a major feature of incomplete healing in tendon and skin of the ADAMTS5 knockout mouse, we propose that ligament failure in DSLD results from a process involving tissue inflammation and the complexation of ADAMTS5.
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cell death associated ADAMTS4 and versican degradation in vascular tissue
Journal of Histochemistry and Cytochemistry, 2009Co-Authors: Richard D Kenagy, Seung Kee Min, Alexander W Clowes, John D. SandyAbstract:High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.
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removal of o linked and n linked oligosaccharides is required for optimum detection of nitege neoepitope on ADAMTS4 digested fetal aggrecans implications for specific n linked glycan dependent aggrecanolysis at glu373 ala374
Osteoarthritis and Cartilage, 2009Co-Authors: J E Frank, Vivian Thompson, M P Brown, John D. SandyAbstract:Summary Objectives We have observed that Western blot analysis with an anti-G1 antibody detects G1-NITEGE product in a d isintegrin a nd m etalloprotease with t hrombo s pondin motifs-4 (ADAMTS4)-digested fetal and mature human and bovine aggrecan, but the neoepitope-specific anti-NITEGE antibody only detects this product in digests of mature aggrecan. Our objective was to determine whether enzymatic removal of O- and/or N-linked oligosaccharides from the fetal products would enable detection of the NITEGE neoepitope with anti-NITEGE antibody. Methods Aggrecan was purified from fetal and mature human and bovine cartilage and digested with: (1) ADAMTS4, (2) ADAMTS4, sialidase II, and N-glycanase, (3) ADAMTS4, sialidase II, and O-glycanase, or (4) ADAMTS4, sialidase II, and both N- and O-glycanases. Western blot analysis was performed using anti-G1 and anti-NITEGE antibodies. Results When fetal G1-NITEGE products were treated with a combination of ADAMTS4, sialidase II, O-glycanase and N-glycanase, the resultant products migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the NITEGE neoepitope was rendered detectable. Conclusions It appears that the NITEGE neoepitope is blocked on Western blots by oligosaccharide structures present on Asn368 and Thr370 of fetal human and bovine aggrecans. Such masking structures do not appear to be present on mature aggrecans from these species. We suggest that when anti-NITEGE antibody is used in Western analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC), removal of oligosaccharides with appropriate glycosidases may unmask reactivity that would otherwise go undetected. The implications of these findings for the much-studied effect of Asn368-linked keratan sulfate (KS)-based structures on ADAMTS4 and ADAMTS5 activity are discussed.
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analysis of ADAMTS4 and mt4 mmp indicates that both are involved in aggrecanolysis in interleukin 1 treated bovine cartilage
Osteoarthritis and Cartilage, 2005Co-Authors: Parth Patwari, John D. Sandy, Gui Gao, Alan J Grodzinsky, Jennifer H LeeAbstract:Summary Objective To investigate the mechanism of aggrecanolysis in interleukin-1 (IL-1)-treated cartilage tissue by examining the time course of aggrecan cleavages and the tissue and medium content of membrane type 4-matrix metalloproteinases (MT4-MMP) and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS)4. Methods Articular cartilage explants were harvested from newborn bovine femoropatellar groove. The effects of IL-1 treatment with or without aggrecanase blockade were investigated by Western analysis of aggrecan fragment generation, ADAMTS4 species (p68 and p53), and MT4-MMP, as well as by realtime PCR (polymerase chain reaction) for ADAMTS4 and 5. Aggrecanase was blocked with mannosamine (ManN), an inhibitor of glycosylphosphatidylinositol anchor synthesis, and esculetin (EST), an inhibitor of MMP-1, MMP-3, and MMP-13 gene expression. Results IL-1 treatment caused a major increase in MT4-MMP abundance in the tissue and medium. ADAMTS4 (p68) was abundant in fresh cartilage and this was retained in the tissue in untreated cartilage. IL-1 treatment for 6 days caused a marked loss of p68 from the cartilage and the appearance of p53 in the medium. Addition of either 1.35mM ManN or 31–500μM EST blocked IL-1-mediated aggrecanolysis and this was accompanied by nearly complete inhibition of the MT4-MMP increase, the p68 loss and the formation of p53. IL-1 treatment increased mRNA abundance for ADAMTS4 (∼3-fold) and ADAMTS5 (∼10-fold) but this was not accompanied by a marked change in enzyme protein abundance. Conclusion These studies support a central role for MT4-MMP in IL-1-induced cartilage aggrecanolysis and are consistent with the identification of p68 as the aggrecanase that cleaves within the CS2 domain, and of p53 as the aggrecanase that generates G1-NITEGE. Since the induction by IL-1 was not accompanied by marked changes in total ADAMTS4 protein, but rather in partial conversion of p68 to p53 and release of both from the tissue, we conclude that aggrecanolysis in this model system results from MT4-MMP-mediated processing of a resident pool of ADAMTS4 and release of the p68 and p53 from their normal association with the cell surface.
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ADAMTS4 aggrecanase 1 activation on the cell surface involves c terminal cleavage by glycosylphosphatidyl inositol anchored membrane type 4 matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on
Journal of Biological Chemistry, 2004Co-Authors: Gui Gao, John D. Sandy, Anna Plaas, Vivian Thompson, Sue Jin, Fengrong ZuoAbstract:Abstract C-terminal truncation of ADAMTS-4 from the p68 form to the p53 form is required for activation of its capacity to cleave the Glu373-Ala374 interglobular domain bond of aggrecan. In transfected human chondrosarcoma cells, this process is not autoproteolytic because the same products form with an inactive mutant of ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin-like motif 4) and truncation is completely blocked by tissue inhibitor of metalloproteinase-1. Instead, activation can be mediated by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase (MT4-MMP, MMP-17) because co-transfection with the active form of MT4-MMP markedly enhanced activation, whereas an inactive mutant of MT4-MMP was ineffective. Treatment of co-transfected cells with phosphatidylinositol-specific phospholipase C liberated the complex of MT4-MMP and p68 ADAMTS4 from the cell membrane, but the p53 ADAMTS4 remained associated. Specific glycosaminoglycan lyase digestions, followed by product analyses using fluorescence-assisted carbohydrate electrophoresis and immunoprecipitation experiments, showed that the p53 form is associated with syndecan-1 through both chondroitin sulfate and heparan sulfate. We conclude that ADAMTS-4 activation in this cell system involves the coordinated activity of both glycosylphosphatidyl inositol-anchored MT4-MMP and the proteoglycan form of syndecan-1 on the cell surface.
Yasunori Okada - One of the best experts on this subject based on the ideXlab platform.
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development of human neutralizing antibody to ADAMTS4 aggrecanase 1 and adamts5 aggrecanase 2
Biochemical and Biophysical Research Communications, 2016Co-Authors: Aya Shiraishi, Kanehisa Kojoh, Akira Miyakoshi, Satsuki Mochizuki, Yasunori OkadaAbstract:ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis.
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hyaluronan inhibits expression of ADAMTS4 aggrecanase 1 in human osteoarthritic chondrocytes
Annals of the Rheumatic Diseases, 2009Co-Authors: Taku Yatabe, Masayuki Takizawa, Aiko Okada, Miyuki Chijiiwa, Tokuhiro Kimura, Yoshinari Fujita, Yoshiaki Toyama, Hideo Matsumoto, Satsuki Mochizuki, Yasunori OkadaAbstract:Background: Intra-articular injection of hyaluronan (HA) has been suggested to have a disease-modifying effect in osteoarthritis, but little is known about the possible mechanisms. Objective: To investigate the effects of HA species of different molecular mass, including 800 kDa (HA800) and 2700 kDa (HA2700), on the expression of aggrecanases (ie, ADAMTS species), which play a key role in aggrecan degradation. Methods: The effects of HA species on the expression of ADAMTS1, 4, 5, 8, 9 and 15 in interleukin 1α (IL1α)-stimulated osteoarthritic chondrocytes were studied by reverse transcription PCR and real-time PCR. Expression of ADAMTS4 protein and aggrecanase activity and signal transduction pathways of IL1, CD44 and intracellular adhesion molecule 1 (ICAM1) were examined by immunoblotting. Results: IL1α treatment of chondrocytes induced ADAMTS4, and HA800 and HA2700 significantly decreased IL1α-induced expression of ADAMTS4 mRNA and protein. IL1α-stimulated aggrecanase activity in osteoarthritic chondrocytes was reduced by treatment with HA2700 or transfection of small interfering RNA for ADAMTS4. A similar result was obtained when HA2700 was added to explant cultures of osteoarthritic cartilage. HA2700 neither directly inhibited nor bound to ADAMTS4. Downregulation of ADAMTS4 expression by HA2700 was attenuated by treatment of IL1α-treated chondrocytes with antibodies to CD44 and/or ICAM1. The increased phosphorylation of IL1 receptor-associated kinase-1 and extracellular signal-regulated protein kinase1/2 induced by the IL1α treatment was downregulated by enhanced IRAK-M expression after HA2700 treatment. Conclusion: These data suggest that HA2700 suppresses aggrecan degradation by downregulating IL1α-induced ADAMTS4 expression through the CD44 and ICAM1 signalling pathways in osteoarthritic chondrocytes.
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calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 aggrecanase 1 in osteoarthritic chondrocytes
FEBS Letters, 2008Co-Authors: Masayuki Takizawa, Taku Yatabe, Aiko Okada, Miyuki Chijiiwa, Satsuki Mochizuki, Peter Ghosh, Yasunori OkadaAbstract:Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1α-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.
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ADAMTS4 aggrecanase 1 interaction with the c terminal domain of fibronectin inhibits proteolysis of aggrecan
Journal of Biological Chemistry, 2004Co-Authors: Gakuji Hashimoto, Masayuki Shimoda, Yasunori OkadaAbstract:Abstract ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693–Lys837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nm), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.
Bruce Caterson - One of the best experts on this subject based on the ideXlab platform.
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human osteoarthritis synovium contains an alternatively spliced transcript of ADAMTS4
Osteoarthritis and Cartilage, 2012Co-Authors: S D Wainwright, Jan Bondeson, Clare Elizabeth Hughes, Bruce CatersonAbstract:Purpose: The characterization of an alternatively spliced transcript of the ADAMTS4 aggrecanase. Methods: In human OA synovial cell cultures, RT-PCR was performed using oligonucleotide primers designed to amplify across the exon 8/9 region of human ADAMTS4. The PCR products were purified using a QIAquick purification kit (Qiagen) and sequenced using in house facilities. A pCEP4 (Invitrogen) mammalian expression vector containing ADAMTS4 plus a FLAG epitope was mutated using the QuikChange II site directed mutagenesis kit (Stratagene) to contain the ADAMTS4 splice variant plus a FLAG epitope. The recombinant proteins were purified from HEK293 transfected cells using Anti-FLAG M2 affinity gel (Sigma). Polyclonal antibodies were raised against synthetic peptides representing sequences within the C-terminal region of the splice variant of ADAMTS4 and the raised antibodies were characterized using the recombinant splice variant of ADAMTS4. The antibodies were used in immunohistochemical analysis of human osteoarthritic synovium. The proteolysis of aggrecan and other proteoglycans by the recombinant spice variant of ADAMTS4 was investigated. Results: The degradation of aggrecan is mainly mediated by the aggrecanases, of which ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) are the best known. We here characterize an alternative splice variant of ADAMTS4. RT-PCR performed as described above resulted in the amplification of normal ADAMTS4, and also a smaller product missing 161 base pairs from the 5’ end of exon 9, the result of alternative splicing in which exon 8 joins to a cryptic 3’ splice site within exon 9. The protein produced by this alternative splicing would lack the spacer domain and have a C-terminus lacking any homologies with the normal ADAMTS4 spacer domain. The alternatively spliced transcript of ADAMTS4 was found in cultured OA synovial cells and in freshly digested OA synovium, but not in human brain, cervix or lung, or in normal bovine synovium. The protein synthesized from this alternatively spliced transcript of ADAMTS4 would lose functions dependent on its spacer domain, like substrate and matrix binding, and inhibition through fibronectin. Removal of the spacer domain from ADAMTS4 has been reported to increase its ability to cleave aggrecan at the Glu373-Ala374 bond, and it may well be that the alternatively spliced transcript produces a protein that is secreted in a more active form. HEK293 cells transfected with a pCEP4 vector containing the cDNA sequence of the splice variant of ADAMTS4 produced the corresponding protein in both the pro and active form. This protein could be found in the media, but mostly associated with the cells, as confirmed using antibodies specific for the splice variant that were produced using synthetic peptides. Immunohistochemical analysis of osteoarthritic synovium using these antibodies showed staining of cells within the synovium. Proteins purified by immunoprecipitation by Anti-FLAG M2 affinity gel from transfected and untransfected HEK293 cells were analysed using the ANASpec SensoLyte 520 Aggrecanase I assay kit. The splice variant had aggrecanase activity comparable to a commercially available ADAMTS4. The splice variant cleaved aggrecan at the G1u373-A1a374 site, as assessed by the neoepitope monoclonal antibody BC3, with activity comparable to ADAMTS4. Conclusions: ADAMTS4 is regulated at multiple levels through control of gene expression, mRNA splicing and protein processing, as well as the expression of naturally occurring inhibitors. We here describe the characteristics of the first known splice variant of ADAMTS4. This alternative splice transcript of ADAMTS4 is expressed as a protein in vivo and can be found in the synovium. It can be speculated that the changes in the C-terminal domain of the protein resulting from this alternatively spliced transcript would have changes in its substrate specificity. The protein produced by the alternative spliced transcript of ADAMTS4 has aggrecanase activity, and the release of low levels of this fully active variant of ADAMTS4 might be a factor in the slow process of superficial zone aggrecan loss in osteoarthritis.
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001 an alternatively spliced transcript of ADAMTS4 in human osteoarthritis synovium
Osteoarthritis and Cartilage, 2010Co-Authors: S D Wainwright, Jan Bondeson, Clare Elizabeth Hughes, Bruce CatersonAbstract:Purpose: To describe and characterize a newly discovered alternatively spliced transcript of the ADAMTS4 aggrecanase. Methods: Human OA synovium was digested and synovial cell cultures established. RT-PCR analysis was performed using oligonucleotide primers designed to amplify across the exon 8/9 region of human ADAMTS4. RT-PCR was performed and PCR products purified using a QIAquick purification kit (Qiagen) and sequenced using in house facilities. A pCEP4 (Invitrogen) mammalian expression vector containing ADAMTS4 plus a FLAG epitope was mutated using QuikChange II site directed mutagenesis kit (Stratagene) to contain the ADAMTS4 splice variant plus a FLAG epitope. The recombinant proteins were purified from HEK293 transfected cells using Anti-FLAG M2 affinity gel (Sigma). Results: Destruction of articular cartilage is a key feature of osteoarthritis. Aggrecan degradation is followed by irreversible collagen degradation. The degradation of aggrecan is mainly mediated by the aggrecanases, multidomain metalloproteinases belonging to the ADAMTS family, in which ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) are the most studied. Although alternative splicing has previously been described in other ADAMTS members, we here describe and characterise the first known alternative splice variant of ADAMTS4. RT-PCR on human OA synovial cells using the above primers resulted in the amplification of two products: normal ADAMTS4 and a smaller product missing 161 base pairs from the 5’ end of exon 9, the result of alternative splicing in which exon 8 joins to a cryptic 3’ splice site within exon 9. The protein produced would lack the spacer domain and have a different C-terminus lacking homologies with the normal human ADAMTS4 C-terminal spacer domain. The alternatively spliced transcript of ADAMTS4 has been found in cultured OA synovial cells and in freshly digested OA synovium, but not in human brain, cervix or lung, or in normal bovine synovium. The predicted protein synthesized from this alternatively spliced transcript of ADAMTS4 would lose functions dependent on its spacer domain, like substrate and matrix binding, and inhibition through fibronectin. Removal of the spacer domain from ADAMTS4 increases its ability to cleave aggrecan, and it may well be that this alternatively spliced transcript produces a protein that is secreted in an active form, capable of cleaving the Glu373-Ala374 bond in the interglobular domain of aggrecan. HEK293 cells transfected with a pCEP4 vector containing the cDNA sequence of the splice variant of ADAMTS4 produced the corresponding protein in both the pro and active form. This protein could be found in the media, but mostly associated with the cells, as confirmed using antibodies specific for the splice variant that were produced using synthetic peptides. Proteins purified by immunoprecipitation by Anti-FLAG M2 affinity gel from transfected and untransfected HEK293 cells were analysed using the ANASpec SensoLyte 520 Aggrecanase I assay kit which showed that the splice variant had aggrecanase activity comparable to a commercially available ADAMTS4. The purified splice variant was also found to cleave
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the regulation of the ADAMTS4 and adamts5 aggrecanases in osteoarthritis a review
Clinical and Experimental Rheumatology, 2008Co-Authors: Jan Bondeson, S D Wainwright, Clare Elizabeth Hughes, Bruce CatersonAbstract:Destruction of articular cartilage is a key feature of a number of arthritides, osteoarthritis prominent among them. Aggrecan degradation, caused by increased activity of proteolytic enzymes that degrade macromolecules in the cartilage extracellular matrix, is followed by irreversible collagen degradation. The degradation of aggrecan is mediated by various matrix proteinases, mainly the aggrecanases, multidomain metalloproteinases belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family. There has been much interest in the possible role of these aggrecanases, mainly ADAMTS4 and ADAMTS5, as therapeutic targets in osteoarthritis. There is still debate which of them is the major aggrecanase in osteoarthritis, however, as well as major issues concerning how they are regulated, with possible discrepancies between murine models and results obtained using human osteoarthritis tissue. This review discusses some recent data regarding the regulation of ADAMTS4 and ADAMTS5 gene expression in osteoarthritis, with emphasis on the role of proinflammatory cytokines in driving these enzymes, and of the transcription factor NFkappaB in mediating their expression.
Jennifer Westling - One of the best experts on this subject based on the ideXlab platform.
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ADAMTS4 aggrecanase 1 cleaves human brain versican v2 at glu405 gln406 to generate glial hyaluronate binding protein
Biochemical Journal, 2004Co-Authors: Jennifer Westling, John D. Sandy, Vivian Thompson, Paul E Gottschall, Amber Cockburn, George Perides, Dieter R ZimmermannAbstract:Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepitope antiserum (JSCNIV) showed that its C-terminal sequence is NIVSFE(405), and digestion of human cerebellum proteoglycans with ADAMTS4 (aggrecanase-1, where ADAMTS, a disintegrin and metalloproteinase with thrombospondin-1-like motifs) indicated that GHAP is a product of cleavage of versican V0 or V2 at the Glu(405)-Gln(406) bond. Since human cerebellum extracts contained multiple forms of ADAMTS4 protein on Western analysis, these data suggest that one or more members of the 'aggrecanase' group of the ADAMTS family (ADAMTS 1, 4, 5 and 9) are responsible for turnover of versican V2 in the adult human brain.
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ADAMTS4 cleaves at the aggrecanase site glu373 ala374 and secondarily at the matrix metalloproteinase site asn341 phe342 in the aggrecan interglobular domain
Journal of Biological Chemistry, 2002Co-Authors: Jennifer Westling, Lisa A Collinsracie, Edward R Lavallie, Vivian Thompson, Amanda J Fosang, Kathy Tomkinson, Tracy Hebert, Thomas Mcdonagh, Elisabeth Morris, John D. SandyAbstract:Abstract Two major proteolytic cleavages, one at NITEGE373↓A374RGSVI and the other at VDIPEN341↓F342FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu373-Ala374 site is cleavedin vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn341-Phe342, is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE373 and G1-VDIPEN341 in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu373-Ala374 site, but also, albeit slowly and secondarily, at the Asn341-Phe342 site. Cleavage at the Asn341-Phe342 site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu373-Ala374 site and secondarily generates G1-VDIPEN341 by removal of the Phe342–Glu373 peptide from G1-NITEGE373. These results show that G1-VDIPEN341 is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.
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ADAMTS4 cleaves at the aggrecanase site glu373 ala374 and secondarily at the matrix metalloproteinase site asn341 phe342 in the aggrecan interglobular domain
Journal of Biological Chemistry, 2002Co-Authors: Jennifer Westling, Lisa A Collinsracie, Edward R Lavallie, Elisabeth A Morris, Vivian Thompson, Amanda J Fosang, Tracy Hebert, Thomas Mcdonagh, Kathy N Tomkinson, John D. SandyAbstract:Two major proteolytic cleavages, one at NITEGE373↓A374RGSVI and the other at VDIPEN341↓F342FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu373-Ala374 site is cleavedin vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn341-Phe342, is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE373 and G1-VDIPEN341in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu373-Ala374 site, but also, albeit slowly and secondarily, at the Asn341-Phe342 site. Cleavage at the Asn341-Phe342 site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu373-Ala374 site and secondarily generates G1-VDIPEN341 by removal of the Phe342–Glu373 peptide from G1-NITEGE373. These results show that G1-VDIPEN341 is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.
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adamts1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors
Biochemical and Biophysical Research Communications, 2002Co-Authors: Juan Carlos Rodriguezmanzaneque, John D. Sandy, Jennifer Westling, Shelley N M Thai, Alfonso Luque, Vera Knauper, Gillian Murphy, Luisa M IruelaarispeAbstract:ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.
Vivian Thompson - One of the best experts on this subject based on the ideXlab platform.
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removal of o linked and n linked oligosaccharides is required for optimum detection of nitege neoepitope on ADAMTS4 digested fetal aggrecans implications for specific n linked glycan dependent aggrecanolysis at glu373 ala374
Osteoarthritis and Cartilage, 2009Co-Authors: J E Frank, Vivian Thompson, M P Brown, John D. SandyAbstract:Summary Objectives We have observed that Western blot analysis with an anti-G1 antibody detects G1-NITEGE product in a d isintegrin a nd m etalloprotease with t hrombo s pondin motifs-4 (ADAMTS4)-digested fetal and mature human and bovine aggrecan, but the neoepitope-specific anti-NITEGE antibody only detects this product in digests of mature aggrecan. Our objective was to determine whether enzymatic removal of O- and/or N-linked oligosaccharides from the fetal products would enable detection of the NITEGE neoepitope with anti-NITEGE antibody. Methods Aggrecan was purified from fetal and mature human and bovine cartilage and digested with: (1) ADAMTS4, (2) ADAMTS4, sialidase II, and N-glycanase, (3) ADAMTS4, sialidase II, and O-glycanase, or (4) ADAMTS4, sialidase II, and both N- and O-glycanases. Western blot analysis was performed using anti-G1 and anti-NITEGE antibodies. Results When fetal G1-NITEGE products were treated with a combination of ADAMTS4, sialidase II, O-glycanase and N-glycanase, the resultant products migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the NITEGE neoepitope was rendered detectable. Conclusions It appears that the NITEGE neoepitope is blocked on Western blots by oligosaccharide structures present on Asn368 and Thr370 of fetal human and bovine aggrecans. Such masking structures do not appear to be present on mature aggrecans from these species. We suggest that when anti-NITEGE antibody is used in Western analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC), removal of oligosaccharides with appropriate glycosidases may unmask reactivity that would otherwise go undetected. The implications of these findings for the much-studied effect of Asn368-linked keratan sulfate (KS)-based structures on ADAMTS4 and ADAMTS5 activity are discussed.
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ADAMTS4 aggrecanase 1 activation on the cell surface involves c terminal cleavage by glycosylphosphatidyl inositol anchored membrane type 4 matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on
Journal of Biological Chemistry, 2004Co-Authors: Gui Gao, John D. Sandy, Anna Plaas, Vivian Thompson, Sue Jin, Fengrong ZuoAbstract:Abstract C-terminal truncation of ADAMTS-4 from the p68 form to the p53 form is required for activation of its capacity to cleave the Glu373-Ala374 interglobular domain bond of aggrecan. In transfected human chondrosarcoma cells, this process is not autoproteolytic because the same products form with an inactive mutant of ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin-like motif 4) and truncation is completely blocked by tissue inhibitor of metalloproteinase-1. Instead, activation can be mediated by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase (MT4-MMP, MMP-17) because co-transfection with the active form of MT4-MMP markedly enhanced activation, whereas an inactive mutant of MT4-MMP was ineffective. Treatment of co-transfected cells with phosphatidylinositol-specific phospholipase C liberated the complex of MT4-MMP and p68 ADAMTS4 from the cell membrane, but the p53 ADAMTS4 remained associated. Specific glycosaminoglycan lyase digestions, followed by product analyses using fluorescence-assisted carbohydrate electrophoresis and immunoprecipitation experiments, showed that the p53 form is associated with syndecan-1 through both chondroitin sulfate and heparan sulfate. We conclude that ADAMTS-4 activation in this cell system involves the coordinated activity of both glycosylphosphatidyl inositol-anchored MT4-MMP and the proteoglycan form of syndecan-1 on the cell surface.
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ADAMTS4 aggrecanase 1 cleaves human brain versican v2 at glu405 gln406 to generate glial hyaluronate binding protein
Biochemical Journal, 2004Co-Authors: Jennifer Westling, John D. Sandy, Vivian Thompson, Paul E Gottschall, Amber Cockburn, George Perides, Dieter R ZimmermannAbstract:Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepitope antiserum (JSCNIV) showed that its C-terminal sequence is NIVSFE(405), and digestion of human cerebellum proteoglycans with ADAMTS4 (aggrecanase-1, where ADAMTS, a disintegrin and metalloproteinase with thrombospondin-1-like motifs) indicated that GHAP is a product of cleavage of versican V0 or V2 at the Glu(405)-Gln(406) bond. Since human cerebellum extracts contained multiple forms of ADAMTS4 protein on Western analysis, these data suggest that one or more members of the 'aggrecanase' group of the ADAMTS family (ADAMTS 1, 4, 5 and 9) are responsible for turnover of versican V2 in the adult human brain.
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ADAMTS4 cleaves at the aggrecanase site glu373 ala374 and secondarily at the matrix metalloproteinase site asn341 phe342 in the aggrecan interglobular domain
Journal of Biological Chemistry, 2002Co-Authors: Jennifer Westling, Lisa A Collinsracie, Edward R Lavallie, Vivian Thompson, Amanda J Fosang, Kathy Tomkinson, Tracy Hebert, Thomas Mcdonagh, Elisabeth Morris, John D. SandyAbstract:Abstract Two major proteolytic cleavages, one at NITEGE373↓A374RGSVI and the other at VDIPEN341↓F342FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu373-Ala374 site is cleavedin vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn341-Phe342, is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE373 and G1-VDIPEN341 in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu373-Ala374 site, but also, albeit slowly and secondarily, at the Asn341-Phe342 site. Cleavage at the Asn341-Phe342 site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu373-Ala374 site and secondarily generates G1-VDIPEN341 by removal of the Phe342–Glu373 peptide from G1-NITEGE373. These results show that G1-VDIPEN341 is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.
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ADAMTS4 cleaves at the aggrecanase site glu373 ala374 and secondarily at the matrix metalloproteinase site asn341 phe342 in the aggrecan interglobular domain
Journal of Biological Chemistry, 2002Co-Authors: Jennifer Westling, Lisa A Collinsracie, Edward R Lavallie, Elisabeth A Morris, Vivian Thompson, Amanda J Fosang, Tracy Hebert, Thomas Mcdonagh, Kathy N Tomkinson, John D. SandyAbstract:Two major proteolytic cleavages, one at NITEGE373↓A374RGSVI and the other at VDIPEN341↓F342FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu373-Ala374 site is cleavedin vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn341-Phe342, is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE373 and G1-VDIPEN341in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu373-Ala374 site, but also, albeit slowly and secondarily, at the Asn341-Phe342 site. Cleavage at the Asn341-Phe342 site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu373-Ala374 site and secondarily generates G1-VDIPEN341 by removal of the Phe342–Glu373 peptide from G1-NITEGE373. These results show that G1-VDIPEN341 is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.