ADAMTS4 Cleaves at the Aggrecanase Site (Glu373-Ala374) and Secondarily at the Matrix Metalloproteinase Site (Asn341-Phe342) in the Aggrecan Interglobular Domain*

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.

[1]  J. Sandy,et al.  Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo , 2001 .

[2]  C. P. Leblond,et al.  Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0–21 days: I. Two groups of proteinases cleave the core protein of aggrecan , 2001, Developmental dynamics : an official publication of the American Association of Anatomists.

[3]  M. Tortorella,et al.  The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation. , 2001, Osteoarthritis and cartilage.

[4]  Douglas K. Anderson,et al.  Intact Aggrecan and Fragments Generated by Both Aggrecanse and Metalloproteinase-Like Activities Are Present in the Developing and Adult Rat Spinal Cord and Their Relative Abundance Is Altered by Injury , 2001, The Journal of Neuroscience.

[5]  R. Mason,et al.  Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice. , 2001, Arthritis and rheumatism.

[6]  M Kashiwagi,et al.  TIMP-3 Is a Potent Inhibitor of Aggrecanase 1 (ADAM-TS4) and Aggrecanase 2 (ADAM-TS5)* , 2001, The Journal of Biological Chemistry.

[7]  K. Tanzawa,et al.  Inhibition of ADAMTS4 (aggrecanase‐1) by tissue inhibitors of metalloproteinases (TIMP‐1, 2, 3 and 4) , 2001, FEBS letters.

[8]  D Heinegård,et al.  The Proteoglycans Aggrecan and Versican Form Networks with Fibulin-2 through Their Lectin Domain Binding* , 2001, The Journal of Biological Chemistry.

[9]  M. Tortorella,et al.  Age-related Changes in Aggrecan Glycosylation Affect Cleavage by Aggrecanase* , 2000, The Journal of Biological Chemistry.

[10]  E. Bartnik,et al.  Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions. , 2000, Matrix biology : journal of the International Society for Matrix Biology.

[11]  T. Hardingham,et al.  Generation and Novel Distribution of Matrix Metalloproteinase-derived Aggrecan Fragments in Porcine Cartilage Explants* , 2000, The Journal of Biological Chemistry.

[12]  T. Burn,et al.  The Thrombospondin Motif of Aggrecanase-1 (ADAMTS-4) Is Critical for Aggrecan Substrate Recognition and Cleavage* , 2000, The Journal of Biological Chemistry.

[13]  C. Little,et al.  Mechanisms involved in cartilage proteoglycan catabolism. , 2000, Matrix biology : journal of the International Society for Matrix Biology.

[14]  T. Burn,et al.  Sites of Aggrecan Cleavage by Recombinant Human Aggrecanase-1 (ADAMTS-4)* , 2000, The Journal of Biological Chemistry.

[15]  P. Patwari,et al.  Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage. , 2000, Archives of biochemistry and biophysics.

[16]  P. Roughley,et al.  Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro. , 1999, The Biochemical journal.

[17]  A. Fosang,et al.  Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase* , 1999, The Journal of Biological Chemistry.

[18]  R. Timpl,et al.  Fibulin-1 Is a Ligand for the C-type Lectin Domains of Aggrecan and Versican* , 1999, The Journal of Biological Chemistry.

[19]  R. Wynn,et al.  Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. , 1999, Science.

[20]  J. V. van Meurs,et al.  Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis. , 1999, Arthritis and rheumatism.

[21]  E B Hunziker,et al.  Physical and Biological Regulation of Proteoglycan Turnover around Chondrocytes in Cartilage Explants: Implications for Tissue Degradation and Repair , 1999, Annals of the New York Academy of Sciences.

[22]  J. Trzăskos,et al.  Generation and Characterization of Aggrecanase , 1999, The Journal of Biological Chemistry.

[23]  A. Poole,et al.  Changes in joint cartilage aggrecan after knee injury and in osteoarthritis. , 1999, Arthritis and rheumatism.

[24]  D. Buttle,et al.  The use of cleavage site specific antibodies to delineate protein processing and breakdown pathways. , 1999, Molecular pathology : MP.

[25]  J. Mort,et al.  Cathepsin B: an alternative protease for the generation of an aggrecan 'metalloproteinase' cleavage neoepitope. , 1998, The Biochemical journal.

[26]  B. Caterson,et al.  Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the 'aggrecanase' and the MMP sites. Characterization of MT1-MMP catabolic activities on the interglobular domain of aggrecan. , 1998, The Biochemical journal.

[27]  A. Grodzinsky,et al.  Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants , 1998, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[28]  J. Fox,et al.  The Interglobular Domain of Cartilage Aggrecan Is Cleaved by Hemorrhagic Metalloproteinase HT-d (Atrolysin C) at the Matrix Metalloproteinase and Aggrecanase Sites* , 1998, The Journal of Biological Chemistry.

[29]  J. Weidner,et al.  Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. , 1997, The Journal of clinical investigation.

[30]  C. Decicco,et al.  Cleavage of Native Cartilage Aggrecan by Neutrophil Collagenase (MMP-8) Is Distinct from Endogenous Cleavage by Aggrecanase* , 1997, The Journal of Biological Chemistry.

[31]  R. Maciewicz,et al.  Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent. , 1996, The Journal of clinical investigation.

[32]  R. Iozzo,et al.  Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  D. Jackson,et al.  Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. , 1995, The Biochemical journal.

[34]  J. Weidner,et al.  Cell-mediated Catabolism of Aggrecan , 1995, The Journal of Biological Chemistry.

[35]  T. Hardingham,et al.  Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E373-A374 in the interglobular domain of cartilage aggrecan. , 1994, The Biochemical journal.

[36]  J. Buckwalter,et al.  Age‐Related changes in cartilage proteoglycans: Quantitative electron microscopic studies , 1994, Microscopy research and technique.

[37]  D. Heinegård,et al.  The cartilage proteoglycan aggregate: assembly through combined protein-carbohydrate and protein-protein interactions. , 1994, Biophysical chemistry.

[38]  L. Lohmander,et al.  The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. , 1993, Arthritis and rheumatism.

[39]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  L. Lohmander,et al.  The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. , 1992, The Journal of clinical investigation.

[41]  T. Hardingham,et al.  Proteoglycans: many forms and many functions , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[42]  M. Lark,et al.  Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. , 1992, The Journal of biological chemistry.

[43]  R. Kaufman,et al.  Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. , 1991, Nucleic acids research.

[44]  J. Sandy,et al.  Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain. , 1991, The Journal of biological chemistry.

[45]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[46]  L. Chasin,et al.  Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[47]  T. Oegema,et al.  Characterization of bovine aorta proteoglycan extracted with guanidine hydrochloride in the presence of protease inhibitors. , 1979, The Journal of biological chemistry.

[48]  A. Hollander,et al.  Degradation of type II collagen, but not proteoglycan, correlates with matrix metalloproteinase activity in cartilage explant cultures. , 1997, Arthritis and rheumatism.