Dr Amanda J Fosang Department of Paediatrics and Murdoch Childrens Research Institute at the Royal Children University of Melbourne Parkville, VIC 3052 Email: amanda.fosang@mcri.edu.au Tel: +61-3-9345-6628 Fax: +61-3-9345-7997 Homepage: http://murdoch.rch.unimelb.edu.au/pages/research/LabResearch.asp Research Our ARTHRITIS research is focused on the MMP and ADAMTS families of metal-dependent proteinases, the products of their activity and the effects of degradation products and enzyme activities on cellular function. We have investigated cartilage degradation at the molecular level, determining the contribution of purified enzymes to discrete catalytic events and identifying specific cleavage sites within the aggrecan core protein. Our studies have led to the development of neoepitope antibodies specific for the newly created N or C-terminus of cleaved proteins, as valuable tools for research and clinical diagnostics. We have developed knock-in and knock-out mice to study the involvement of MMP and ADAMTS enzymes in each of the initiation, progression and repair phases of arthritis. One approach has been to mutate the structural matrix molecule, aggrecan, making it proteinase resistant. We have developed two strains of knock-in mice; one resistant to MMP cleavage and one resistant to ADAMTS cleavage, in a proteinase-sensitive region of aggrecan. These knock-in mice will enable us to differentiate between MMP and ADAMTS activity in cartilage pathology following a challenge with experimental arthritis. A second approach has been to mutate ADAMTS-4 and ADAMTS-5, making them constitutively inactive. ADAMTS-4 and -5 are considered major proteinases in arthritis pathology. We have mice with inactive ADAMTS-4 and -5 in all tissues or in cartilage only. Because only the ADAMTS catalytic site is disrupted, our studies will show potential effects of active site inhibitors, without affecting the biological functions invested in the C-terminal domains of the enzymes. Our in vitro studies focus on regulators of MMP and ADAMTS activity. One important regulator is the glycosaminoglycan, keratan sulphate (KS), which is substituted in the proteinase-sensitive region of aggrecan. Proteolysis in this region is influenced by KS substitution. We are analysing the variable patterns of sulphation, fucosylation and terminal sialylation on KS in regions of the core protein that are proteinase-sensitive or insensitive, with a view to determining candidate structures for biological recognition. Other potential regulators of cartilage homeostasis are the products of proteinase activity. We are investigating the effects of naturally occurring aggrecan fragments. One interesting fragment is a 32mer that results from dual cleavage by MMPs and ADAMTS enzymes. Fortuitously, the proteinase-resistant knock-in mice can be used to assess the in vivo functions of the catalytic fragments, since the 32mer fragment is effectively "knocked-out" in these animals. Collaborations Prof John Sandy, Florida, USA Dr Christopher Little, Sydney, Australia Dr Warren Knudson, Chicago, USA Dr Melanie Pritchard, Melbourne, Australia Dr Elizabeth Arner, St. Louis, USA Prof Ian Wicks, Melbourne, Australia Dr John Mort, Quebec, Canada Dr Ian Campbell, Melbourne, Australia Dr Vera Knuper, York, UK Dr Eric Morand, Melbourne, Australia Dr Anna Plaas, Florida, USA Prof John Bateman, Melbourne, Australia Publications Fosang, AJ., Last, K., Gardiner, P., Jackson, DC., & Brown, L. (1995) Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. Biochem. J. 310, 337-343 Fosang, AJ., Last, K. & Maciewicz, RA. (1996) Aggrecan is degraded by matrix metalloproteinases in human arthritis: Evidence that matrix metalloproteinase and aggrecanase activities can be independent. J. Clin. Invest. 98, 2292-2299 Fosang AJ., Last, K., Weeks, DB., Stanton, H., Campbell, IK., Hardingham, TE., Hembry, RM. (2000) Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants. J. Biol. Chem. 275, 33,027-33,037 Mercuri, FA., Maciewicz, RA., Tart, J., Last, K., Fosang, AJ. (2000) Mutations in the interglobular domain of aggrecan alters matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity. J. Biol. Chem. 275, 33,038-33,045 Fosang, AJ & Smith, PJ. (2001) To clot or not. Nature 413, 475-476 Questions/suggestions? Please contact the Network Administrator. Copyright © by International Protease Network All Right Reserved.