TIMP-3 interaction with aggrecanase 1 (ADAMTS-4) and aggrecanse 2 (ADAMTS-5)investigated by site-directed mutagenesis

• Main Author: Lim, Ngee Han
• Published: Imperial College London 2008
• Subjects: 572.76
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506803

Degradation of the proteoglycan aggrecan, one ofthe major components ofthe cartilage matrix, is thought to be a critical step in the development of osteoarthritis (OA). Both matrix metalloproteinases (MMPs) and aggrecanases [members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family have been shown to degrade aggrecan in vitro. However, there is debate as to their relative roles in vivo. The tissue inhibitor of metalloproteinases 3 (TIMP-3), which inhibits the MMPs, is the most potent tissue inhibitor of the aggrecanases, ADAMTS-4 and ADAMTS-S. The purpose of this thesis is to investigate the key elements of TIMP-3 interaction with the ADAMTS enzymes with the aim of engineering an inhibitor that would discriminate between the MMPs and the ADAMTSs, which would provide insight into the relative contributions of MMPs and ADAMTSs in aggrecan degradation in the OA disease process. Initially I describe the characterisation of a new recombinant substrate based on the interglobular domain of aggrecan, designed to enable rapid quantification of aggrecanase activity. This assay was used to determine which region of the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3) is responsible for the inhibition of ADAMTS-4 and ADAMTS-S. I determined this by expressing chimeras of N-TIMP-I which does not inhibit the aggrecanases and N-TIMP-3. I shall then describe the kinetic analysis of aggrecanase inhibition by several N-TIMP-3 mutants that have previously been shown to be poor inhibitors of MMPs, but are potent inhibitors of a related proteinase, ADAM-I7 (tumor necrosis factor a converting enzyme). These mutants were found to be good inhibitors of both ADAMTS-4 and ADAMTS-S, and effectively protected aggrecan.