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Title: Design of a TIMP-3 mutant with an increased half-life for improved protection against cartilage breakdown
Author: Doherty, Christine
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Osteoarthritis (OA) is a degenerative joint disease for which there are currently no disease-modifying drugs. OA is characterised by degradation of cartilage extracellular matrix (ECM) components by catabolic metalloproteinases. Tissue inhibitor of metalloproteinase 3 (TIMP-3) is the endogenous inhibitor of these enzymes, and thus a target for the development of novel OA therapies. Extracellular levels of TIMP-3 are regulated by endocytosis via the scavenger receptor LDL receptor-related protein-1 (LRP-1), reducing its in vivo half-life and chondroprotective activity. The aim of this study was to generate a TIMP-3 mutant resistant to LRP-1 binding but with unaltered affinity for target metalloproteinases and ECM glycosaminoglycans, since we hypothesised such a mutant would have increased chondroprotective activity. LRP-1 ligand-binding motifs have been proposed to consist of a pair of surface lysine residues separated by 21Å. Using in silico molecular modeling of a proposed TIMP-3 structure, we identified 10 pairs of lysine residues potentially satisfying this motif. Site-directed mutagenesis of all of these lysine pairs to alanine generated TIMP-3 mutants with resistance to LRP-1-mediated endocytosis, localising the LRP-1-binding residues to an extended basic patch on the TIMP-3 surface. TIMP-3 K26A/K45A and TIMP-3 K42A/K110A were further analysed. TIMP-3 K42A/K110A had unaltered affinity for target metalloproteinases, but markedly reduced affinity for ECM glycosaminoglycans. It displayed improved chondroprotection over 24 hours, but was no more effective than Wild-type TIMP-3 over longer time periods, possibly due to its inability to be retained on the ECM. TIMP-3 K26A/K45A, on the other hand, retained high affinity for both target metalloproteinases and ECM glycosaminoglycans, and was found to have a longer half-life in cartilage and improved chondroprotective activity relative to Wild-type TIMP-3. This study thus identified an LRP-1-resistant mutant of TIMP-3 with an increased half-life and protective activity in cartilage, and showed that such behaviour was dependent on maintenance of interaction with the ECM. A similar paradigm may apply to other ECM-associated LRP-1 ligands.
Supervisor: Troeberg, Linda ; Nagase, Hideaki Sponsor: Kennedy Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available