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Localization of the Functional Domains of Human Tissue Inhibitor of Metalloproteinases-3 and the Effects of a Sorsby's Fundus Dystrophy Mutation \ud

By K.P. Langton, M.D. Barker and N. McKie

Abstract

A transient COS-7 cell expression system was used to\ud investigate the functional domain arrangement of tissue\ud inhibitor of metalloproteinases-3 (TIMP-3), specifically\ud to assess the contribution of the amino- and carboxylterminal\ud domains of the molecule to its matrix metalloproteinase\ud (MMP) inhibitory and extracellular matrix\ud (ECM) binding properties. Wild type TIMP-3 was entirely\ud localized to the ECM in both its glycosylated (27\ud kDa) and unglycosylated (24 kDa) forms. A COOH-terminally\ud truncated TIMP-3 molecule was found to be a non-\ud ECM bound MMP inhibitor, whereas a chimeric TIMP\ud molecule, consisting of the NH2-terminal domain of\ud TIMP-2 fused to the COOH-terminal domain of TIMP-3,\ud displayed ECM binding, albeit with a lower affinity than\ud the wild type TIMP-3 molecule. Thus the functional domain\ud arrangement of TIMP-3 is analogous to that seen in\ud TIMP-1 and -2, namely that the NH2-terminal domain is\ud responsible for MMP inhibition whereas the COOH-terminal\ud domain is most important in mediating the specific\ud functions of the molecule. A mutant TIMP-3 in\ud which serine 181 was changed to a cysteine, found in\ud Sorsby’s fundus dystrophy, a hereditary macular degenerative\ud disease, was also expressed in COS-7 cells. This\ud gave rise to an additional 48-kDa species (possibly a\ud TIMP-3 dimer) that retained its ability to inhibit MMPs\ud and localize to the ECM. These data favor the hypothesis\ud that the TIMP-3 mutations seen in Sorsby’s fundus dystrophy\ud contribute to disease progression by accumulation\ud of mutant protein rather than by the loss of functional\ud TIMP-3.\u

Year: 1998
OAI identifier: oai:eprints.whiterose.ac.uk:440

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