Abstract
A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino- and carboxyl-terminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non-ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH2-terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH2-terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby's fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby's fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3.
Highlights
The matrix metalloproteinases (MMPs)1 are a family of zincdependent endopeptidases that exist in both secreted and membrane bound forms
We have shown that the three NH2-terminal domains are sufficient for MMP inhibition, whereas the three COOH-terminal domains confer most of the unique ability of the molecule to localize to the extracellular matrix (ECM)
Wild type tissue inhibitors of metalloproteinases (TIMPs)-3 was solely localized to the ECM and was undetectable in the conditioned medium, whereas the reverse was true for ⌬tissue inhibitor of metalloproteinases-3 (TIMP-3), lacking the three COOH-terminal domains
Summary
The matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases that exist in both secreted and membrane bound forms. The activity of MMPs is tightly regulated by the tissue inhibitors of metalloproteinases (TIMPs), a family of secreted proteins currently comprising four members (TIMP-1–TIMP-4) (1– 4). Likewise TIMP-2 shows MMP independent inhibition of endothelial tube formation (15) These differences in TIMP specificities, together with the finding that TIMPs are regulated differently at the transcriptional level It has been reported that TIMP-1 and -2 consist of 2 functional domains, their NH2-terminal domains being responsible for MMP inhibition and their COOH-terminal domains performing their specific functions (23, 24) Structural studies on these molecules have shown that their NH2-terminal domains have very similar tertiary structures, both being members of the oligonucleotide/oligosaccharide binding fold family (25, 26). The aim of this study was to determine whether TIMP-3 exhibits a similar functional domain structure to that of TIMP-1 and -2 and to begin to investigate the effects of SFD mutations on TIMP-3 function
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