Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby's fundus dystrophy mutation.
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.
Langton
K P
KP
Krebs Institute for Biomolecular Research, Department of Pathology, University of Sheffield, Medical School, Sheffield S10 2RX, United Kingdom.
Barker
M D
MD
McKie
N
N
D000818:Animals; D019556:COS Cells; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics*; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D019716:Tissue Inhibitor of Metalloproteinase-2