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

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 carboxylterminal 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 NH 2 -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 NH 2 -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.The matrix metalloproteinases (MMPs) 1 are a family of zincdependent endopeptidases that exist in both secreted and membrane bound forms. The enzymes are initially expressed as inactive pro-enzymes becoming activated by proteolytic cleavage of their amino termini. 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). The balance between MMPs and TIMPs regulates the integrity of the proteinacious extracellular matrix (ECM) and thus plays a key role in a wide range of physiological processes that include embryonic development, connective tissue remodeling, wound healing, glandular morphogenesis, and angiogenesis. An imbalance in MMP/ TIMP expression has been implicated in various diseases such as erosive joint disease, cardiovascular disease, and cancer (reviewed in Refs. 5-7).The TIMPs form high affinity 1:1 complexes with the active forms of most MMPs (reviewed in Ref. 8) but show varying specificity for different pro-MMPs allowing TIMPs to control the activation of specific MMPs (9 -12). Activities have also been ascribed to the TIMPs that are independent of their ability to inhibit MMPs; for example, anti-angiogenic and erythroid-potentiating activities have been described for TIMP-1 that are independent of MMP inhibition (13,14). Likewise TIMP-2 shows MMP independent inhibition of endothelial tube formation (15). These differences in TIMP ...

S156C Mutation in Tissue Inhibitor of Metalloproteinases-3 Induces Increased Angiogenesis

“…The discrepancy between previous reports and ours is most likely because of differences in cell type examined. Reduced binding of mutant TIMP-3 to the ECM could be a result of an excessive glycosylation, which can potentially decrease its apparent affinity for the ECM, presumably by masking basic residues (16). Excessive glycosylation might also lead to a reduction of MMP inhibitory activity of mutant TIMP-3, presumably by a conformation change or reduced ECM binding (48).…”

“…TIMP-3 protein is produced constitutively by the retina pigment epithelium (RPE) and choroidal endothelial cells (9,10) in the eye and is a component of the normal Bruch membrane (11). Sorsby fundus dystrophy (SFD) (12), a dominantly inherited, degenerative disease of the macula, is caused by specific mutations in the TIMP-3 gene (13)(14)(15)(16)(17)(18)(19)(20), most of which introduce an unpaired cysteine at the C terminus of the protein. SFD is of considerable interest as it is the only genetic disorder in which choroidal neovascularization occurs in the majority of affected patients (21)(22)(23).…”

SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis

“…ADAM10 has been shown to be inhibited by tissue inhibitor of metalloproteases (TIMPs) 24 through the activity of their NTR module 24,25 . Because Sfrp1/2 contain an NTR module 8 , we postulated that Sfrp1/2 may normally down-regulate the activity of ADAM10.…”

Promoter Hypermethylation of Multiple Genes in Hydatidiform Mole and Choriocarcinoma

“…Interestingly, both TFPI-2 and TIMP3 effectively decreased the activation of metalloproteinases, thus inhibiting degradation of the extracellular matrix and reducing the invasive potential of tumor cells. 15,33 In addition, TIMP3 also has some unique properties in human cancer development, such as growth suppression, 34 inhibition of angiogenesis, 35,36 and induction of apoptosis. 37,38 Transcriptional inactivation of TIMP3 by hypermethylation has been found to associated with a more malignant and invasive phenotype in some human cancer cells.…”