Mouse models targeted for the tissue inhibitor of metalloproteinases-3 (TIMP3) - molecular and functional dissection of Sorsby fundus dystrophy (SFD)

Sorsby fundus dystrophy (SFD) is an autosomal dominant disorder of the central retina (Sorsby et al., 1949). It is characterized by subretinal neovascularization, atrophy of the retinal pigment epithelium and the choriocapillaris. It is a progressive disorder leading ultimately to blindness. Mutations in the TIMP3 molecule have been linked to SFD (Weber et al., 1994). So far, several point mutations have been identified in the TIMP3 molecule, all localized at the C-terminal domain of the inhibitor, the majority of which result in a single unpaired cysteine residue in the mature protein. TIMP3 belongs to a family of secreted proteins comprising four members (TIMP1, -2, -3, -4). TIMP3 is ECM (extracellular matrix) bound inhibitor with a molecular mass of 24 kDa having 12 conserved Cys residues consisting of two domains which fold independently with 3 disulfide bonds in each domain. TIMP3 is a multifunctional protein involved in the inhibition of matrix metalloproteinases (MMPs) (Murphy et. al., 1994). It can block the TACE (tumour necrosis factor (TNF)-alpha converting enzyme) (or ADAM17 - a disintegrin and metalloproteinase) mediated ectodomain shedding of pro-TNF- (Amour et al., 1998), Syndecan-1 and Syndecan-4 (Fitzgerald et al., 2000), L-selectin (Borland et al., 1999), and interleukin-6 receptor (Hargreaves et al., 1998). TIMP3 is also involved in the inhibition of the ADAMTSs (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif) especially ADAMTS4, ADAMTS5 (Kashiwagi et al., 2001) and ADAMTS2 (Wang et al., 2006). TIMP3 also blocks angiogenesis by binding to VEGFR2 (vascular endothelial growth factor receptor 2) (Qi et al., 2003) and also interacts with EFEMP1 (epithelial growth factor-containing fibulin-like extracellular matrix protein 1) (Klenotic et al., 2004). TIMP3 is unique among the TIMPs in that it can inhibit TACE mediated TNF- shedding. We have measured TACE activity in liver extracts from mice deficient for the TIMP3 gene using cleavage of a fluorogenic substrate and found increased TACE activity. We were also interested to analyse the influence of the SFD mutation on the inhibition of TACE activity. The S156C mutation in the C-terminal domain of TIMP3 does not lead to significant modification of the inhibitory activity towards TACE as measured in this assay. Another biological function of TIMP3 is its ability to block ADAMTS4/ADAMTS5 (aggrecanase-1, aggrecanase-2) and MMPs mediated cleavage of aggrecan (Kashiwagi et al., 2001). TIMP3 influence on Aggrecan cleavage can be monitored by so-called neoepitope antibodies – which recognize the newly created epitope after cleavage of the aggrecan by ADAMTS4/ADAMTS5 or MMPs. In a cell-based ELISA using chondrocytes it was found that cleavage activity of ADAMTS4/ADAMTS5 and MMPs towards aggrecan are not affected either in TIMP3 knock-out (KO) chondrocytes or S156C-TIMP3 knock-in (KI) chondrocytes compared to WT counterparts. It has been also demonstrated that TIMP3 is able to block angiogenesis by competing with VEGF for binding to VEGFR2 (Qi et al., 2003). We have also used recombinant proteins to analyse the antiagiogenic property of TIMP3 via its known function of competing with VEGF ligand for its receptor (VEGFR2). The results demonstrate that the S156C mutation in the TIMP3 molecule does not have a significant effect on its capacity to bind VEGFR2 compared to WT recombinant protein. As the pathomechanism of SFD is still unknown it seemed reasonable to search for new interacting partners which might help gain insight into the molecular basis of the disease. In this context, the ability of TIMP3 to bind several different types of collagens was tested. In an ELISA, recombinant TIMP3 was able to bind to five different types of collagens in a concentration dependence manner. In parallel, TIMP3 interacted with BSA and another unrelated protein (TTR), indicating that the interactions are apparently unspecific. These interactions are mediated by the C-terminal domain of TIMP3. Cysteine residues are required for the formation of disulfide bonds which stabilize the folded conformation of proteins (Raina et. al., 1997). Therefore, a free cysteine residue in the TIMP3 molecule could potentially disturb folding of the protein. To analyse this hypothesis we have performed western blotting and detected higher molecular weight complexes caused by intermolecular disulfide bonds. As fibroblasts from the S156C KI mouse were available we quantified the amount of mutant TIMP3 in heterozygous and homozygous immortalized cell lines. ELISA results showed increased amounts of the mutant protein compared to the WT in both the ECM and cells. We next tested if the accumulation of mutant protein is due to increased resistance of the misfolded protein to proteolytic degradation. TIMP3 turnover in the ECM, determined by ELISA, revealed that S156C-TIMP3 mutant does indeed have a slower turnover compared to the WT. These results indicate that the pathomechanism of SFD is not due to a loss-of-function, but rather a toxic gain-of-function caused by accumulation of misfolded mutant TIMP3 in the ECM. Therefore, it is reasonable to conclude that the oligomeric form of TIMP3 is the major cause of the SFD disorder. This would classify SFD in the category of conformational diseases specific for the neurodegenerative disorders.