Background / aims: Sorsby's fundus dystrophy is a degenerative retinopathy that is characterised by the accumulation of mutant TIMP-3 protein in Bruch's membrane and apart from initiating earlier in life, is clinically similar to ARMD. The aim of this project was to compare the stability of matrix-bound SFD-mutant TIMP-3s with wild-type TIMP-3.
Methods: COS-7 cells were transfected with plasmids containing wild-type, Ser 181, Gly-167, Ser-156 and Tyr-168 TIMP-3 cDNA. The cells and their matrices were subsequently harvested and homogenised. After measuring the bound wild-type and SFD-mutant TIMP-3 concentrations by ELISA, aliquots of the homogenates were heated to 100oC. The rates of denaturation of the TIMP proteins at this temperature were monitored by reverse zymography.
Results: Over a period of 24hr at 100oC the biological activity of both wild-type and SFD mutant TIMP-3 was lost. Over a period of 6hr at this temperature the biological activity of the SFD-mutant TIMP-3s was fully retained whereas that of the wild-type TIMP-3 was lost.
Conclusion: Matrix-bound SFD-mutant TIMP-3s are thermodynamically more stable than wild-type. This may explain why SFD initiates earlier in life than ARMD.
- Age Related macular degeneration (ARMD)
- Bruch's membrane
- Sorsby's fundus dystrophy (SFD)
- TIMP-3 gene transfer