Matrix bound SFD mutant TIMP-3 is more stable than wild type TIMP-3.

Abstract

Sorsby's fundus dystrophy (SFD) is a degenerative retinopathy characterised by accumulation of mutant TIMP-3 protein in Bruch's membrane. To compare the stability of matrix bound SFD mutant TIMP-3s with wild type TIMP-3. 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 100 degrees C. The rates of denaturation of the TIMP proteins at this temperature were monitored by reverse zymography. Over a period of 24 h at 100 degrees C the biological activity of both wild type and SFD mutant TIMP-3 was lost. Over a period of 6 h 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. Matrix bound SFD mutant TIMP-3s are thermodynamically more stable than wild type. This may explain why SFD starts earlier in life than age related macular degeneration.