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Matrix-bound SFD-mutant TIMP-3 is more stable than wild-type TIMP-3
  1. Mohammed A Majid,
  2. Valerie A Smith (val.smith{at}bristol.ac.uk),
  3. Andrew C Newby,
  4. Andrew D Dick
  1. University of Bristol, United Kingdom
  2. University of Bristol, United Kingdom
  3. University of Bristol, United Kingdom
  4. University of Bristol, United Kingdom

    Abstract

    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

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