Abstract
Purpose :
Sorsby Fundus Dystrophy (SFD) is a macular dystrophy, caused by mutations in tissue inhibitor of metalloproteinases 3 (TIMP-3). Mutant TIMP-3 accumulates extracellularly in the macula of SFD patients through an unknown mechanism. We investigated whether low-density lipoprotein receptor-related protein 1 (LRP1) is responsible for endocytosis of TIMP-3 in retinal pigment epithelial (RPE) cells, and whether SFD mutations delay TIMP-3 endocytosis.
Methods :
Wild-type (WT) and SFD TIMP-3 mutants (Tyr191Cys and Ser204Cys) were expressed in HEK293 cells and purified. WT or mutant TIMP-3 was added to ARPE19 cells and their rate of disappearance from the medium over 24 hours was monitored by western blotting. The role of LRP family receptors in endocytosis was investigated using receptor associated protein (RAP) treatment and LRP1-null mouse embryonic fibroblasts (MEFs). Expression of LRP family members in ARPE19 cells was investigated by qPCR. One-way ANOVA was used for statistical analysis.
Results :
WT TIMP-3 (N=6) was taken up readily by ARPE19 cells with a half-life of 2.6±1.1 hours, with only 27±10% of the added TIMP-3 remaining in the conditioned medium after 6 hours. Significantly more Tyr191Cys (N=5) and Ser204Cys (N=6) TIMP-3 remained in the medium after 6 hours (88±19% and 80±26%, respectively), to the extent that calculation of half-lives was not possible. Uptake of WT and SFD mutant TIMP-3 by ARPE19 cells was significantly inhibited by RAP treatment. Uptake of WT and mutant TIMP-3 was also higher in WT MEFs than LRP1-null MEFs (p=0.04 for WT; p=0.0004 for Ser204Cys; p=0.07 for Tyr191Cys). LRP2, 4, 5, 6, 8, 10, 11, 12 and the LDL and VLDL receptors were expressed in ARPE19 cells.
Conclusions :
WT TIMP-3 was endocytosed rapidly by RPE cells, primarily via the endocytic scavenger receptor LRP1, with a minor contribution from other LRP family members. The SFD mutants Tyr191Cys and Ser204Cys were endocytosed more slowly by RPE cells, providing a potential molecular mechanism for extracellular accumulation of these proteins in SFD. Endocytosis of the mutants was effectively inhibited by RAP, but only partially reduced in LRP1-null cells, indicating the involvement of LRP1 and other LRP family members in their uptake. Several LRPs were found to be expressed in RPE cells and could potentially mediate this slower endocytic rate.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.