Abstract
Purpose :
An increasing number of studies identify genetic mutations encoding amino acid changes in proteins expressed by the retinal pigment epithelium (RPE) leading to increased risk of developing age-related macular degeneration (AMD). Approximately half of the overall AMD risk is now attributed to genetic variations, but the functional effects of these mutations are largely unexplored. This study demonstrates an efficient and versatile framework for intracellular interactome mapping of AMD-linked protein variants in RPE tissue using the wet AMD-associated A25T cystatin C as a model protein.
Methods :
Cystatin C protein coding sequence subcloned into the pHTC HaloTag® CMV-neo vector, encoding a C-terminally tagged cystatin C Halotag fusion protein, was used to generate a construct encoding the AMD-linked variant B cystatin C Halotag fusion by site-directed mutagenesis. Transient transfection of ARPE19 cells was achieved through Neon electroporation (n=3 for each construct), followed by cell lysis in mild detergent buffer and selective Halo-protein immobilisation on HaloLink sepharose resin. Interacting proteins were eluted in mass spectrometry compatible Rapigest SF buffer and analysed by nanoLC-ESI-MS/MS following trypsin digestion, as well as immunoblotting in independent samples for validation.
Results :
We identified a total of 28 proteins interacting with at least one of the WT or AMD-linked variant B cystatin C in RPE cells. The majority of interacting proteins, including all high-abundance proteins eluted, was identical for both WT and variant B cystatin C and dominated by cysteine proteases from the cathepsin family of proteins, indicating that the mutated processed protein retains its native protease-inhibiting function. Interestingly, 8 other proteins were exclusively pulled down by variant B cystatin C, indicating previously unknown gain of functions of the mutation.
Conclusions :
We demonstrate novel interaction partners exclusively binding the AMD-associated variant B form of cystatin C. This type of RPE interactome analysis can be scaled up and serve as a high throughput and cost-effective strategy for identifying proteins or cellular pathways affected by AMD-associated protein coding mutations. In extension, it may serve as a tool for testing novel therapeutic approaches for altering the impacts of protein variants with degenerative effects on the RPE.
This is a 2020 ARVO Annual Meeting abstract.