A novel interaction of human Bestrophin1 and mesencephalic astrocyte-derived neurotrophic factor in the retinal pigment epithelium

Abstract

Purpose : Specific mutations in human Bestrophin1 (Best1) gene causes Best Vitelliform Macular Dystrophy (BVMD), as well as other ocular phenotypes, collectively termed Bestrophinopathies. Best1 expression is confined to the retinal pigment epithelium (RPE), responsible for phagocytosis of photoreceptors outer segments (POS). As a hallmark of BVMD is lipofuscin accumulation, we suggest that Best1 modulates POS phagocytosis by interacting with regulatory proteins. Here we aim to identify and further characterize proteins interacting with Best1

Methods : Human RPE cDNA library was screened with Wt Best1 using the Ras recruitment system, a cytoplasmic-based yeast two hybrid system. The identified interaction was validated by co-immunoprecipitation (co-IP) and Duolink proximity ligation assay (DPLA) kit, and tested for dependency upon 3 Best1 mutations and upon Cl^- and Ca⁺² concentrations

Results : One of the clones identified was mesencephalic astrocyte-derived neurotrophic factor (MANF), that is expressed and secreted in response to endoplasmic reticulum stress. Lysates from HEK293 cells expressing Wt Best1 and MANF were subjected to co-IP, and Best1 was detected in the precipitated immunocomplexes, confirming Wt Best1-MANF interaction in mammalian cells. The effect of Best1 mutations on Best1-MANF interaction depended upon the type of mutation: Glu300Asp and Arg218Ser mutated-Best1, associated with BVMD, failed to interact with MANF, while Val86Met mutated-Best1, associated with Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC) exhibited weaker interaction compared to Wt Best1. DPLA experiments with stable lines prepared from ARPE-19, expressing either Wt Best1, or mutated variants, confirmed the results obtained by co-IP. Known Best1 functions, as Ca⁺² regulated Cl^- channel and regulator of intracellular Ca⁺² homeostasis, were tested for effects on Best1-MANF interaction. Applying 500µM DIDS, a non-specific Cl^- channel blocker, diminished the Wt Best1-MANF interaction, but did not disrupt the weak interaction of ADVIRC mutation. Adding 100µM BAPTA-AM, a permeable Ca⁺² buffer, did not affect Wt Best1-MANF interaction, but eliminated the weak interaction of ADVIRC mutation

Conclusions : We describe a novel interaction between Best1 and MANF in RPE cells. This interaction depends on Best1 mutations and the Cl^- channel activity of Best1, but not on intracellular Ca⁺² levels

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.