Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1

Abstract

BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ~174 amino acids of Best1 are sufficient for oligomerization to occur.

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

“…6 Monomers of Best1 have four transmembrane domains as well as intracellular N-and C-termini, with the latter being at the terminus of a large, cytosolic domain. [7][8][9] Monomers of Best1 oligomerize [10][11][12][13][14] to form a highly conserved homopentameric Ca 2þ -activated ion channel. 8,9 Data from heterologous systems, 15,16 primary human RPE cell culture, 17,18 mouse models, 19,20 the structurally similar paralog bestrophin-2, 21,22 as well as recently published crystal structures 8,9 all unambiguously demonstrate that mammalian Best1 is a calcium-activated anion channel.…”

“…We maintained HEK293 and MDCK II cells (American Type Culture Collection, Manassas, VA, USA) as previously described 10,11 : in a 95% air 5% CO 2 environment at 378C. Transfections were carried out as before 10,11,17 using a commercial reagent (Lipofectamine; Invitrogen, Carlsbad, CA, USA).…”

“…10,11 We synthesized the cDNA for the novel Best1 mutant synthetically (Integrated DNA Technologies, Coralville, IA, USA) and spliced into the NheI and AgeI restriction sites of pECFP-N1 (Clontech Laboratories, Inc., Mountain View, CA, USA) in-frame with CFP. To generate a replication-defective adenovirus encoding for this mutant fused to CFP, Best1…”

“…10,11,17 Immunofluorescence of filters (Transwell; Corning, Inc.) 48 hours after infection with specified adenovirus vectors was performed with the following modifications: a rabbit, polyclonal antibody (Life Technologies, Frederick, MD, USA) was used to stain ZO-1 (1:100). We stained CFP and YFP using mouse, monoclonal (JL-8; Clontech Laboratories, Inc.) or rabbit, polyclonal (Clontech Laboratories, Inc.) antibodies to GFP at a dilution of 1:1000.…”

“…10,11 For immunoprecipitation and blotting of Best1, the rabbit, polyclonal Pab-125 and the mouse, monoclonal E6-6 were used, respectively. For immunoprecipitation and blotting of cellular retinaldehyde binding protein (CRALBP), a rabbit, polyclonal antibody (Novus Biologicals, Cambridge, UK) and a mouse, monoclonal antibody (ab15051; Abcam, Cambridge, UK) were used.…”