Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein

Jordan Doumanov,Christina Zeitz,Emeline Nandrot,Shomi Bhattacharya,Veselina Moskova-Doumanova,Abhay Krishna,Maria Diaz,José-Alain Sahel,Isabelle Audo,Marie-Elise Lancelot,Paloma Gimenez,Giovanna Alfano

doi:10.3390/ijms140715121

Abstract

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.

Highlights

Best vitelliform macular dystrophy (BVMD) is an autosomal dominant inherited disorder that affects central vision starting generally around teenage years [1]
The level of BEST1 transcription in MDCK cells was ~60% of the level observed in retinal pigment epithelium (RPE)-J cells (Figure 1b)
They express BEST1 mRNA, RPE cell lines do not synthesize the Best1 protein. This is true for MDCK cells, as they do not produce any Best1 protein either (Figure 1c)

Summary

Introduction

Best vitelliform macular dystrophy (BVMD) is an autosomal dominant inherited disorder that affects central vision starting generally around teenage years [1]. BVMD has been shown to manifest as a generalized retinal pigment epithelium (RPE) abnormality associated with lipofuscin accumulation, regions of geographic atrophy, and deposition of abnormal fibrillar material beneath the RPE [2]. BVMD is linked to mutations in the Bestrophin-1 gene (BEST1, formerly VMD2) located on chromosome 11 [5,6]. Mutations in BEST1 are associated with phenotypic heterogeneity and cause five clinically distinct human diseases—the classical BVMD, adult-onset vitelliform macular dystrophy (AVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), autosomal recessive bestrophinopathy (ARB) [7] and retinitis pigmentosa (RP) [8,9,10]. The protein is expressed predominantly in the RPE and localizes mostly to the basolateral plasma membrane of the cells [11]

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