Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium

Abstract

Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease.

Figure 1

Specificity of antibestrophin antibodies. The specificity of polyclonal antibody Pab125 (A--C) and monoclonal antibody E6-6 (D–F) was determined by immunoblot. Both antibodies recognize a band of ≈68 kDa, the predicted mass of bestrophin, in human RPE and in RPE-J cells transduced with an adenovirus vector encoding human bestrophin (AdBest) but not in untransduced RPE-J cells (A and D). To determine the crossreactivity of our antibodies with bestrophin in other species, we performed Western blots by using lysates derived from RPE or RPE/choroid preparations from various species (B, C, E, and F). Pab-125 (B) recognized an ≈68kDa band in human-, macaque-, and porcine-derived lysates, as well as an ≈45kDa band in bovine and porcine lysates that was not present in a control blot (C) using only the goat anti-rabbit secondary antibody. Monoclonal antibody E6-6 (E) recognized a single band of ≈68 kDa in human, macaque, and porcine lysates. Extraneous bands were caused by nonspecific reactivity of the secondary goat antimouse antibody (F).

Figure 2

Expression of bestrophin in RPE-derived cell lines. To examine the expression of bestrophin mRNA in RPE-derived cell lines, we probed total RNA derived from RPE-J, ARPE-19, and D407 cells by RT-PCR (A). A portion of the PCR reaction samples (1/5) was resolved on a 2% agarose gel. In PCR reactions using primer set 1, a 310-bp band is visible for all three cell lines. In PCR reactions using primer set 2, a 773-bp band is visible for ARPE-19 and D407, but not RPE-J. This finding is consistent with the lack of immunoreactivity of our antibodies with rat bestrophin, because the antigen used to generate the antibodies is a peptide derived from the C terminus of human bestrophin, which would overlap with the reverse primer in primer set 2. To examine the expression of bestrophin protein (B) in RPE-derived cell lines, we probed 250 μg each of lysates derived from RPE-J, ARPE-19, and D407 cells, and as a positive control, 25 μg of a human RPE lysate, by Western blot with monoclonal antibody E6-6 and Pab-125. Bestrophin was not detected in any of the cell lines. However, because our antibodies do not detect rat bestrophin, we cannot at this time conclude that RPE-J cells do not express bestrophin.

Figure 3

Expression of bestrophin in human ocular tissues. To examine the distribution of bestrophin in the human eye we performed Western blots (A) by using monoclonal antibody E6-6 on lysates derived from RPE, RPE/choroid (RPE/Ch), neural retina, ciliary body and iris (cil./iris), cornea, and lens. All were loaded at 400 μg per lane except RPE, which was loaded at 50 μg per lane. As shown in A, bestrophin was detected only in RPE and RPE/choroid preparations. The intensity of the bands is similar even though the protein load in the RPE/Ch lane is 8-fold greater.

Figure 4

Biochemical localization of bestrophin in human RPE. To test whether bestrophin is a membrane protein we performed Triton X-114 (Tx-114) phase extraction (A) on human RPE cells. Cells were lysed in 1% Tx-114 and bestrophin was immunoprecipitated from either the aqueous or detergent phase by using either Pab-125, E6-6, or a control primary antibody. A strong bestrophin band was present in the detergent phase, indicating that bestrophin is a membrane protein. To determine whether bestrophin is an integral plasma membrane protein, we labeled the surface of human RPE cells with sulfo-N-hydroxysuccinimide-long chain-biotin. After lysis bestrophin was immunoprecipitated with Pab-125 and as a control, the endoplasmic reticulum protein Grp78/bip was immunoprecipitated by using a rabbit anti-Grp78 antibody. Streptavidin blotting of the immunoprecipitates revealed that bestrophin had indeed been biotinylated, in contrast to Grp78, which demonstrates that the cells were intact. To ensure that Grp78 immunoprecipitation worked, we stripped and blotted the membranes with either rabbit anti-Grp78 or Pab-125. Both bestrophin and Grp78 were efficiently immunoprecipitated.

Figure 5

Immunohistochemical localization of bestrophin in macaque and porcine RPE. Macaque (I) or porcine (II) eyes fixed by immersion in 4% paraformaldehyde were stained for bestrophin by using monoclonal antibody E6-6 (A and C), or to control for specificity with monoclonal antibody E6-6 preincubated with the peptide antigen (B and D) as described in Methods. Inspection of sections at low magnification (A and B) revealed that bestrophin expression is restricted to the RPE in both macaque and porcine eyes. Inspection at higher magnification (C and D) revealed that bestrophin is concentrated along the basal surface and the lateral borders of the cells.

Figure 6

Localization of bestrophin in RPE-J cells. Confluent polarized monolayers of RPE-J cells were transduced with AdBest as described in Methods. Cells were fixed in methanol at −20°C and stained with monoclonal antibody E6-6 and goat anti-mouse IgG conjugated to FITC (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (red). Confocal microscopy was used to produce a section series with planes spaced ≈0.5 μm apart. A montage is shown (A) that depicts the average fluorescence in four adjacent images, covering a 2-μm depth of the cells. Inspection of the planes revealed staining along the basal surface and lateral borders as well as in a perinuclear compartment and intracellular vesicular structures. Analysis of average pixel intensities (B) in each plane indicated a pattern of bestrophin immunoreactivity consistent with its localization in the basolateral plasma membrane of the cells. This basolateral localization in RPE-J cells transduced with AdBest was reinforced by the greater streptavidin reactivity of bestrophin immunoprecipitates from cells biotinylated selectively on either their apical or basolateral surface (C).