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Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes with Emphasis on Transporters

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Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes with Emphasis on Transporters Physiology and Pharmacology Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes With Emphasis on Transporters Laura Hellinen,1 Kazuki Sato,2 Mika Reinisalo,1,3 Heidi Kidron,4 Kirsi Rilla,5 Masanori Tachikawa,2 Yasuo Uchida,2 Tetsuya Terasaki,2 and Arto Urtti1,4,6 1School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland 2Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 3Institute of Clinical Medicine, Department of Ophthalmology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland 4Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 5School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland 6Laboratory of Biohybrid Technologies, Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russian Federation Correspondence: Arto Urtti, Univer- PURPOSE. Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic sity of Eastern Finland, Faculty of blood stream to the eye. RPE surface transporters can be important in ocular drug Health Sciences, School of Pharmacy, distribution, but it has been unclear whether they are expressed on the apical, basal, or both Yliopistonranta 1, P.O. Box 1627, cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral 70211 Kuopio, Finland; RPE surface proteomes. arto.urtti@uef.fi. LH and KS contributed equally to the METHODS. We separated the apical and basolateral membranes of differentiated human fetal work presented here and should RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient therefore be regarded as equivalent centrifugation. The membrane fractions were analyzed with quantitative targeted absolute authors. proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra Submitted: June 20, 2019 mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell Accepted: October 11, 2019 surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. Citation: Hellinen L, Sato K, Reinisalo M, et al. Quantitative protein expres- RESULTS. Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport sion in the human retinal pigment were expressed on the hfRPE plasma membranes. Most drug transporters showed similar epithelium: comparison between api- abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p- cal and basolateral plasma membranes glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical with emphasis on transporters. Invest enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on Ophthalmol Vis Sci. 2019;60:5022– both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 5034. https://doi.org/10.1167/ iovs.19-27328 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0þ) on the apical membrane. CONCLUSIONS. Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics. Keywords: blood–retinal barrier, retinal pigment epithelium, retinal cell culture, proteomics, transporter etinal pigment epithelium (RPE) is a polarized cell of some transporter and channel proteins: the RPE provides R monolayer located between the photoreceptors of the nutrients for the photoreceptors from the choroidal blood flow retina and choroid. The apical microvilli of the RPE face the and removes metabolic waste products and excess water from photoreceptors whereas the basal surface faces Bruch’s the subretinal space.1,2 membrane on the choroidal side. The RPE is polarized; thus, The RPE is also an important tissue in ocular pharmacoki- many of its functions have specific direction either from the netics because it serves as the outer blood–retinal barrier. Tight apical-to-basolateral (from the retina into the choroidal blood junctions between the RPE cells limit the nonspecific diffusion flow) or basolateral-to-apical (from choroid into the retina) of molecules, thereby protecting the eye from xenobiotics direction. These include important vision supporting functions present in the systemic blood flow. As the RPE cells are Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 5022 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/27/2021 Transporter Protein Localization in the RPE IOVS j December 2019 j Vol. 60 j No. 15 j 5023 damaged in ocular disorders, such as age-related macular TABLE 1. Antibodies Used in the Immunocytochemical Analysis degeneration (AMD) and diabetic retinopathy,3 the RPE itself is also an important drug target. It has been proposed that efflux Catalogue proteins on the RPE surface would serve as a functional Antibody Vendor Number Dilution component for the outer blood–retinal barrier by preventing 4 MCT1 Abcam ab90582 1:100 the ocular entry of their substrates. However, it is unclear LAT1 Cell Signaling Technology 5347S 1:50 which efflux transporters contribute and what is the extent of MDR1/P-gp Sigma Aldrich P7965-100UL 1:100 their impact. Many studies show conflicting results on the MRP1 Abcam ab3368 1:100 expression of the drug transporters, presumably due to the use 5 CD81 Abcam ab59477 1:100 of different cell models and antibody-based methods. In most MCT4 EMD Millipore AB3316P 1:100 cases, the localization of the transporters (apical or basal BEST1 Novus Biologicals NB300-164 1:50 surface) is unclear, and their transport directionality has not been investigated. In addition, the functional assays of efflux transport have been performed mostly in the RPE cell lines4,6 this study, and the schematic presentation is displayed in that may differ from the situation in vivo. Knowledge on Figure 1. The membrane separations were performed on two transporter localization on the RPE surface is important for understanding the ocular pharmacokinetics, because the separate assay days on which two individual membrane transporters may contribute to the inward (from the choroid separations were performed. This resulted in a total of four across the RPE into the eye) or outward (from the subretinal individually separated apical and basolateral plasma membrane space across the RPE into the choroid) transport depending on fractions and two whole cell lysates. The membrane fractions their localization. (n ¼ 4) were analyzed with both QTAP and SWATH, whereas Quantitative targeted absolute proteomics (QTAP) enables the whole cell lysates (n ¼ 2) were analyzed with QTAP. the quantitative assessment of protein expression. Thus, the The cells were rinsed twice with membrane-preserving expression of proteins can be compared between tissues and buffer (1 mM MgCl2 and 0.1 mM CaCl2 in PBS; Gibco BRL, cell models quantitatively, as in our previous study that Grand Island NY, USA). A nitrocellulose membrane (GE compared the transporter expression in the plasma mem- Healthcare, Chicago, IL, USA) was prewetted with sterile branes of the ARPE19 cell line and human fetal RPE (hfRPE).7 water and placed on top of the cell monolayer (Fig. 1A). However, our previous study did not provide information on Suction was used to remove excess water, and the cell plates transporter localization (apical or basolateral plasma mem- with nitrocellulose membranes were placed in the incubator brane), and the localization has remained mostly unclear in (þ378C, 5% CO2) for 5 minutes. After the incubation, the apical other earlier studies. In this paper, we quantified the membrane was peeled by lifting the nitrocellulose membrane expression of the previously studied 36 proteins in the apical from the cell monolayer (Fig. 1B). The peeling efficiency was and basolateral plasma membranes of primary RPE cells. These confirmed visually with light microscope. The apical mem- proteins include important drug transporting proteins (e.g., brane was scraped from the nitrocellulose membrane and multidrug resistance-associated proteins [MRPs], p-glycopro- collected with sterile water. The remaining cellular fraction tein [P-gp]) and other transporters that are important in RPE containing the basolateral membranes was rinsed twice with functions (e.g., GLUT1, MCTs). Because 15 of the detected PBS (Gibco BRL) and then scraped from the cell plates. The proteins were quantitated also in nonfractionated cell samples, membrane fractions were isolated with differential centrifuga- we scaled the expression of those 15 transporters to the RPE tion (Fig. 1C). The whole cells were removed with 1000g tissue level. In addition, we show the relative expression of centrifugation from both membrane fractions, and the >1300 proteins detected with SWATH-MS (sequential window supernatants were collected for further purification. The acquisition of all theoretical fragment ion spectra mass membrane fractions were purified
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