TY - JOUR
T1 - Quantitative protein expression in the human retinal pigment epithelium
T2 - Comparison between apical and basolateral plasma membranes with emphasis on transporters
AU - Hellinen, Laura
AU - Sato, Kazuki
AU - Reinisalo, Mika
AU - Kidron, Heidi
AU - Rilla, Kirsi
AU - Tachikawa, Masanori
AU - Uchida, Yasuo
AU - Terasaki, Tetsuya
AU - Urtti, Arto
N1 - Funding Information:
The authors thank laboratory technician Lea Pirskanen for valuable help in the hfRPE cell culture and fractionation and Anna Poutiainen, MSc, for important contributions in the membrane separation method development. Supported by funding from the Academy of Finland (311122) and Doctoral Programme in Drug Research (University of Eastern Finland). AU acknowledges support from Government of Russian Federation Mega-Grant 14.W03.031.0025 ‘‘Biohybrid technologies for modern biomedicine.’’ This study was also supported in part by a Grant-in-Aid from the Japanese Society for the Promotion of Science for Bilateral Open Partnership Joint Research Program (between Finland and Japan).
Publisher Copyright:
© 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783
PY - 2019/12/1
Y1 - 2019/12/1
N2 - PURPOSE: Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. METHODS: We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. RESULTS: Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0+) 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.
AB - PURPOSE: Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. METHODS: We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. RESULTS: Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0+) 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.
KW - Blood-retinal barrier
KW - Proteomics
KW - Retinal cell culture
KW - Retinal pigment epithelium
KW - Transporter
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U2 - 10.1167/iovs.19-27328
DO - 10.1167/iovs.19-27328
M3 - Article
C2 - 31791063
AN - SCOPUS:85075940451
VL - 60
SP - 5022
EP - 5034
JO - Investigative Ophthalmology
JF - Investigative Ophthalmology
SN - 0146-0404
IS - 15
ER -