Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Large-Scale Quantitative Comparison of Plasma Transmembrane Proteins between Two Human Blood−Brain Barrier Model Cell Lines, hCMEC/D3 and HBMEC/ciβ Takeshi Masuda,†,‡,§,∥ Toshiki Hoshiyama,† Tatsuki Uemura,† Mio Hirayama-Kurogi,†,‡,§ Seiryo Ogata,† Arisu Furukawa,§ Pierre-Olivier Couraud,⊥ Tomomi Furihata,# Shingo Ito,†,‡,§,∥ and Sumio Ohtsuki*,†,‡,§,∥ Downloaded via UNIV AUTONOMA DE COAHUILA on April 16, 2019 at 08:28:18 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, ‡Department of Pharmaceutical Microbiology, Faculty of Life Sciences, and §Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan ∥ AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda, Tokyo 100-0004, Japan ⊥ Institut Cochin, Paris Descartes University, Inserm U1016, CNRS UMR8104, Paris 75014, France # Department of Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670 Japan S Supporting Information *
ABSTRACT: Transmembrane (TM) proteins localized at the plasma membrane, such as transporters and receptors, play important roles in regulating the selective permeability of the blood−brain barrier (BBB). The purpose of the present study was to clarify the differences in the expression levels of TM proteins in the plasma membrane between two established human BBB model cell lines, hCMEC/D3 and HBMEC/ciβ, in order to assist researchers in selecting the most appropriate cell line for particular purposes. We first confirmed that plasma membranes could be enriched sufficiently for a quantitative proteomics study by using the Plasma Membrane Protein Extraction Kit provided by BioVision with a modified protocol. This method was applied to hCMEC/D3 and HBMEC/ciβ cells, and fractions were used for untargeted quantitative proteomics based on sequential window acquisition of all theoretical fragment-ion spectra. In the plasma membrane fractions, 345 TM proteins were quantified, among which 135 showed significant expression differences between the two cell lines. In hCMEC/D3 cells, amino acid transporters SNAT1, SNAT2, SNAT5, ASCT1, CAT1, and LAT1; adenosine 5′-triphosphate-binding cassette transporters P-gp and MRP4; and GLUT1 were more highly expressed. The transferrin receptor expression was also 4.56-fold greater in hCMEC/ D3 cells. In contrast, HBMEC/ciβ cells expressed greater levels of IgG transporter neonatal Fc receptor, as well as tight-junction proteins PECAM1, JAM1, JAM3, and ESAM. Our results suggest that hCMEC/D3 cells have greater efflux transport, amino acid transport, and transferrin receptor-mediated uptake activities, whereas HBMEC/ciβ cells have greater IgG-transport activity and tight-junction integrity. KEYWORDS: blood−brain barrier, human brain capillary endothelial cells, plasma membrane enrichment, quantitative proteomics, transporter, tight-junction protein, receptor
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INTRODUCTION The blood−brain barrier (BBB) consists of a monolayer of endothelial cells linked by tight-junctions and serves to passively or actively regulate the transfer of compounds between the blood and brain tissues. In particular, it incorporates efflux pumps that restrict the entry of therapeutic drugs into the central nervous system (CNS), and these are major obstacles to the development of drugs targeting CNS diseases.1,2 It also contains various directional transport systems for endogenous compounds, such as glucose and amino acids, required to maintain brain functions.3,4 Because transporters, receptors, and tight-junction proteins function at the plasma membrane, it is important to know their expression © XXXX American Chemical Society
levels and functional activities at the plasma membrane of human brain microvascular endothelial cells in order to understand the characteristics of the BBB. Studies of plasma membrane proteins at the BBB usually employ cultured brain microvascular endothelial cells because large numbers of cells are necessary to prepare a plasma membrane fraction (e.g., 109 cells for preparation by sucrose density gradient centrifugation).5 hCMEC/D3 and HBMEC/ Received: January 24, 2019 Revised: March 30, 2019 Accepted: April 3, 2019
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DOI: 10.1021/acs.molpharmaceut.9b00114 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
B were from Santa Cruz Biotechnology (Dallas, TX). Anticalnexin and COX4 antibodies were obtained from Abcam (Cambridge, UK). Anti-LDHA and GM130 antibodies were from Cell Signaling Technology (Danvers, MA). Plasma Membrane Protein Extraction Kit was purchased from BioVision (Milpitas, CA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). Hydrocortisone, bFGF, lithium chloride, and protease inhibitor cocktail were from Sigma-Aldrich (St. Louis, MO). Polyvinylidene difluoride membrane was purchased from Bio-Rad Laboratories (Hercules, CA). Cell Culture. HEK293 cells were cultured to confluence in DMEM supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1% penicillin−streptomycin in an atmosphere of 95% air and 5% CO2 at 37 °C. hCMEC/D3 and HBMEC/ciβ cells were obtained from Dr. Pierre-Olivier Couraud (Institut Cochin) and Dr. Tomomi Furihata (Chiba University), respectively, and cultured in EBM-2 (Lonza, Walkersville, MD) supplemented with 5% fetal bovine serum, 1.4 μM hydrocortisone, 1 μg/mL of bFGF, 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Dojindo, Kumamoto, Japan), lithium chloride, and 1% penicillin−streptomycin in an atmosphere of 95% air and 5% CO2 at 37 or 33 °C for hCMEC/D3 or HBMEC/ciβ, respectively. hCMEC/D3 and HBMEC/ciβ cells were cultured at different temperatures because of the different immortalization methods used to generate these lines. HBMEC/ciβ cells were immortalized by the introduction of tsSV40T and hTERT genes,7 whereas SV40T gene was used for immortalization of hCMEC/D3 cells.6 It was reported that tsSV40T is activated at 33 °C7,14,15 so that HBMEC/ciβ cells can be cultured at this temperature. On the other hand, tsSV40T is inactivated at 35 °C or higher and the cellular morphology is changed.7,14,15 hCMEC/D3 (passages 32−34) and HBMEC/ ciβ (passages 60−62) cells were seeded onto culture dishes coated with collagen-I at 4000 cells/cm2 and grown for 4 and 6 days to reach confluence, respectively. For hCMEC/D3 cells, all experiments were performed in the passage range (−35th) recommended by Weksler et al.16 Plasma Membrane Enrichment. Cytosol, crude membrane, and plasma membrane fractions were prepared from 107 HEK293 cells or 5 × 106 hCMEC/D3 or HBMEC/ciβ cells by using the Plasma Membrane Protein Extraction Kit. The fractionation was performed according to the instruction manual with some modifications. Harvested cells were washed with 3 mL of cold phosphate-buffered saline (PBS) three times, suspended in 500 μL of homogenization buffer containing protease inhibitor cocktail, transferred to a cold Dounce homogenizer, and lysed on ice (50 passes). The homogenate was used as whole-cell lysate (WCL). The debris was removed by centrifugation at 700g for 10 min at 4 °C, and the supernatant was collected in a 1.5 mL tube. Debris removal was performed twice. The final supernatant was centrifuged at 10 000g for 30 min at 4 °C. The supernatant and pellet were collected as the cytosol and crude membrane fractions, respectively. The crude membrane fraction was resuspended in 200 μL of upper-phase solution, and 200 μL of lower-phase solution was added. The upper-phase and lower-phase solutions are included in the Plasma Membrane Extraction Kit. The two phases were mixed by vortexing and incubated on ice for 5 min. The upper-phase solution after centrifugation at 1000g for 5 min was collected in a new 1.5 mL tube, and this step was repeated once more. The combined upper-phase
ciβ cells are brain microvascular endothelial cell lines. hCMEC/D3 cells were established by immortalization through transduction of a human telomerase reverse transcriptase (hTERT) subunit and a simian virus 40 large T antigen (SV40T).6 Immortalization of HBMEC/ciβ cells was done by introduction of temperature-sensitive SV40T (tsSV40T) and hTERT genes.7 The cultured cells show barrier properties, having similar transendothelial electrical resistance values (5− 20 Ω cm2) and sodium fluorescein permeability values (1−3 × 10−5 cm/s).8,9 The expression of endothelial junctional markers and transporters including PECAM-1, VE-cadherin, claudin-5, GLUT1, and P-gp has been confirmed by immunofluorescence and quantitative polymerase chain reaction studies in both cell lines6,7 and by quantitative proteomics in hCMEC/D3 cells.10 Knowledge of the largescale protein expression profile in the plasma membrane of the BBB model cell lines would be helpful for researchers to select the most appropriate cell line. Indeed, we have already compared the absolute amounts of transporters and tightjunction proteins in hCMEC/D3 cells with those in human microvessels11 by using targeted proteomics approaches.10 Recent advances in liquid chromatography−mass spectrometry (LC−MS) systems and sample preparation techniques have reduced the amount of sample required to a few micrograms of peptides, and thus quantification of transmembrane (TM) proteins has become possible.10 We have developed a phase-transfer surfactant (PTS)-aided sample preparation protocol,12 in which TM proteins are quantitatively extracted and digested in the presence of PTSs.13 However, for quantifying proteins in plasma membrane by LC−MS, enrichment of the plasma membrane fraction from cells is necessary. Several protocols have been reported. For example, sucrose density gradient centrifugation, as reported by Boone et al.,5 has been applied to various cell types and tissues. However, this ultracentrifugation method needs large numbers of cells. In the present study, we first validated the subcellular fractionation of the Plasma Membrane Extraction Kit (BioVision, USA) by using western blotting. This kit employs a simple protocol and yields highly enriched plasma membrane fractions. Sufficient plasma membrane proteins for quantitative proteomics could be collected as a pellet from as few as 5 × 106 cells by using this kit with a modified protocol. We employed the modified method to compare the quantitative expression levels of TM proteins in plasma membrane fractions of hCMEC/D3 and HBMEC/ciβ cells, using untargeted proteomics combined with the PTS protocol, with which it is possible to identify the proteins localized in the plasma membrane of the BBB model cell lines on a large scale. These data suggest that hCMEC/D3 cells have greater expression of efflux transporters and amino acid transporters, whereas HBMEC/ciβ cells have greater expression of tight-junction proteins.
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MATERIALS AND METHODS Materials. Dulbecco’s modified Eagle’s medium (DMEM), penicillin−streptomycin, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, sodium deoxycholate (SDC), sodium lauroyl sarcosinate (SLS), dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), ethyl acetate, and lysyl endopeptidase were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Antibodies against Na+/K+ ATPase and lamin B
DOI: 10.1021/acs.molpharmaceut.9b00114 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
were filtered at FDR less than 1% for identification and quantification. Targeted peptide peaks were extracted from the SWATH data by PeakView Software version 2.1 (Sciex) using the identified protein data from ProteinPilot, and the sum of the area values of specific peptide peaks from each protein was calculated as the protein expression level. A heat map was generated by using Morpheus (https://software.broadinstitute. org/morpheus/). DAVID version 6.7 (https://david.ncifcrf. gov/summary.jsp) was used for the gene ontology (GO) enrichment analysis. Statistical Analysis. Hierarchical clustering with one minus Pearson correlation was performed in Morpheus. Two-tailed Student’s t-test was performed by using Excel (Microsoft, WA) or GraphPad Prism 6 (GraphPad Software, CA). For proteomics data, statistical significance was considered for fold change >2 or
