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Article

Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro

1
Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
2
Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
3
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
4
Institut Cochin, Universite de Paris, Inserm U1016, CNRS UMR8104, 22 rue Méchain, 75014 Paris, France
*
Author to whom correspondence should be addressed.
Pharmaceutics 2020, 12(6), 579; https://doi.org/10.3390/pharmaceutics12060579
Received: 30 April 2020 / Revised: 15 June 2020 / Accepted: 18 June 2020 / Published: 23 June 2020
(This article belongs to the Special Issue New Drug Delivery across the Blood–Brain Barrier)
Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood–brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases. View Full-Text
Keywords: blood–brain barrier; cell-surface biotinylation; internalization; podocalyxin; proteomics blood–brain barrier; cell-surface biotinylation; internalization; podocalyxin; proteomics
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MDPI and ACS Style

Ito, S.; Oishi, M.; Ogata, S.; Uemura, T.; Couraud, P.-O.; Masuda, T.; Ohtsuki, S. Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro. Pharmaceutics 2020, 12, 579. https://doi.org/10.3390/pharmaceutics12060579

AMA Style

Ito S, Oishi M, Ogata S, Uemura T, Couraud P-O, Masuda T, Ohtsuki S. Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro. Pharmaceutics. 2020; 12(6):579. https://doi.org/10.3390/pharmaceutics12060579

Chicago/Turabian Style

Ito, Shingo, Mariko Oishi, Seiryo Ogata, Tatsuki Uemura, Pierre-Olivier Couraud, Takeshi Masuda, and Sumio Ohtsuki. 2020. "Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro" Pharmaceutics 12, no. 6: 579. https://doi.org/10.3390/pharmaceutics12060579

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