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

• Main Authors: Shingo Ito (Author), Mariko Oishi (Author), Seiryo Ogata (Author), Tatsuki Uemura (Author), Pierre-Olivier Couraud (Author), Takeshi Masuda (Author), Sumio Ohtsuki (Author)
• Format: Book
• Published: MDPI AG, 2020-06-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.3390/pharmaceutics12060579; 1999-4923
• Keywords: blood-brain barrier; cell-surface biotinylation; internalization; podocalyxin; proteomics; Pharmacy and materia medica; RS1-441
• Source: Pharmaceutics, Vol 12, Iss 6, p 579 (2020)

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.