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

Tatsuki Uemura,Sumio Ohtsuki,Mariko Oishi,Shingo Ito,Pierre-Olivier Couraud,Seiryo Ogata,Takeshi Masuda

doi:10.3390/pharmaceutics12060579

Abstract

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.

Highlights

The blood–brain barrier (BBB) constitutes the dynamic interface between the systemic circulation and the brain parenchyma
Detection of Biotinylated Proteins in hCMEC/D3 Cells and human umbilical vein endothelial cells (HUVECs) hCMEC/D3 cells were used as a model of human brain microvascular endothelial cells, and HUVECs were used as a model of peripheral microvascular endothelial cells
To internalize the biotinylated cell-surface proteins into the cells, the cell-surface biotinylated cells were treated with 20% human serum for 5 min at 37 ◦C, and the cells were washed with mercaptoethane sulfonate Na (MESNA) buffer to remove residual biotin on the cell surface (Internalization fraction)

Summary

Introduction

The blood–brain barrier (BBB) constitutes the dynamic interface between the systemic circulation and the brain parenchyma. The BBB selectively supplies macromolecules such as transferrin and insulin to the brain by receptor-mediated transcytosis (RMT), an endogenous system for macromolecule transport across the cell via receptor endocytosis, membrane trafficking, and exocytosis. Transferrin receptor (TFRC) functions as a major RMT receptor at the luminal side of the BBB [1]. An anti-human TFRC antibody supported the successful delivery of recombinant iduronate-2-sulfatase protein to the brain for the treatment of mucopolysaccharidosis II in an animal model and in humans [3,4]. Insulin receptor (INSR) and low-density lipoprotein receptor-related protein 1 (LRP1) are alternative RMT receptors at the BBB [7,8]. The delivery of biopharmaceuticals to the brain across the BBB remains a major challenge in central nervous system (CNS) drug development

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