Large-Scale Quantitative Comparison of Plasma Transmembrane Proteins between Two Human Blood–Brain Barrier Model Cell Lines, hCMEC/D3 and HBMEC/ciβ

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.