Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier

Toshiki Kurosawa; Yuma Tega; Yasuo Uchida; Kei Higuchi; Hidetsugu Tabata; Takaaki Sumiyoshi; Yoshiyuki Kubo; Tetsuya Terasaki; Yoshiharu Deguchi

doi:10.3390/pharmaceutics14081683

Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier

Pharmaceutics. 2022 Aug 12;14(8):1683. doi: 10.3390/pharmaceutics14081683.

Authors

Toshiki Kurosawa¹, Yuma Tega^{1

2}, Yasuo Uchida³, Kei Higuchi⁴, Hidetsugu Tabata⁵, Takaaki Sumiyoshi⁶, Yoshiyuki Kubo¹, Tetsuya Terasaki³, Yoshiharu Deguchi¹

Affiliations

¹ Laboratory of Drug Disposition and Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University, Tokyo 173-8605, Japan.
² Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA.
³ Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.
⁴ Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan.
⁵ Laboratory of Medicinal Chemistry, Faculty of Pharma-Sciences, Teikyo University, Tokyo 173-8605, Japan.
⁶ Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Osaka 564-8680, Japan.

Abstract

A proton-coupled organic cation (H⁺/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H⁺/OC antiporter. We synthesized a novel H⁺/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H⁺/OC antiporter.

Keywords: SWATH-MS (sequential window acquisition of all theoretical-mass spectra); blood–brain barrier; photo-affinity labeling; proteomics; proton-coupled organic cation antiporter.

Abstract

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