A Study of Lipid-Protein Binding Assays With α-Tocopherol Transfer Protein Using Surface Plasmon Resonance

Abstract

Vitamin E is an essential micronutrient required both in early embryonic development, as well as into adulthood for proper neurological function and to prevent cell damage due to oxidative stress. Of its eight naturally occurring forms, the RRR-α-tocopherol isomer is selectively retained and used as an antioxidant in mammalian tissues. The α-tocopherol transfer protein (α-TTP) localized in the liver is responsible for the uptake and transport of RRR-α-tocopherol to the plasma membrane of hepatocytes to be secreted and delivered to target tissues throughout the body via systemic circulation. Despite knowing the bioactive role of vitamin E for centuries, the complete cyclic transport of α-tocopherol within hepatocytes remains unknown. This study investigates the binding interactions between recombinantly expressed wildtype α-TTP and unilamellar plasma membrane lipid vesicles containing various phosphatidylinositol phosphates to elucidate the mechanism of binding between α-TTP and its proposed secondary ligand – PI(4,5)P2. Using surface plasmon resonance binding assays, wildtype α-TTP alone or preincubated with α-tocopherol was injected onto lipid vesicles containing either PI(4,5)P2, PI(3,4)P2, or PI(3)P. Based on association rate constants (kon, s-1), wtTTP preferentially bound to lipid vesicles containing 4% PI(3,4)P2 (0.01896 s-1) and 4% PI(4,5)P2 (0.01174 s-1) instead of 4% PI(3)P (0.00750 s-1). In addition, concentration-dependence was observed for the rate of binding to lipid vesicles containing 2%, 4%, or 6% PI(4,5)P2. All binding assays with wtTTP preincubated with α-tocopherol produced rate constants almost three times greater than their non-preincubated counterparts. However, while wtTTP preincubated with α-tocopherol still associated fastest to lipid vesicles containing PI(3,4)P2, there was no correlation between rate of binding and increased PI(4,5)P2 concentrations. The results found in this study provide further insight into the mode of α-tocopherol transport and ligand exchange facilitated by α-TTP in hepatocytes.