Engineering of a functional γ-tocopherol transfer protein

alpha-tocopherol transfer protein (TTP) was previously reported to self-aggregate into 24-merit spheres (alpha-TTPs) and to possess transcytotic potency across mono-layers of human umbilical vein endothelial cells (HUVECs). In this work, we describe the characterisation of a functional TTP variant with its vitamer selectivity shifted towards gamma-tocopherol. The shift was obtained by introducing an alanine to leucine substitution into the substrate-binding pocket at position 156 through site directed mutagenesis. We report here the X-ray crystal structure of the gamma-tocopherol specific particle (gamma-TTPs) at 2.24 angstrom resolution. gamma-TTPs features full functionality compared to its alpha-tocopherol specific parent including self-aggregation potency and transcytotic activity in trans-well experiments using primary HUVEC cells. The impact of the A156L mutation on TTP function is quantified in vitro by measuring the affinity towards gamma-tocopherol through micro-differential scanning calorimetry and by determining its ligand-transfer activity. Finally, cell culture experiments using adherently grown HUVEC cells indicate that the protomers of gamma-TTP, in contrast to alpha-TTP, do not counteract cytokine-mediated inflammation at a transcriptional level. Our results suggest that the A156L substitution in TTP is fully functional and has the potential to pave the way for further experiments towards the understanding of alpha-tocopherol homeostasis in humans.