Engineering of a functional ?-tocopherol transfer protein.

ABSTRACT: ?-tocopherol transfer protein (TTP) was previously reported to self-aggregate into 24-meric spheres (?-TTP_S) 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 ?-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 ?-tocopherol specific particle (?-TTP_S) at 2.24 Å resolution. ?-TTP_S features full functionality compared to its ?-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 ?-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 ?-TTP, in contrast to ?-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 ?-tocopherol homeostasis in humans.

SUBMITTER: Aeschimann W 

PROVIDER: S-EPMC7677715 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature