Engineering of a functional γ-tocopherol transfer protein

• Published in: Redox biology 2021-01, Vol.38, p.101773, Article 101773
• Main Authors: Aeschimann, Walter, Kammer, Stephan, Staats, Stefanie, Schneider, Petra, Schneider, Gisbert, Rimbach, Gerald, Cascella, Michele, Stocker, Achim
• Format: Article
• Language: English
• Subjects: Antioxidant; Cytokine; Nanoparticle; Research Paper; Transcytosis; Vitamin E
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2020.101773

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