Engineering of a functional γ-tocopherol transfer protein

Aeschimann, Walter; Kammer, Stephan; Staats, Stefanie; Schneider, Petra; Schneider, Gisbert; Rimbach, Gerald; Cascella, Michele; Stocker, Achim (2021). Engineering of a functional γ-tocopherol transfer protein. Redox biology, 38, p. 101773. Elsevier 10.1016/j.redox.2020.101773

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Aeschimann, Walter; Kammer, Stephan Martin and Stocker, Achim

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

2213-2317

Publisher:

Elsevier

Language:

English

Submitter:

Achim Stocker

Date Deposited:

16 Nov 2020 14:52

Last Modified:

22 Nov 2020 02:50

Publisher DOI:

10.1016/j.redox.2020.101773

BORIS DOI:

10.7892/boris.148152

URI:

https://boris.unibe.ch/id/eprint/148152

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