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
|
Text
1-s2.0-S2213231720309782-main.pdf - Published Version Available under License Creative Commons: Attribution-Noncommercial-No Derivative Works (CC-BY-NC-ND). Download (7MB) | Preview |
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 > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
UniBE Contributor: |
Aeschimann, Walter, Kammer, Stephan Martin, 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: |
05 Dec 2022 15:42 |
Publisher DOI: |
10.1016/j.redox.2020.101773 |
BORIS DOI: |
10.7892/boris.148152 |
URI: |
https://boris.unibe.ch/id/eprint/148152 |
Actions (login required)
 |
Edit item |