Mechanisms of Ligand Transfer by the Hepatic Tocopherol Transfer Protein

Samantha Morley,Matt Cecchini,Wendy Zhang,Alessandro Virgulti,Noa Noy,Jeffrey Atkinson,Danny Manor

doi:10.1074/jbc.m800121200

Samantha Morley, Matt Cecchini + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m800121200

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Abstract

alpha-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic alpha-tocopherol transfer protein (alphaTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of alphaTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to alphaTTP increases with increasing alphaTTP concentration. This concentration dependence indicates that ligand transfer by alphaTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable alphaTTP-bilayer complex. The physical association of alphaTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in alphaTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.

Highlights

JUNE 27, 2008 VOLUME 283 NUMBER 26 vitro, ␣TTP binds tocopherol with high selectivity and affinity and catalyzes transfer of the vitamin between membrane vesicles [7,8,9]
The role of ␣TTP in regulating whole-body levels of tocopherol is underscored by the fact that mutations in the ttpA gene cause hereditary vitamin E deficiency (ataxia with vitamin E deficiency, AVED [12])
To gain further insight into the molecular mechanisms of ligand transfer, we examined the catalytic efficacy of ␣TTP mutants associated with the AVED disorder

Summary

Mechanisms of Ligand Transfer by the Hepatic Tocopherol Transfer Protein*

Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to ␣TTP increases with increasing ␣TTP concentration This concentration dependence indicates that ligand transfer by ␣TTP involves direct protein-membrane interaction. Substitution mutations such as R59W, E141K, and R221W cause an early onset, severe form of the AVED syndrome, whereas the H101Q, A120T, and R192H substitutions are associated with a later onset, milder form of the disorder [13,14,15,16,17,18,19] Because these mutations have different effects on the protein intermembrane transfer and secretion activities [20, 21], they represent useful tools with which to study the molecular mechanisms underlying ␣TTP function. Ligand was extracted, minimizing contribution from back-reactions (re-association of NBD-tocopherol with the bilayer)

EXPERIMENTAL PROCEDURES

Wild typeb

Findings

DISCUSSION