Mechanisms of ligand transfer by the hepatic tocopherol transfer protein

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.

FIGURE 1.

Sequestration of membrane-bound tocopherol by αTTP. A, time-dependent changes in FRET between NBD-tocopherol and fluorescent liposomes (25 μm in SET buffer) in the presence of 5 or 30 μm αTTP. Fluorescence of TRITC-DHPE was monitored at 575 nm upon excitation at 466 nm. Data collection was initiated upon mixing of the two samples in the stopped-flow device. B, dependence of the rate of tocopherol sequestration on αTTP concentrations. Rate constants were extracted from the time-dependent decrease in FRET as described under “Experimental Procedures.” Shown are averages and S.D. from >5 independent measurements at each protein concentration.

FIGURE 2.

Physical association of αTTP with lipid vesicles. A, purified recombinant αTTP (or glutathione S-transferase (GST); 1.5 μm) was incubated with the indicated concentration of sonicated unilamellar vesicles (SUV) for 30 min. Free protein was separated from membrane-bound protein by centrifugal filtration as described under “Experimental Procedures.” Shown is a representative of three independent experiments. B, fluorescence emission spectrum of αTTP (0.8 μm; excitation = 295 nm) was monitored in the presence or absence of 15 mm concentrations of sonicated vesicles. Fluorescence intensities were normalized at the wavelength of maximal emission and corrected for scattering. These results are representative of three experiments.

FIGURE 3.

Effect of vitamin E on the interaction between αTTP and membranes. A, filtration experiments were performed as in Fig. 2A in the presence or absence of RRR-α-tocopherol (6 mol %). Shown is a representative of three independent experiments. SUV, sonicated unilamellar vesicles. (B, DPI. Shown is the specific mass of αTTP adsorbed to immobilized phospholipid layers containing (or not) RRR-α-tocopherol (6 mol %) at different protein concentrations. C, maximum specific adsorbed mass observed with 500 nm αTTP flowed over adsorbed phospholipids containing 6 mol % of either cholesterol, RRR-α-tocopherol, or RRR-δ-tocopherol.

FIGURE 4.

Sequestration of membrane-bound tocopherol by αTTP mutants. A, location of AVED-affected residues relative to the ligand-binding pocket of αTTP. Plotted after Meier et al. ((42) Protein Data Bank code 1OIP). B, time-dependent changes in FRET between NBD-tocopherol and fluorescent liposomes in the presence of wild type (WT), R192H, E141K, H101Q, or R59W variants of αTTP (30 μm active protein). Conditions are as in Fig. 1.

FIGURE 5.

Dependence of tocopherol sequestration rates on protein concentration. Rates of αTTP-induced sequestration of NBD-tocopherol were measured as described in Fig. 1 using various concentrations of the indicatedαTTP variants. Shown are averages and S.D. from at least five independent measurements at each protein concentration.

FIGURE 6.

Association of AVED variants with membranes. Maximum specific adsorbed mass observed with 500 nm concentrations of the indicated form ofαTTP to phospholipid membranes lacking or containing 6 mol% RRR-α-tocopherol was measured using DPI as described under “Experimental Procedures.” Data are representative of at least three independent measurements. p values were calculated with unpaired t tests, and highly significant differences (p < 0.03) are denoted by an asterisk. WT, wild type.