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Originally published In Press as doi:10.1074/jbc.M800121200 on May 5, 2008

J. Biol. Chem., Vol. 283, Issue 26, 17797-17804, June 27, 2008
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Mechanisms of Ligand Transfer by the Hepatic Tocopherol Transfer Protein*

Samantha Morley{ddagger}, Matt Cecchini§, Wendy Zhang§, Alessandro Virgulti§, Noa Noy, Jeffrey Atkinson§, and Danny Manor1

From the {ddagger}Cornell University, Ithaca, New York, 14853, §Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada, and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received for publication, January 7, 2008 , and in revised form, May 2, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{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 ({alpha}TTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of {alpha}TTP 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 {alpha}TTP increases with increasing {alpha}TTP concentration. This concentration dependence indicates that ligand transfer by {alpha}TTP 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 {alpha}TTP-bilayer complex. The physical association of {alpha}TTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in {alpha}TTP 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin E is the major lipid-soluble antioxidant in numerous species. By virtue of its radical-trapping activity, vitamin E is thought to alleviate oxidative damage in cells, and thus, to prevent various pathologies related to oxidative stress. Vertebrates selectively accumulate only one form of vitamin E from dietary mixtures, namely RRR-{alpha}-tocopherol (1, 2). This preferential retention is achieved through degradation of other forms of vitamin E (e.g. Refs. 3 and 4) and the selective, high affinity binding of RRR-{alpha}-tocopherol (herein abbreviated tocopherol) by the hepatic {alpha}-tocopherol transfer protein ({alpha}TTP)2 (5, 6). In vitro, {alpha}TTP binds tocopherol with high selectivity and affinity and catalyzes transfer of the vitamin between membrane vesicles (7–9). In cultured hepatocytes, expression of {alpha}TTP enhances secretion of vitamin E to the culture media (10, 11). It is generally believed that in vivo {alpha}TTP is critical for the incorporation of dietary RRR-{alpha}-tocopherol into circulating lipoproteins, which deliver the vitamin to target cells.

The role of {alpha}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)). AVED patients present progressive neurodegeneration and low plasma tocopherol levels. Multiple mutations in the ttpA gene were identified in AVED patients, which are thought to impair the cellular activities of the protein. 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 (13–19). 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 {alpha}TTP function.

The molecular mechanisms that underlie intermembrane tocopherol transfer by {alpha}TTP are not known. Sec14p, a related protein from the CRAL-TRIO family, was proposed to facilitate lipid transfer by interacting directly with membranes and actively "extracting" the ligand from the bilayer (22). However, no experimental evidence was put forward in support of this model. Other lipid transfer proteins are thought to employ multiple mechanisms to facilitate ligand transfer, such as bringing donor and acceptor particles to close proximity in the case of phospholipid transfer protein (23, 24), physical association with the bilayer in the case of adipocyte and heart fatty acid-binding proteins (25), or by binding lipids that have diffused from membranes into the aqueous milieu, such as in the case of the liver fatty acid-binding protein (26). We report on our investigations into the mechanism by which {alpha}TTP facilitates the intermembrane transfer of tocopherol. Specifically, we address the role of physical association between the protein and the membrane during the ligand transfer reaction.


Figure 1
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  		FIGURE 1.
Sequestration of membrane-bound tocopherol by {alpha}TTP. 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 {alpha}TTP. 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 {alpha}TTP 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.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Details of the synthesis and fluorescent characteristics of NBD-tocopherol were previously reported (11, 27, 43). Wild-type and mutant {alpha}TTP proteins were expressed and purified as previously described (20, 27). Protein expression was induced with 0.25 mM isopropyl-β-D-thiogalactopyranoside overnight at 25 °C. All proteins were stored in 20 mM Tris, pH 8.0, 150 mM NaCl, 50% glycerol at –20 °C.

The affinity of {alpha}TTP for NBD-tocopherol was determined by fluorescence titrations as previously described (27). Briefly, 0.1–1 µM {alpha}TTP was titrated with increasing concentrations of the fluorescent ligand NBD-tocopherol, and NBD fluorescence was monitored at 526 nm (excitation = 466 nm) with a Quanta-Master 4 fluorimeter (Photon Technologies International).

Sonicated unilamellar vesicles were prepared from egg yolk phosphatidylcholine, NBD-tocopherol, TRITC-DHPE (Molecular Probes), and butylated hydroxytoluene (molar ratio 98.2: 0.8:0.5:0.5) by sonication in SET buffer (0.25 M sucrose, 1 mM EDTA, 50 mM Tris, pH 8.0) and used at 25 µM in a stopped-flow apparatus (TGK Scientific). Tocopherol-containing lipid vesicles were prepared from egg yolk phosphatidylcholine, RRR-{alpha}-tocopherol, and butylated hydroxytoluene (molar ratio 93.5:6: 0.5) by sonication in SET buffer. Sonicated vesicles were centrifuged at 100,000 x g at 4 °C for 1 h, and the resultant supernatant was purged with argon and stored at 4 °C in the dark for up to 2 weeks. Large unilamellar vesicles for dual polarization interferometry were prepared by 13 repeated extrusion steps through a 100-nm polycarbonate filter (LipoFast, Avestin, Ottawa, CA) in 10 mM potassium phosphate, 137 mM sodium chloride, pH 7.4.

Movement of NBD-tocopherol from membranes into the TTP binding pocket was examined by monitoring the fluorescence resonance energy transfer (FRET) between NBD-tocopherol and the fluorescent lipid TRITC-DHPE as described earlier (27). Briefly, small unilamellar vesicles containing NBD-tocopherol and TRITC-DHPE were prepared as described above and mixed with purified recombinant {alpha}TTP. Release of FRET between NBD-tocopherol and the fluorescent lipid was measured by monitoring the time-dependent change in TRITC-DHPE fluorescence (excitation = 466 nm, emission = 575 nm). Raw fluorescence data were fitted to the sum of a single exponential process and a linear term (representing chromophore bleaching), and the obtained pseudo first-order half-life values are reported. FRET experiments were done at protein concentrations > 5 µM, where more than 95% of the ligand was extracted, thus minimizing contribution from back-reactions (re-association of NBD-tocopherol with the bilayer).

To evaluate the association of {alpha}TTP with membranes, the protein was incubated with sonicated vesicles for 30 min at room temperature and filtered through a centrifugal concentrator (Micro-con YM-100, Millipore, 11,000 x g for 30 min). Lipid vesicles and associated protein were retained above the filter, whereas buffer and free protein flow through the filter to the lower chamber (28). Vesicle-associated protein was recovered with SET buffer supplemented with 150 µM Triton X-100. Samples were resolved on SDS-PAGE, visualized by Coomassie staining, and quantitated by densitometry.

Protein-membrane interactions were also measured using dual polarization interferometry (DPI) on an Analight Bio 200 with an unmodified sensor chip (Farfield Scientific Ltd.). Extruded vesicles were deposited onto the surface of the sensor chip at a flow rate of 25 µl/min for 8 min. The deposited phospholipids were allowed to equilibrate under flow conditions until a stable layer formed with a final thickness 3–10 nm before protein was injected. The association of {alpha}TTP to adsorbed phospholipids in the first 450 s was fit to a one-site exponential equation, which yielded the maximum specific mass of bound protein adsorbed at a given concentration of {alpha}TTP. After each injection of protein the sensor chip was regenerated with 80% ethanol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The defining biochemical activity of {alpha}TTP is catalysis of tocopherol transfer between lipid bilayers (7, 8, 29). To understand the molecular mechanisms by which the protein exerts this activity, we examined the first step of the transfer reaction, namely, transfer of tocopherol from lipid bilayers into the TTP binding pocket. Two possible mechanisms could be envisioned to underlie ligand transfer process. One involves physical association of {alpha}TTP with the lipid bilayer followed by extraction of tocopherol into the protein binding pocket (Reaction 1).

Formula 1REACTION 1
Alternatively, the protein may facilitate transfer by binding the vitamin following its passive diffusion from membranes, thereby shifting the equilibrium partitioning of tocopherol toward the aqueous milieu (Reaction 2),

Formula 2REACTION 2
To distinguish between these two mechanisms, we examined the rate of ligand sequestration from its FRET partner in the presence of different {alpha}TTP concentrations. If {alpha}TTP directly interacts with the bilayer (Reaction 1), the rate of ligand sequestration is expected to depend on protein concentration. On the other hand, if the protein binds tocopherol in the aqueous buffer (Reaction 2), the reaction rate will be limited by the rate of spontaneous ligand dissociation from the membrane. In this case the rate of tocopherol extraction will be independent of protein concentration. We utilized a FRET assay that we recently developed to monitor ligand transfer. In this system FRET occurs between NBD-tocopherol and a fluorescently labeled lipid, TRITC-DHPE, when both are present in the same bilayer (27). Sequestration of NBD-tocopherol by {alpha}TTP results in loss of FRET between the lipid and NBD-tocopherol, which is readily detected fluorimetrically (see "Experimental Procedures").


Figure 2
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  		FIGURE 2.
Physical association of {alpha}TTP with lipid vesicles. A, purified recombinant {alpha}TTP (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 {alpha}TTP (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.

 
Fig. 1 shows the kinetics of FRET release at various protein concentrations. We observed a pronounced dependence of the reaction half-life on {alpha}TTP concentrations from 8.7 ± 0.4 s (5 µM {alpha}TTP; n = 10) to 3.9 ± 0.2 s (30 µM {alpha}TTP; n = 9, Fig. 1B). The concentration-dependent nature of this process suggests that {alpha}TTP extracts tocopherol from membranes through a direct, collision-mediated mechanism.

To further examine a direct {alpha}TTP-membrane interaction, we used a size-exclusion filtration assay that allows for the detection of stable protein-vesicle complexes (28). Purified {alpha}TTP was incubated with varying concentrations of sonicated unilamellar vesicles, and the mixture was passed through a centrifugal filtration device (molecular weight cut-off = 100 kDa). Vesicles and associated protein were retained by the filter, whereas free protein passed through the filter to the lower chamber. As shown in Fig. 2A, the fraction of lipid-bound {alpha}TTP increased with increasing vesicle concentration. This effect was not because of nonspecific "trapping" of the protein by the vesicles, as another soluble protein (GST) did not associate with the vesicles under identical conditions (Fig. 2A). A physical interaction between {alpha}TTP and lipid bilayers was also evident from the fluorescence properties of the protein. The presence of five tryptophans in the {alpha}TTP primary structure gives rise to a characteristic intrinsic fluorescence spectrum when the protein is excited at 295 nm ({lambda}max emission = 328 nm; Fig. 2B). In the presence of lipid vesicles, however, the emission spectrum is redshifted by 4 nm ({lambda}max emission = 332 nm). Such a spectral shift is often observed when the local environment of tryptophan residues is changed to one of a more polar nature (30). These observations indicate that {alpha}TTP undergoes a distinct conformational change upon association with lipid bilayers. We also evaluated the interaction between {alpha}TTP and bilayers using DPI measurements. Specifically, we used this method to probe the interactions between soluble {alpha}TTP and a lipid bilayer adsorbed onto the surface of a sensor chip (see "Experimental Procedures"). {alpha}-TTP formed a distinct layer on the immobilized phospholipids, the specific mass of which was dependent on protein concentration (Fig. 3B).


Figure 3
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  		FIGURE 3.
Effect of vitamin E on the interaction between {alpha}TTP and membranes. A, filtration experiments were performed as in Fig. 2A in the presence or absence of RRR-{alpha}-tocopherol (6 mol %). Shown is a representative of three independent experiments. SUV, sonicated unilamellar vesicles. (B, DPI. Shown is the specific mass of {alpha}TTP adsorbed to immobilized phospholipid layers containing (or not) RRR-{alpha}-tocopherol (6 mol %) at different protein concentrations. C, maximum specific adsorbed mass observed with 500 nM {alpha}TTP flowed over adsorbed phospholipids containing 6 mol % of either cholesterol, RRR-{alpha}-tocopherol, or RRR-{delta}-tocopherol.

 
The presence of vitamin E greatly affected the partitioning of {alpha}TTP between the aqueous and the lipid phases; in filtration experiments, the apparent affinity of {alpha}TTP to lipid vesicles was markedly reduced by the presence of RRR-{alpha}-tocopherol (Fig. 3A). In DPI measurements we found that the amount of lipid-adsorbed {alpha}TTP was reduced by >2-fold in the presence of vitamin E (Fig. 3, B and C). Importantly, this effect was specific, since another neutral lipid, cholesterol, did not influence the {alpha}TTP-lipid interaction (Fig. 3C). Further evidence for the specificity of the ligand effect is the observation that {delta}-tocopherol exhibited a significantly weaker effect on the affinity of {alpha}TTP for membranes as compared with the effect of {alpha}-tocopherol (Fig. 3C). This difference likely reflects the reduced affinity of {alpha}TTP for {delta}-tocopherol (5, 6). We conclude from these data that the sensitivity of the {alpha}TTP-lipid interaction to ligands is a specific attribute of vitamin E, which is most attenuated by the natural ligand of the protein.

To gain further insight into the molecular mechanisms of ligand transfer, we examined the catalytic efficacy of {alpha}TTP mutants associated with the AVED disorder. Table 1 summarizes the effects of these mutations on the binding affinity of {alpha}TTP for NBD-tocopherol. Both the glutathione S-transferase-fused and the "naked" (untagged) forms of {alpha}TTP bind tocopherol with high affinity (Kd ~ 10 nM), demonstrating that the presence of the amino-terminal tag does not perturb the native conformation of the protein. Although none of the AVED-affected residues is present in the ligand binding pocket of the protein (Fig. 4A), the naturally occurring R59W and H101Q mutations cause a significant reduction in {alpha}TTP affinity for NBD-tocopherol (Kd = 39 and 71 nM, respectively, Table 1). The affinities of the remaining mutants for NBD-tocopherol are similar to that of the wild-type protein (Kd = 3–27 nM, Table 1).


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  		TABLE 1
Affinity of mutant {alpha}TTP proteins for NBD-tocopherol

 


Figure 4
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  		FIGURE 4.
Sequestration of membrane-bound tocopherol by {alpha}TTP mutants. A, location of AVED-affected residues relative to the ligand-binding pocket of {alpha}TTP. 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 {alpha}TTP (30 µM active protein). Conditions are as in Fig. 1.

 
The efficacy of wild-type {alpha}TTP and several AVED variants in catalyzing the sequestration of tocopherol from membranes is shown in Fig. 4B. The rate at which the R192H or H101Q mutants facilitated ligand removal was essentially indistinguishable from the wild-type protein (t1/2 = 4.1 and 4.8 s for the R192H and H101Q mutants, respectively, at 30 µM protein). Loss of FRET induced by the E141K and R59W mutants, on the other hand, occurred at a significantly slower rate (t1/2 = 7.9 and 7.01 s, respectively). These observations indicate that the clinical phenotype associated with a particular mutation in {alpha}TTP (i.e. mutations associated with the severe versus the mild forms of AVED) is correlated with the competence of the protein in sequestering tocopherol from lipid bilayers.

Another correlation between in vitro activity and clinical severity is evident when inspecting the concentration dependence of the ligand transfer rate for the different mutants (Fig. 5). All {alpha}TTP variants tested facilitated the sequestration of tocopherol to a similar extent (~20% change in emission at 575 nm). However, the kinetic characteristics of this change were markedly different. {alpha}-TTP variants associated with the late-onset, mild phenotype of AVED (i.e. harboring the R192H, H101Q, or A120T substitutions) exhibited tocopherol transfer rates that are highly dependent on protein concentration, similar to the wild-type protein (i.e. a 2-fold decrease in t1/2 between 5 and 30 µM protein; Fig. 5). In contrast, the rate for protein-induced FRET loss decreased by only 20% for the R221W mutant and did not exhibit any significant concentration dependence in the case of the E141K and R59W variants of {alpha}TTP, associated with the severe form of AVED (Fig. 5). We previously reported similar kinetic distinctions between these {alpha}TTP mutant classes in their ability to catalyze the vesicle-to-vesicle transfer of radiolabeled tocopherol (20). These observations raise the possibility that naturally occurring mutations in {alpha}TTP alter the ability of the protein to interact with membranes. To directly address this issue, we quantitated the interaction of various AVED mutants with lipids using DPI. As shown in Fig. 6, all proteins associated with phospholipids to a similar extent as the wild-type protein (~1 ng of protein adsorbed per mm2). However, the effect of vitamin E on the protein-lipid interaction was strikingly different among the different mutant classes. {alpha}-TTP variants associated with the mild, late-onset form of AVED (i.e. R192H, H101Q) exhibited pronounced sensitivity to the presence of vitamin E, similar to the wild-type protein; the mass of lipid-adsorbed {alpha}TTP was reduced by ~50% in the presence of tocopherol, as would be expected if {alpha}TTP-tocopherol has reduced affinity for the membrane. Mutant proteins associated with the severe, early onset form of AVED (i.e. R221W, R59W), on the other hand, were not affected by the presence of tocopherol in the bilayer. That the severe AVED mutations diminish the ligand sensitivity of the {alpha}TTP-membrane interaction was also observed in filtration experiments (data not shown). These data raise the possibility that a decline in tocopherol sensitivity, rather than differences in affinity for membranes per se, gives rise to the functional defects associated with the severe AVED phenotype.


Figure 5
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  		FIGURE 5.
Dependence of tocopherol sequestration rates on protein concentration. Rates of {alpha}TTP-induced sequestration of NBD-tocopherol were measured as described in Fig. 1 using various concentrations of the indicated{alpha}TTP variants. Shown are averages and S.D. from at least five independent measurements at each protein concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The critical role of {alpha}TTP in regulating whole-body status of vitamin E is evident from the observations that mutations in the human ttpA gene lead to vitamin E deficiency with ataxia. Similarly, mice in which expression of the {alpha}TTP gene is disrupted display reduced vitamin E levels and neurological disorders (31–33). Two biochemical activities are ascribed to {alpha}TTP; that is, stimulation of tocopherol secretion from cultured hepatocytes (10, 11, 21, 34) and catalysis of tocopherol transfer between membrane vesicles in vitro (6, 20, 29, 35). Neither the molecular mechanism underlying tocopherol transfer nor the relationship between this activity and the physiological function of {alpha}TTP are known at present.

We showed that the rate by which {alpha}TTP sequesters membrane-bound tocopherol increases with the increasing protein:vesicles ratio, suggesting that the ligand transfer activity is mediated by direct protein-membrane interactions. In support of this notion, equilibrium measurements utilizing filtration, DPI, and intrinsic fluorescence spectroscopy demonstrated that a stable complex is formed between {alpha}TTP and membranes. These observations suggest that intermembrane transfer of tocopherol by {alpha}TTP involves at least three principal steps: 1) association between {alpha}TTP and the lipid bilayer, 2) extraction of tocopherol from the bilayer into the TTP binding pocket, and 3) dissociation of the holoprotein from the membrane, similar to the proposed mechanism for ligand transfer by Sec14 (22). Ligand transfer is not likely to be limited by association between {alpha}TTP and membranes, since in solution this step is likely to be too fast (diffusion-controlled). Possibly, sequestration of tocopherol by {alpha}TTP (step 2) is the rate-determining step of the ligand transfer reaction, which is monitored in our kinetic FRET measurements.


Figure 6
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  		FIGURE 6.
Association of AVED variants with membranes. Maximum specific adsorbed mass observed with 500 nM concentrations of the indicated form of{alpha}TTP to phospholipid membranes lacking or containing 6 mol% RRR-{alpha}-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.

 
An important feature of the interaction between {alpha}TTP and lipid vesicles is its marked sensitivity to the presence of vitamin E. It is possible that this "bias" contributes to the directionality of tocopherol distribution within the hepatocyte, i.e. that preferential affinity results in net fluxes of tocopherol from vitamin E-containing to vitamin E-poor membranes. The molecular mechanism by which {alpha}TTP "senses" the presence of tocopherol in lipid bilayers is not known. The protein may be sensitive to ligand-induced changes in the physical properties of the bilayer, such as fluidity and surface tension, as is the case for other lipid binding and transfer proteins (36–41). Our observations that cholesterol does not influence {alpha}TTP-lipid interactions argue against this mechanism. Alternatively, the presence of tocopherol in the binding pocket may stabilize {alpha}TTP in solution, as predicted from a structural comparison of the apo and holo conformations (42). The reduced sensitivity of the "severe" {alpha}TTP mutants (e.g. R59W and R221W) mutants to the presence of tocopherol supports this explanation. Thus, we propose that ligand-induced protein conformation rather than changes in physical properties of the bilayer underlie the ligand sensitivity of the {alpha}TTP-membrane interaction.

We previously observed that {alpha}TTP variants that cause the severe form of AVED in humans (i.e. the R59W, E141K, or R221W substitutions) are impaired in catalyzing vesicle-to-vesicle transfer of radiolabeled tocopherol (20). In the present study we observed that the rate at which these mutants sequester membrane-bound tocopherol is independent of protein concentration, unlike the wild-type {alpha}TTP or mutants associated with the mild form of AVED. It is possible that the insensitivity of the severe mutants to tocopherol is at the root of the functional defect, since it could lead to altered partitioning of {alpha}TTP between different membranes and compromise the directionality of tocopherol transfer. This explanation could also account for the inability of such mutants to facilitate the transfer of NBD-tocopherol from lysosomes to the plasma membranes in intact cells (e.g. the R221W mutant (21)).

As shown by the observations presented here and in previous studies (20), {alpha}TTP mutations that are associated with the mild AVED pathology (R192H, H101Q, A120T) do not impede the ability of the protein to catalyze tocopherol transfer in vitro. These observations suggest that additional physiological factors may contribute to {alpha}TTP actions in vivo. The nature of these factors and their role in regulating tocopherol status remain to be clarified.


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grant DK067494 (to D. M.) and Training Grant T32-DK715827 (to S. M.). This work was also supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Case Western Reserve University School of Medicine, WG-48, Cleveland, OH, 44106. Tel.: 216-368-6230; Fax: 216-368-6644; E-mail: dxm178{at}case.edu.

2 The abbreviations used are: {alpha}TTP, {alpha}-tocopherol transfer protein; AVED, ataxia with vitamin E deficiency; tocopherol, RRR-{alpha}-tocopherol; NBD-tocopherol, (R)-2,5,7,8-tetramethyl-chroman-2-oxadiazol-4-ylamino)-nonyl]-chroman-6-ol; TRITC-DHPE, N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; FRET, fluorescence resonance energy transfer; DPI, dual polarization interferometry. Back


   ACKNOWLEDGMENTS
 
We are grateful to Valerie Cross and Nikhil Shyam for technical help in the initial stages of the study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Traber, M. G., and Kayden, H. J. (1989) Am. J. Clin. Nutr. 49, 517–526[Abstract/Free Full Text]
  2. Behrens, W. A., and Madere, R. (1986) J. Am. Coll. Nutr. 5, 91–96[Abstract]
  3. Sontag, T. J., and Parker, R. S. (2002) J. Biol. Chem. 277, 25290–25296[Abstract/Free Full Text]
  4. Swanson, J. E., Ben, R. N., Burton, G. W., and Parker, R. S. (1999) J. Lipid Res. 40, 665–671[Abstract/Free Full Text]
  5. Panagabko, C., Morley, S., Hernandez, M., Cassolato, P., Gordon, H., Parsons, R., Manor, D., and Atkinson, J. (2003) Biochemistry 42, 6467–6474[CrossRef][Medline] [Order article via Infotrieve]
  6. Hosomi, A., Arita, M., Sato, Y., Kiyose, C., Ueda, T., Igarashi, O., Arai, H., and Inoue, K. (1997) FEBS Lett. 409, 105–108[CrossRef][Medline] [Order article via Infotrieve]
  7. Mowri, H., Nakagawa, Y., Inoue, K., and Nojima, S. (1981) Eur. J. Biochem. 117, 537–542[Medline] [Order article via Infotrieve]
  8. Verdon, C. P., and Blumberg, J. B. (1988) Anal. Biochem. 169, 109–120[CrossRef][Medline] [Order article via Infotrieve]
  9. Arita, M., Sato, Y., Miyata, A., Tanabe, T., Takahashi, E., Kayden, H. J., Arai, H., and Inoue, K. (1995) Biochem. J. 306, 437–443[Medline] [Order article via Infotrieve]
  10. Arita, M., Nomura, K., Arai, H., and Inoue, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12437–12441[Abstract/Free Full Text]
  11. Qian, J., Morley, S., Wilson, K., Nava, P., Atkinson, J., and Manor, D. (2005) J. Lipid Res. 46, 2072–2082[Abstract/Free Full Text]
  12. Ouahchi, K., Arita, M., Kayden, H., Hentati, F., Ben Hamida, M., Sokol, R., Arai, H., Inoue, K., Mandel, J. L., and Koenig, M. (1995) Nat. Genet. 9, 141–145[CrossRef][Medline] [Order article via Infotrieve]
  13. Cavalier, L., Ouahchi, K., Kayden, H. J., Di Donato, S., Reutenauer, L., Mandel, J. L., and Koenig, M. (1998) Am. J. Hum. Genet. 62, 301–310[CrossRef][Medline] [Order article via Infotrieve]
  14. Gotoda, T., Arita, M., Arai, H., Inoue, K., Yokota, T., Fukuo, Y., Yazaki, Y., and Yamada, N. (1995) N. Engl. J. Med. 333, 1313–1318[Abstract/Free Full Text]
  15. Yokota, T., Shiojiri, T., Gotoda, T., and Arai, H. (1996) N. Engl. J. Med. 335, 1770–1771[Free Full Text]
  16. Yokota, T., Shiojiri, T., Gotoda, T., Arita, M., Arai, H., Ohga, T., Kanda, T., Suzuki, J., Imai, T., Matsumoto, H., Harino, S., Kiyosawa, M., Mizusawa, H., and Inoue, K. (1997) Ann. Neurol. 41, 826–832[CrossRef][Medline] [Order article via Infotrieve]
  17. Yokota, T., Uchihara, T., Kumagai, J., Shiojiri, T., Pang, J. J., Arita, M., Arai, H., Hayashi, M., Kiyosawa, M., Okeda, R., and Mizusawa, H. (2000) J. Neurol. Neurosurg. Psychiatry 68, 521–525[Abstract/Free Full Text]
  18. Pang, J., Kiyosawa, M., Seko, Y., Yokota, T., Harino, S., and Suzuki, J. (2001) Jpn. J. Ophthalmol. 45, 672–676[CrossRef][Medline] [Order article via Infotrieve]
  19. Hentati, A., Deng, H. X., Hung, W. Y., Nayer, M., Ahmed, M. S., He, X., Tim, R., Stumpf, D. A., Siddique, T., and Ahmed, A. (1996) Ann. Neurol. 39, 295–300[CrossRef][Medline] [Order article via Infotrieve]
  20. Morley, S., Panagabko, C., Shineman, D., Mani, B., Stocker, A., Atkinson, J., and Manor, D. (2004) Biochemistry 43, 4143–4149[CrossRef][Medline] [Order article via Infotrieve]
  21. Qian, J., Atkinson, J., and Manor, D. (2006) Biochemistry 45, 8236–8242[CrossRef][Medline] [Order article via Infotrieve]
  22. Sha, B., Phillips, S. E., Bankaitis, V. A., and Luo, M. (1998) Nature 391, 506–510[CrossRef][Medline] [Order article via Infotrieve]
  23. Ponsin, G., Qu, S. J., Fan, H. Z., and Pownall, H. J. (2003) Biochemistry 42, 4444–4451[CrossRef][Medline] [Order article via Infotrieve]
  24. Desrumaux, C., Labeur, C., Verhee, A., Tavernier, J., Vandekerckhove, J., Rosseneu, M., and Peelman, F. (2001) J. Biol. Chem. 276, 5908–5915[Abstract/Free Full Text]
  25. Wootan, M. G., Bernlohr, D. A., and Storch, J. (1993) Biochemistry 32, 8622–8627[CrossRef][Medline] [Order article via Infotrieve]
  26. Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317–13323[Abstract/Free Full Text]
  27. Morley, S., Cross, V., Cecchini, M., Nava, P., Atkinson, J., and Manor, D. (2006) Biochemistry 45, 1075–1081[CrossRef][Medline] [Order article via Infotrieve]
  28. Huang, H., Ball, J. M., Billheimer, J. T., and Schroeder, F. (1999) Biochem. J. 344, 593–603[CrossRef][Medline] [Order article via Infotrieve]
  29. Sato, Y., Hagiwara, K., Arai, H., and Inoue, K. (1991) FEBS Lett. 288, 41–45[CrossRef][Medline] [Order article via Infotrieve]
  30. Lackowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, pp. 354–363, Plenum Press, New York
  31. Terasawa, Y., Ladha, Z., Leonard, S. W., Morrow, J. D., Newland, D., Sanan, D., Packer, L., Traber, M. G., and Farese, R. V., Jr. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13830–13834[Abstract/Free Full Text]
  32. Yokota, T., Igarashi, K., Uchihara, T., Jishage, K., Tomita, H., Inaba, A., Li, Y., Arita, M., Suzuki, H., Mizusawa, H., and Arai, H. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15185–15190[Abstract/Free Full Text]
  33. Leonard, S. W., Terasawa, Y., Farese, R. V., Jr., and Traber, M. G. (2002) Am. J. Clin. Nutr. 75, 555–560[Abstract/Free Full Text]
  34. Horiguchi, M., Arita, M., Kaempf-Rotzoll, D. E., Tsujimoto, M., Inoue, K., and Arai, H. (2003) Genes Cells 8, 789–800[Abstract]
  35. Murphy, D. J., and Mavis, R. D. (1981) J. Biol. Chem. 256, 10464–10468[Free Full Text]
  36. Radhakrishnan, A., Sun, L. P., Kwon, H. J., Brown, M. S., and Goldstein, J. L. (2004) Mol. Cell 15, 259–268[CrossRef][Medline] [Order article via Infotrieve]
  37. Rao, C. S., Chung, T., Pike, H. M., and Brown, R. E. (2005) Biophys. J. 89, 4017–4028[CrossRef][Medline] [Order article via Infotrieve]
  38. Weinberg, R. B., Cook, V. R., Jones, J. B., Kussie, P., and Tall, A. R. (1994) J. Biol. Chem. 269, 29588–29591[Abstract/Free Full Text]
  39. Gargouri, Y., Moreau, H., and Verger, R. (1989) Biochim. Biophys. Acta 1006, 255–271[Medline] [Order article via Infotrieve]
  40. Lowe, M. E. (2002) J. Lipid Res. 43, 2007–2016[Abstract/Free Full Text]
  41. Wu, F., Corsico, B., Flach, C. R., Cistola, D. P., Storch, J., and Mendelsohn, R. (2001) Biochemistry 40, 1976–1983[CrossRef][Medline] [Order article via Infotrieve]
  42. Meier, R., Tomizaki, T., Schulze-Briese, C., Baumann, U., and Stocker, A. (2003) J. Mol. Biol. 331, 725–734[CrossRef][Medline] [Order article via Infotrieve]
  43. Nava, P., Cecchini, M., Chirico, S., Gordon, H., Morley, S., Manor, D., and Atkinson, J. (2006) Bioorg. Med. Chem. 14, 3721–3736[CrossRef][Medline] [Order article via Infotrieve]

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