Molecular Determinants of Heritable Vitamin E Deficiency

Morley, Samantha; Panagabko, Candace; Shineman, Diana W.; Mani, Bernhard; Stocker, Achim; Atkinson, Jeffrey; Manor, Danny

doi:10.1021/bi0363073

Cited by 36 publications

(45 citation statements)

References 37 publications

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“…These observations confirm previous reports that demonstrated a ca. 5-fold lower affinity of this mutant to tocopherol, as compared to wild-type TTP (25). Moreover, these data illustrate the utility of NBD-TOH for the detection of subtle perturbations to TTP's ligand binding pocket.…”

Section: Affinity Of Nbd-toh To Ttpmentioning

confidence: 65%

“…We have previously shown that several TTP variants associated with the AVED pathology are impaired in facilitating vesicle-to-vesicle tocopherol transfer in vitro (25). We asked whether we could detect analogous differences in the kinetics of the isolated partial reaction (tocopherol dissociation from the bilayer).…”

Section: Kinetic Resolution Of Ttp's Ligand Transfer Activitymentioning

confidence: 96%

“…Traditionally, the transfer of radioactively labeled TOH between two lipid vesicle populations was used to describe TTP activity (e.g. 5,7,8,25,26). This assay is limited by cumbersome physical manipulations, poor kinetic resolution, and low signal-to-noise.…”

Section: Nih Public Accessmentioning

confidence: 99%

“…We have shown previously that the latter class of mutations in TTP does not significantly impair the protein's ligand transfer activity, while those associated with the severe phenotype exhibit a ca. 3-fold impairment in the protein's in vitro transfer activity (25). Thus, the AVED mutants represent valuable tools for exploring the mechanism of TTP-facilitated tocopherol transfer in vitro, and secretion of the vitamin in vivo.…”

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Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

et al. 2006

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The tocopherol transfer protein (TTP1) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analog, R) -2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5] oxadiazol-4-ylamino)-nonyl]-chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue-shifted and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analog binds to TTP reversibly and with high affinity (Kd=8.5 ± 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the Ataxia with Vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (Kd = 71 ± 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction. Keywords affinity; fluorescence; kinetics; tocopherol; vitamin E; lipid transfer; TTP Vitamin E is a lipophilic antioxidant, initially identified as a plant-derived factor that prevented fetal resorption in diet-restricted rats (1). The term "Vitamin E" refers to a class of structurally related molecules, all of which show potent radical trapping activity in vitro (2). The ability of 1 ABBREVIATIONS NBD-TOH, (R) -2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-nonyl]-chroman-6-ol; TTP, alpha tocopherol transfer protein; AVED, ataxia with vitamin E deficiency; TOH, RRR-α-tocopherol; GST, glutathione-S-transferase; IPTG, isopropyl-β-D-thiogalactopyranoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid, PMSF, phenylmethylsulfonyl fluoride; TRIS, tris(hydroxymethyl)aminomethane; FPLC, fast performance liquid chromatography; ABC, ATP-binding cassette transporter; Marina Blue-DHPE, Marina Blue 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; TRITC-DHPE, N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; PC, phosphatidylcholine; IgG, immunoglobulin G. § To whom correspondence should be addressed at The Division of Nutritional Sciences, 228 Savage Hall, Cornell University, Ithaca, New York, 14853. Phone: 607-255-6085; Fax: 6...

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Section: Affinity Of Nbd-toh To Ttpmentioning

confidence: 65%

Section: Kinetic Resolution Of Ttp's Ligand Transfer Activitymentioning

confidence: 96%

Section: Nih Public Accessmentioning

confidence: 99%

mentioning

confidence: 99%

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Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

et al. 2006

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“…In humans, mutations in the TTPA gene cause systemic vitamin E deficiency accompanied by neurological compromise, primarily spinocerebellar ataxia (10 -17). Patients afflicted with this disorder (ataxia with vitamin E deficiency; OMIM 277460) present with debilitating neurodegeneration, the severity of which depends on the specific mutation's effect on TTP's biochemical activity (18,19). Lifelong vitamin E supplementation can delay or reverse disease progression, especially when patients are treated in earlier stages (20,21).…”

mentioning

confidence: 99%

Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein

Chung¹,

Ghelfi

Atkinson

et al. 2016

Journal of Biological Chemistry

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␣-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, ␣-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic ␣-tocopherol transfer protein (TTP) preferentially selects dietary ␣-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of ␣-tocopherol. The mechanisms by which TTP facilitates ␣-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of ␣-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with ␣-tocopherol, TTP-and ␣-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to ␣-tocopherol and is time-and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of ␣-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of ␣-tocopherol through hepatocytes.Vitamin E is a plant-derived lipid that was discovered as a dietary component vital for female fertility in rodents (1) and for neuronal health in humans (2). Eight vitamin E forms are synthesized by plants, differing in the degree of methylation of the chromanol ring and the saturation of the isoprenoid side chain (3). Of the eight naturally occurring forms of vitamin E, ␣-tocopherol exhibits the most potent biological activity in preventing deficiency-induced reproductive failure in rodents (4, 5). ␣-Tocopherol's efficacy in scavenging free radicals (i.e. functioning as an antioxidant) is thought to underlie its critical roles in health (6 -8), but additional redox-independent actions have been recently proposed (9).The major regulator of vitamin E status is the ␣-tocopherol transfer protein (TTP), 2 and mutations in the TTPA gene cause heritable vitamin E deficiency. In humans, mutations in the TTPA gene cause systemic vitamin E deficiency accompanied by neurological compromise, primarily spinocerebellar ataxia (10 -17). Patients afflicted with this disorder (ataxia with vitamin E deficiency; OMIM 277460) present with debilitating neurodegeneration, the severity of which depends on the specific mutation's effect on TTP's biochemical activity (18,19). Lifelong vitamin E supplementation can delay or reverse disease progression, especially when patients are treated in earlier stages (20,21). Disruption of the TtpA gene in mice recapitulated the human ataxia with vitamin E deficiency disorder (22).TTP is expressed at high levels in parenchymal cells of the liver (23, 24), but low level expression has b...

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The establishment of tocopherol reference intervals for Hungarian adult population using a validated HPLC method

Veres

Szpisjak

Bajtai

et al. 2017

Biomedical Chromatography

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Evidence suggests that decreased α-tocopherol (the most biologically active substance in the vitamin E group) level can cause neurological symptoms, most likely ataxia. The aim of the current study was to first provide reference intervals for serum tocopherols in the adult Hungarian population with appropriate sample size, recruiting healthy control subjects and neurological patients suffering from conditions without symptoms of ataxia, myopathy or cognitive deficiency. A validated HPLC method applying a diode array detector and rac-tocol as internal standard was utilized for that purpose. Furthermore, serum cholesterol levels were determined as well for data normalization. The calculated 2.5-97.5% reference intervals for α-, β/γ- and δ-tocopherols were 24.62-54.67, 0.81-3.69 and 0.29-1.07 μm, respectively, whereas the tocopherol/cholesterol ratios were 5.11-11.27, 0.14-0.72 and 0.06-0.22 μmol/mmol, respectively. The establishment of these reference intervals may improve the diagnostic accuracy of tocopherol measurements in certain neurological conditions with decreased tocopherol levels. Moreover, the current study draws special attention to the possible pitfalls in the complex process of the determination of reference intervals as well, including the selection of study population, the application of internal standard and method validation and the calculation of tocopherol/cholesterol ratios.

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Molecular Determinants of Heritable Vitamin E Deficiency

Cited by 36 publications

References 37 publications

Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein

The establishment of tocopherol reference intervals for Hungarian adult population using a validated HPLC method

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