Influence of dietary vitamin E on the intermembrane transfer of alpha-tocopherol as mediated by an alpha-tocopherol binding protein

The effect of dietary vitamin E on the intermembrane transfer of (3R)-alpha-tocopherol, a spontaneous process accelerated in the presence of an alpha-tocopherol binding protein (alpha TBP), was examined. The transfer activity of this cytosolic liver protein was assayed via in vitro transfer of (3R)-alpha-[3H]tocopherol (alpha[3H]T) from egg lecithin liposomes to human erythrocyte ghosts (EG). Male Fisher 344 rats (1 and 20 months old) were fed diets containing 0, 30, and 500 mg/kg vitamin E (dl-alpha-tocopheryl acetate) for 15 weeks. Liver cytosol fractions were assayed for alpha[3H]T transfer activity (alpha TTA). Among young rats, those fed vitamin E-deficient diets had the highest alpha TTA, 5.02 +/- 3.10 pmole alpha[3H]T/min (mean +/- SD), which was different (P less than 0.05) from the spontaneous transfer rate of 2.10 pmole/min. Neither young rats fed 30 and 500 mg/kg vitamin E diets nor any of the aged rats showed alpha TTA which differed significantly from the spontaneous transfer rate. To examine the relationship between hepatic alpha-tocopherol levels and alpha TTA, alpha-tocopherol concentration per gram of wet liver was assayed by HPLC. A steep positive slope (6.39 +/- 1.46 pmole min-1 nmole g-1) and strong correlation (r = 0.873) between hepatic alpha-tocopherol and alpha TTA were observed (P less than 0.005) among young vitamin E-deficient rats. The data indicates that alpha TTA varies directly with hepatic alpha-tocopherol concentration when total liver vitamin E stores are very low. Thus, alpha TBP-mediated transfer of alpha-tocopherol may be manifest only when vitamin E status is compromised.     

The molecular basis of vitamin E retention: structure of human alpha-tocopherol transfer protein

Alpha-tocopherol transfer protein (alpha-TTP) is a liver protein responsible for the selective retention of alpha-tocopherol from dietary vitamin E, which is a mixture of alpha, beta, gamma, and delta-tocopherols and the corresponding tocotrienols. The alpha-TTP-mediated transfer of alpha-tocopherol into nascent VLDL is the major determinant of plasma alpha-tocopherol levels in humans. Mutations in the alpha-TTP gene have been detected in patients suffering from low plasma alpha-tocopherol and ataxia with isolated vitamin E deficiency (AVED). The crystal structure of alpha-TTP reveals two conformations. In its closed tocopherol-charged form, a mobile helical surface segment seals the hydrophobic binding pocket. In the presence of detergents, an open conformation is observed, which probably represents the membrane-bound form. The selectivity of alpha-TTP for RRR-alpha-tocopherol is explained from the van der Waals contacts occurring in the lipid-binding pocket. Mapping the known mutations leading to AVED onto the crystal structure shows that no mutations occur directly in the binding pocket.     

Expression and refolding of recombinant human alpha-tocopherol transfer protein capable of specific alpha-tocopherol binding

alpha-Tocopherol transfer protein (alpha-TTP) is a cytosolic protein found predominantly in mammalian liver that is proposed to be responsible for the stereoselective uptake of alpha-tocopherol from the diet. Although recombinant alpha-TTP has been reported previously, little detail has been provided about the yields and competency of the recovered protein at binding tocopherols and other ligands. In this work, we report the successful expression and refolding of a recombinant human alpha-TTP. Ligation-independent cloning generated a construct in pET-30 encoding an alpha-TTP fusion protein (pET-30/ttp) containing a six-histidine tag and an S-tag, each cleavable by a separate protease upon expression in Escherichia coli. Overexpression of the protein led to the formation of inclusion bodies that were solubilized in 8 M urea and purified by metal chelate affinity chromatography. Another construct in pET-28b (pET-28b/ttp) provided a soluble protein product after expression that contained a 40-amino-acid N-terminal extension, which can be reduced to 21 amino acids by cleavage with thrombin. The success of different refolding experiments was assessed using a Lipidex gel-based tocopherol binding assay. The best recovery of refolded recombinant alpha-TTP fusion capable of binding alpha-tocopherol was provided by matrix-assisted refolding in the presence of 0.5 M arginine. Cleavage of the fusion protein with Factor Xa successfully generated the full-length wild-type protein with no additional N-terminal amino acids. The resulting purification scheme provides recombinant alpha-TTP in good yield and purity for investigation of both its structure and its binding affinities for different ligands including natural and synthetic tocols.     

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.     

An alpha-tocopherol binding protein in rat liver cytoplasm.

Gel filtration and sucrose density gradient analysis of rat liver high speed supernatant revealed the presence of a protein capable of binding [³H] α-tocopherol. The protein sedimented with an S value of 3.0. Gel filtration yielded an estimated molecular weight of 31,000. Specificity for α-tocopherol was demonstrated by competition for binding of [³H]α-tocopherol with unlabeled α-tocopherol, but not with α-tocopheryl quinone or α-tocopheryl acetate. Pronase digestion completely abolished binding.     

A sensitive competitive protein binding assay for vitamin D in plasma

A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.     

An improved protein binding assay for cyclic AMP

A simplified and inexpensive protein binding assay for cAMP has been developed for the rapid measurement of subpicomole quantities of the nucleotide. Conditions of assay were established in which interferences from salts and nucleotides present in biological samples were eliminated. Sodium chloride was demonstrated to reduce cAMP binding by denaturing the regulatory subunit of the protein kinase.     

Glycolipid transfer protein mediated transfer of glycosphingolipids between membranes: a model for action based on kinetic and thermodynamic analyses

Glycolipid transfer protein (GLTP) catalyzes the intermembrane transfer of lipids that have sugars beta-linked to either diacylglycerol or ceramide backbones, including simple glycosphingolipids (GSLs) and gangliosides. The present study provides a quantitative understanding of GLTP action involving bilayer vesicles that have high and low curvature stress, i.e., small and large unilamellar vesicles (SUVs and LUVs). When the GSL intervesicular transfer was monitored in real time using an established fluorescence resonance energy approach, the initial GSL transfer rates (v(0)) and net transfer equilibrium (K(eq)) were determined for GLTP-mediated transfer from SUVs and LUVs over the temperature range of 30-44 degrees C. v(0) exhibited a linear dependence with respect to varying GLTP concentrations (0-143 nM range) in SUVs and LUVs, suggesting a first order dependence on the GLTP bulk concentration. Thermodynamic parameters associated with the GLTP-GSL transition-state complex and GSL net transfer were determined from linear Arrhenius and van't Hoff plots, respectively. Although initial transfer rates were lower for LUVs than for SUVs, the activation energy barriers were higher for LUVs, while the Gibbs's free energy of the transition states were similar. The formation of a transition-state complex was predominantly enthalpy driven, whereas the net transfer of GSLs was mainly entropy driven. The rate-limiting step for GLTP action appeared to be the surface processes leading to the GLTP-GSL complex formation and release associated with a shuttle/carrier mode of action. Because surface processes leading to the GLTP-GSL complex formation were limiting for GLTP action with SUVs and LUVs, it was concluded that GLTP is likely to be a valuable tool to probe and manipulate GSL environments in membranes.     

An activation-collision mechanism for cholesterol transfer between membranes

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.     

Non-antioxidant molecular functions of alpha-tocopherol (vitamin E)

alpha-Tocopherol (the major vitamin E component) regulates key cellular events by mechanisms unrelated with its antioxidant function. Inhibition of protein kinase C (PKC) activity and vascular smooth muscle cell growth by alpha-tocopherol was first described by our group. Later, alpha-tocopherol was shown to inhibit PKC in various cell types with consequent inhibition of aggregation in platelets, of nitric oxide production in endothelial cells and of superoxide production in neutrophils and macrophages. alpha-Tocopherol diminishes adhesion molecule, collagenase and scavenger receptor (SR-A and CD36) expression and increases connective tissue growth factor expression.     

Effects of alpha-tocopherol (vitamin E) on the stability and lipid dynamics of model membranes mimicking the lipid composition of plant chloroplast membranes

Tocopherol (vitamin E) is widely recognized as a cellular antioxidant. It is essential for human and animal health, but only synthesized in photosynthetic organisms, where it is localized in chloroplast membranes. While many studies have investigated non-antioxidative effects of tocopherol on phospholipid membranes, nothing is known about its effects on membranes containing chloroplast glycolipids. Here, liposomes resembling plant chloroplast membranes were used to investigate the effects of alpha-tocopherol on vesicle stability during freezing and on lipid dynamics. alpha-Tocopherol had a pronounced influence on membrane dynamics and showed strong interactions in its effects on membrane stability during freezing with the cryoprotectant sucrose. alpha-Tocopherol showed maximal effects at low concentrations (around 2mol%), close to its contents in chloroplast membranes.     

Exhaustive exercise affects fluidity and alpha-tocopherol levels in brain synaptosomal membranes of normal and vitamin E supplemented rats

Alpha-tocopherol level and fluidity were studied in the neuronal membrane of rat brain after exhaustive exercise. The order parameter, 5-doxyl-stearic acid (5-DS), which is utilized for assessing the fluidity of the lipid bilayer closer to the hydrophilic face of the membrane, decreased in the pons-medulla oblongata, and the motion parameter, 16-doxyl-stearic acid (16-DS) for the core of the lipid bilayer, decreased in the cortex, hippocampus, hypothalamus and striatum, whereas it increased in the cerebellum after exercise. The w/s ratio of n-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimido (maleimido-TEMPO) for the conformation of SH-protein also decreased in the hippocampus and midbrain after exercise. These changes were not observed in alpha-tocopheryl acetate supplemented rats after exercise. Although the levels of 5-DS, 16-DS and maleimido-TEMPO were affected by alpha-tocopheryl acetate in rat neuronal membranes, fluidity changes were reversible with exercise.     

Stabilization of synaptic membranes with alpha-tocopherol against the damaging effect of phospholipases. Possible mechanism of the biological action of vitamin E

Using fluorescent and EPR spin probing techniques, the effects of phospholipases A2, C and D on rat brain synaptosomal membranes were investigated. It was shown that treatment of synaptosomal membranes with phospholipases A2, C and D results in their depolarization and increase of their surface negative charge. In case of phospholipases A2 and C, these changes are also accompanied by a decrease of the microviscosity of the synaptosomal membrane lipid bilayer. alpha-Tocopherol protects synaptosomal membranes against the damaging action of phospholipases. The stabilization of synaptosomes by vitamin E consists in the reconstitution of the transmembrane potential and in an increased microviscosity of phospholipase-treated membranes. The stabilizing effect of alpha-tocopherol is due to the binding of phospholipid hydrolysis products rather than to the inhibition of phospholipases. The observed stabilization of synaptosomal membranes by alpha-tocopherol is interpreted as a feasible mechanism of biological effects of vitamin E on biological membranes.     

Biochemical consequences of heritable mutations in the alpha-tocopherol transfer protein

Tocopherol transfer protein (TTP) regulates vitamin E status by facilitating the secretion of tocopherol from liver to circulating lipoproteins. Heritable mutations in the ttpA gene, encoding for TTP, result in ataxia with vitamin E deficiency (AVED) syndrome, typified by low vitamin E levels and a plethora of neurological disorders. The molecular mechanisms by which TTP facilitates tocopherol secretion are presently unknown. We recently showed that vitamin E is taken up by hepatocytes through an endocytic process and that, shortly following uptake, the vitamin is found primarily in lysosomes. We showed further that TTP is localized to late endocytic vesicles and that it facilitates the intracellular trafficking of tocopherol from lysosomes to the plasma membrane. To gain insight into the molecular mechanisms that underlie TTP actions, we studied the physiological impact of three naturally occurring heritable mutations in the ttpA gene (the R59W, R221W, and A120T substitutions). We found that these mutations impair the ability of TTP to facilitate the secretion of vitamin E from cells. Furthermore, the degree of impairment corresponded to the severity of the AVED pathology associated with each mutation. In cells that express mutated TTP proteins, vitamin E did not traffic to the plasma membrane and remained "trapped" in lysosomes. In addition, we observed that substitution mutations that cause the AVED syndrome impart a marked instability on the TTP protein. These observations suggest that the physiological role of TTP is anchored in its ability to direct vitamin E trafficking from the endocytic compartment to transport vesicles that deliver the vitamin to the site of secretion at the plasma membrane.     

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.     

A protein binding assay for hyaluronate

A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronection was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with [3H]hyaluronate, followed by an equal volume of saturated (NH4)2SO4, which precipitated the hyaluronectin and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. The mixture was then centrifuged, and the amount of bound [3H]hyaluronate in the precipitate was determined. Using this assay, we found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity (Kd = 5.7 X 10(-10) M), and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, we used a competition assay in which the binding of a set amount of [3H]hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. We have found that this method is sensitive to 1 microgram or less of hyaluronate, and is unaffected by the presence of proteins.