Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity

Abstract

The tocopherol transfer protein (TTP) 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 analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[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 analogue binds to TTP reversibly and with high affinity (K(d) = 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 (K(d) = 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.

Figure 1. Chemical and spectral characteristics of NBD-TOH

A) Structure of NBD-TOH. B) Excitation and emission spectra of NBD-TOH in methanol. Fluorescence excitation (solid line, emission monitored at 533 nm) and emission (dashed line, excitation at 466 nm) of 0.22 μM NBD-TOH in methanol were monitored. Readings were taken at 1nm and 1 second intervals. Spectral resolution = 1nm.

Figure 2. Spectral changes of NBD-TOH upon binding to TTP

A) Fluorescence excitation and emission spectra of 0.22 μM NBD-TOH in Buffer A were recorded before (dotted line) or after (solid line) the addition of 5μM TTP. Note different scales for the free (right y-axis) and TTP-bound (left y-axis) fluorophores.

Figure 3. Affinity and specificity of NBD-TOH to TTP

A) NBD-TOH was titrated into SET buffer lacking or containing 1 μM of the indicated protein. Fluorescence was excited at 466 nm, and intensity at 526 nm was recorded. Solid lines represent fit of the data to a simple association model. B) Increasing amounts of the indicated unlabeled competitor were added to pre-loaded TTP·NBD-TOH, and fluorescence at 526 nm was recorded. Analyses of NBD-TOH displacement revealed an apparent affinity of ca. 50 nM between TTP and native tocopheol. C) Titration data are shown for 1 μM wild-type or R59W TTP proteins. Average values and standard deviations were obtained from > 7 titrations, utilizing at least 3 independent protein preparations.

Figure 4. Fluorescence energy transfer from NBD-TOH to TRITC-DHPE

A) Fluorescence emission at 533 nm (excitation = 466 nm) was monitored upon titration with NBD-TOH to liposomes lacking or containing TRITC-DHPE in SET buffer. B) Fluorescence emission (excitation = 466 nm) was monitored upon titration of liposomes containing 100 μM phosphatidylcholine / 0.1 μM TRITC-DHPE with NBD-TOH in SET buffer. C) Fluorescence emission (excitation = 466 nm) of liposomes containing both TRITC-DHPE and NBD-TOH was monitored in SET buffer, before (dotted line) or after (solid line) the addition of 10 μM TTP.

Figure 5. Energy transfer from Marina Blue-DHPE to NBD-TOH

A) Quenching of Marina Blue fluorescence by NBD-TOH. Emission spectra (excitation = 367nm) were collected upon the addition of 0, 0.24, 0.48, 0.71, 0.95, 1.12, 1.42, 1.66, and 1.89 μM NBD-TOH (upper to lower spectra, respectively) to liposomes containing 100μM phosphatidylcholine / 0.1 μM Marina Blue-DHPE in SET buffer. Inset: Linearity of Marina Blue fluorescence quenching by NBD-TOH. Fluorescence intensity at 464nm was plotted as a function of NBD-TOH concentration; data were fit to a line function. B) Release of FRET upon binding of NBD-TOH by TTP. Fluorescence emission was monitored before (upper trace) and after (lower trace) the addition of 1.2 μM NBD-TOH. Following the addition of 5μM TTP, Marina-Blue fluorescence is restored (middle trace). Excitation: 367 nm; spectral resolution = ± 7 nm.

Figure 6. TTP-induced tocopherol release from membrane bilayers

A) Time-dependent changes in FRET between NBD-TOH and TRITC-DHPE (lower curve) or Marina-Blue-DHPE (upper curve), upon mixing with TTP. Five μM TTP (or 30μM GST as a control protein) were mixed with sonicated vesicles containing 25μM phosphatydilcholine, 0.2μM NBD-TOH, and 0.125 μM fluorescent lipid (TRITC-DHPE or Marina-Blue-DHPE). FRET from Marina Blue-DHPE to NBD-TOH was followed by monitoring the recovery of Marina-Blue fluorescence (excitation: 365 nm; emission: 464 nm). FRET from NBD-TOH to TRITC-DHPE was followed by monitoring TRITC emission (575 nm) while exciting NBD-TOH (466 nm). Raw fluorescence values were normalized to the value of fluorescence change under each condition. Raw data (dots) were fitted to a function summing an exponential and a linear term (solid line). B) Impaired tocopherol transfer activity of TTP(R59W). Liposomes containing 25 μM phosphatydilcholine, 0.125 μM TRITC-DHPE, and 0.2 μM NBD-TOH were mixed with 30 μM active TTP (wild-type or R59W), and the fluorescence changes recorded as described in panel A.

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