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

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 vitamin E to protect cells against oxidative stress is thought to render it essential for membrane, protein, and DNA integrity. Vertebrates evolved efficient mechanisms for the selective retention of RRR- α-tocopherol (TOH) over other dietary tocols. This discrimination results from the combined effects of enzymatic degradation of other forms of the vitamin (3), and the high affinity of RRR-α-tocopherol to the hepatic tocopherol transfer protein (TTP; 4, 5). This 32 kDa protein is a member of the CRAL-TRIO family of proteins that bind small lipids such as phosphoinositides and retinoids (6). TTP was initially identified as a protein in liver cytosol that stimulates TOH transfer between membranes (7, 8). The transfer protein was subsequently shown to enhance TOH secretion into culture media when over-expressed in cultured hepatocytes (9-11). It is generally accepted that by virtue of its ability to facilitate the secretion of TOH from hepatocytes to circulating lipoproteins, TTP functions as a key regulator of vitamin E status. The critical role of TTP in maintaining normal TOH levels is evident in patients with the syndrome Ataxia with Isolated Vitamin E Deficiency (AVED). This autosomal recessive disorder is characterized by low plasma levels of TOH, presumably giving rise to elevated oxidative stress that, in turn, lead to neurodegeneration (12). All known AVED patients bear mutations in the gene encoding TTP (13). The syndrome is thus thought to arise from structural defects in TTP that compromise its tocopherol transfer activity. Indeed, mice with targeted disruption of the TTP gene exhibit low plasma TOH levels, demonstrate signs of elevated oxidative stress, and eventually develop the neurological symptoms associated with AVED (14-17). A number of mutations in TTP have been reported, most of which disrupt the primary structure of the protein (18-22). Six naturally occurring mutations that result in single amino acid substitutions in TTP are associated with the AVED pathology. Human carriers of the R59W, E141K, and R221W mutations develop an early-onset, severe form of the disease. Notably, mutations in homologous residues in other CRAL-TRIO proteins abolish their ligand transfer function (23, 24). In contrast, carriers of the three other missense mutations, occurring in non-conserved residues (H101Q, A120T, and R192H), develop a later-onset, milder form of the disease. 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. While facilitation of inter-membrane tocopherol transfer in vitro represents the “signature” activity of TTP, the molecular mechanisms underlying this activity are presently not clear. To date, thorough understanding of TTP's mechanism of action has been hampered by intrinsic limitations in the assays used to monitor the protein's transfer activity. 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. To overcome these experimental problems, we devised an assay that relies on the optical properties of a fluorescent TOH analog to monitor TTP activity. The availability of this novel experimental tool opens the door for detailed mechanistic studies of TTP action, and will greatly expand our understanding of this important regulatory protein.