A novel human tocopherol-associated protein - Cloning, in vitro expression, and characterization

Vitamin E (alpha-tocopherol) is an essential dietary nutrient for humans an d animals. The mechanisms involved in cellular regulation as well as in the preferential cellular and tissue accumulation of alpha-tocopherol are not yet well established. We previously reported (Stocker, A., Zimmer, S., Spyc her, S. E., and Azzi, A. (1999) IUBMB Life 48, 49-55) the identification of a novel 46-kDa tocopherol-associated protein (TAP) in the cytosol of bovin e liver. Here, we describe the identification, the molecular cloning into E scherichia coil, and the in vitro expression of the human homologue of bovi ne TAP, hTAP. This protein appears to belong to a family of hydrophobic lig and binding proteins, which have the CRAL (cis-retinal binding motif) seque nce in common. By using a biotinylated alpha-tocopherol derivative and the IASys resonant mirror biosensor, the purified recombinant protein was shown to bind tocopherol at a specific binding site with K-d 4.6 x 10(-7) M. Nor thern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed. The highest amounts of hTAP message ar e found in liver, brain, and prostate. In conclusion, hTAP has sequence hom ology to proteins containing the CRAL_TRIO structural motif. TAP binds to a lpha-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14.