A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization.
Vitamin E (alpha-tocopherol) is an essential dietary nutrient for humans and 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., Spycher, 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 bovine liver. Here, we describe the identification, the molecular cloning into Escherichia coli, and the in vitro expression of the human homologue of bovine TAP, hTAP. This protein appears to belong to a family of hydrophobic ligand binding proteins, which have the CRAL (cis-retinal binding motif) sequence 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. Northern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed. The highest amounts of hTAP message are found in liver, brain, and prostate. In conclusion, hTAP has sequence homology to proteins containing the CRAL_TRIO structural motif. TAP binds to alpha-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14.
Zimmer
S
S
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Stocker
A
A
Sarbolouki
M N
MN
Spycher
S E
SE
Sassoon
J
J
Azzi
A
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D050356:Lipid Metabolism; D008074:Lipoproteins; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D015534:Trans-Activators; D014810:Vitamin E / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008074:Lipoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C432422:SEC14L2 protein, human; D015534:Trans-Activators; C017763:alpha-tocopherol transfer protein; D014810:Vitamin E; D003488:Cyanogen Bromide