CAROTENOID BINDING PROTEIN AND CAROTENOID METABOLISM

George Washington University, Washington DC

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Abstract

Our hypothesis is as follows: In analogy with retinoid binding proteins, if carotenoid action is mediated through cellular carotenoid binding protein (CCBP), its molecular characterization, organ distribution, regulation and possible function are very important physiologically. Our ongoing studies in ferrets showed the following: 1. 0.5 percent taurocholate (TC) and 13.4 percent fat in the diet increased the absorption and tissue distribution of dietary beta-carotene by 3-fold, whereas 1 percent TC and 23 percent fat increased its conversion to vitamin A by 10-fold. 2. CCBP was purified to homogeneity as a 67kD protein with a Kd of 56 nM and a polyclonal anti-CCBP was prepared. PI has the following preliminary studies: i. Western- blot showed CCBP existence in the intestine and other tissues; ii. Anti-CCBP was shown to quantitatively precipitate labeled CCBP from liver extract and was used to determine in vivo [3H]leucine incorporation into immunoprecipitable liver [3H]CCBP; iii. A 800bp cDNA containing the internal sequence of CCBP has been synthesized by RT-PCR and has been successfully subcloned into a plasmid vector. Northern blot analysis of ferret liver total RNA using the 800bp cDNA probe showed the existence of CCBP mRNA in the ferret liver; iv. CCBP was shown to be different from CD36 or fatty acid transport proteins; and v. CCBP-bound beta- carotene served as a substrate for carotene dioxygenase (CDO) that was stimulated by CRBP II. The experimental approaches in this grant proposal should help to accomplish the following specific aims: 1. Further Characteristics of purified native CCBP. 2. Cloning of the cDNA for CCBP and its deduced amino acid sequence. 3. Effect of dietary beta-carotene on the tissue concentration of CCBP, its mRNA and hepatic relative synthetic rate of CCBP. 4. Effects of moderate Taurocholate and Fat and high Taurocholate and Fat diets on tissue concentration of CCBP, its mRNA and its hepatic relative synthetic rate. 5. Role and characteristics of CCBP and CRBP II in CDO reaction. The PI has expertise in the following systems and techniques to accomplish the above specific aims: 1. Affinity and immuno-affinity chromatography, gel electrophoresis and specific binding assays for the characterization of CCBP. 2. Molecular biology techniques to measure CCBP mRNA levels in different tissues and to clone its cDNA. 3. HPLC procedures for quantification of various carotenoids and retinoids.