Characterization of 25-Hydroxyvitamin D Binding Protein from Intestinal Cells Dorothy Teegarden, 1 Kwang Park Nickel, and Lingling Shi Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907 Received July 30, 2000 We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat entero- cytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we dem- onstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxy- vitamin D (25OHD) with at least a 1000-fold greater affinity than 1,25-dihydroxyvitamin D (1,25(OH) 2 D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH) 2 D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respec- tively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nu- clear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH) 2 D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH) 2 D and cBDP is distinct from the nuclear VDR. © 2000 Academic Press Key Words: 25-hydroxyvitamin D; vitamin D; intes- tine; steroid hormone; binding protein. The active vitamin D metabolite, 1,25 dihydroxyvi- tamin D (1,25(OH) 2 D), has well known effects in regu- lating calcium absorption and maintaining serum cal- cium levels within a narrow range. In addition, 1,25(OH) 2 D affects cellular metabolism, including cell growth and differentiation (1). The metabolic precursor to 1,25(OH) 2 D, 25 hydroxyvitamin D (25OHD) is present in serum in high levels, absorbed from the diet and may have metabolic effects of its own in regulating cell growth and calcium metabolism (2–7). Several proteins have been identified that regulate vitamin D metabolism and action. 25OHD as well as 1,25(OH) 2 D are transported via a well characterized serum vitamin D binding protein (sDBP) and in some mammals, albumin. In addition, a nuclear vitamin D receptor (VDR) mediates gene regulation events trig- gered by 1,25(OH) 2 D which can up- or down-regulate the transcription of genes encoding a variety of pro- teins, including calbindins, osteopontin, osteocalcin, c-Fos and c-Myc through the vitamin D response ele- ment (VDRE) on specified genes (1). In addition, sev- eral lines of evidence demonstrate that 1,25(OH) 2 D regulates cellular events, including absorption, via pathways independent of the nuclear VDR (8 –11). Ev- idence suggests that these actions are mediated via a potential membrane receptor for 1,25(OH) 2 D (12–14) or via the nuclear VDR translocating to acceptor sites in the membrane (15). Finally an intracellular vitamin D binding protein (IDBP) which has recently been pu- rified and partially sequenced which preferentially binds 25OHD and is proposed to interact with and regulate nuclear VDR actions (16 –21). This protein is expressed at high levels in New World monkeys and hypothesized to play a role in the resistance to 1,25(OH) 2 D noted in this species (16). Thus, there are several proteins which have been identified which can bind vitamin D metabolites. Our laboratory has partially purified a cytosolic pro- tein from rat enterocytes which may play a role in 25OHD action or metabolism (22). The protein was identified in rat enterocyte cytosol (cDBP) as a mole- cule which binds 25OHD, and whose known character- istics appear to be distinct from sDBP, albumin, the membrane VDR and the nuclear VDR. Cytosolic bind- ing activity for 25OHD was exploited to purify the protein to greater than 95% homogeneity from rat en- terocyte cytosol. The N-terminal sequence of the pro- tein was found to be distinct from all other vitamin D metabolite binding proteins with published sequence (sDBP, albumin, IDBP and nuclear VDR). In the cur- rent studies, cDBP is further characterized in terms of the binding characteristics, regulation by 1,25(OH) 2 D and similarity to other known vitamin D metabolite binding proteins. 1 To whom correspondence should be addressed at Department of Foods and Nutrition, Stone Hall-1264, Purdue University, West Lafayette, IN 47907. Fax: 765-494-0906. E-mail: Teegarden@CFS. purdue.edu. Biochemical and Biophysical Research Communications 275, 845– 849 (2000) doi:10.1006/bbrc.2000.3397, available online at http://www.idealibrary.com on 845 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.