FUNCTIONAL-CHARACTERIZATION AND PURIFICATION OF ALL INTRACELLULAR VITAMIN-D-BINDING PROTEIN IN VITAMIN-D-RESISTANT NEW-WORLD PRIMATE CELLS - AMINO-ACID-SEQUENCE HOMOLOGY WITH PROTEINS IN THE HSP-70 FAMILY

Most genera of New World primates exhibit resistance to vitamin D. The se monkeys harbor high circulating concentrations of the prohormone 25 -hydroxyvitamin D and the active vitamin D hormone 1,25-dihydroxyvitam in D, Previous work from this laboratory indicated that resistance is associated with the overexpression of a 60-65-kDa intracellular protei n that binds vitamin D metabolites competitively, In the current studi es 25-[H-3]hydroxyvitamin D-3 (25-OHD3) was used as a competitive liga nd to investigate the ability of a number of small lipid molecules to interact with this intracellular vitamin D-binding protein (IDBP) in p ost-nuclear extracts of a prototypical lymphoblast cell line from the common marmoset, a vitamin D-resistant New World primate, Only those v itamin D metabolites with a hydroxyl moiety in the C-25 position were bound by IDBP, Disruption of the C-25 hydroxyl obviated binding, where as more proximal alterations in the vitamin D side chain did not. Modi fications in the A-ring of 25-hydroxylated vitamin D metabolites, most specifically hydroxylation of C-l, diminished but did not abolish lig and binding, Of more than two dozen other small lipid molecules examin ed, only the C-19 17-hydroxysteroids 17 beta-estradiol and testosteron e, and the C-21 steroid progesterone were found to be capable of bindi ng specifically to IDBP. Using a combination of physical and serial ch romatographic techniques, we enriched IDBP 25-OHD, binding activity 17 ,588-fold in extracts of B95-8 cells. Two-dimensional sodium dodecyl s ulfate-polyacrylamide gel electrophoresis of this purified fraction de monstrated a predominant 65-kDa molecular species with a pI similar to 4.5, Seven different peptide fragments were isolated from the 65-kDa protein, each possessing sequence similarity to the hsp-70 family of p roteins, Ligand binding analyses confirmed that human inducibly expres sed hsp-70-bound 25-OHD, with approximately similar affinity (similar to 10(-7) M) as did purified IDBP. In summary, these results suggest a novel action for the hsp-70 family of proteins as intracellular vitam in D- and gonadal steroid hormone-binding molecules.