Functional characterization and purification of an 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. These monkeys harbor high circulating concentrations of the prohormone 25-hydroxyvitamin D and the active vitamin D hormone 1, 25-dihydroxyvitamin D. Previous work from this laboratory indicated that resistance is associated with the overexpression of a 60-65-kDa intracellular protein that binds vitamin D metabolites competitively. In the current studies 25-[3H]hydroxyvitamin D3 (25-OHD3) was used as a competitive ligand to investigate the ability of a number of small lipid molecules to interact with this intracellular vitamin D-binding protein (IDBP) in post-nuclear extracts of a prototypical lymphoblast cell line from the common marmoset, a vitamin D-resistant New World primate. Only those vitamin D metabolites with a hydroxyl moiety in the C-25 position were bound by IDBP. Disruption of the C-25 hydroxyl obviated binding, whereas more proximal alterations in the vitamin D side chain did not. Modifications in the A-ring of 25-hydroxylated vitamin D metabolites, most specifically hydroxylation of C-1, diminished but did not abolish ligand binding. Of more than two dozen other small lipid molecules examined, only the C-19 17-hydroxysteroids, 17beta-estradiol and testosterone, and the C-21 steroid progesterone were found to be capable of binding specifically to IDBP. Using a combination of physical and serial chromatographic techniques, we enriched IDBP 25-OHD3 binding activity 17,588-fold in extracts of B95-8 cells. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified fraction demonstrated a predominant 65-kDa molecular species with a pI approximately 4.5. Seven different peptide fragments were isolated from the 65-kDa protein, each possessing sequence similarity to the hsp-70 family of proteins. Ligand binding analyses confirmed that human inducibly expressed hsp-70-bound 25-OHD3 with approximately similar affinity ( approximately 10(-7) M) as did purified IDBP. In summary, these results suggest a novel action for the hsp-70 family of proteins as intracellular vitamin D- and gonadal steroid hormone-binding molecules.
Gacad
M A
MA
Cedars-Sinai Burns and Allen Research Institute, UCLA School of Medicine, Los Angeles, California 90048, USA.
Chen
H
H
Arbelle
J E
JE
LeBon
T
T
Adams
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002112:Calcifediol / Q000378:metabolism; D002117:Calcitriol / Q000378:metabolism; D002427:Cebidae; D002762:Cholecalciferol / Q000031:analogs & derivatives / Q000378:metabolism; D002845:Chromatography; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism; D014809:Vitamin D-Binding Protein / Q000737:chemistry* / Q000302:isolation & purification
D018840:HSP70 Heat-Shock Proteins; D013256:Steroids; D014809:Vitamin D-Binding Protein; D002762:Cholecalciferol; D002117:Calcitriol; D002112:Calcifediol