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

Mercedes A. Gacad, Hong Chen, Jonathan E. Arbelle, Thomas LeBon, John S. Adam

Research output: Contribution to journalArticlepeer-review

55 Scopus citations

Abstract

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, 17β-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 ~ 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 (~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.

Original languageEnglish
Pages (from-to)8433-8440
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number13
DOIs
StatePublished - 28 Mar 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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