Abstract

Vitamin D binding protein (DBP), a major serum protein, binds to vitamin D and its metabolites with high affinity, and in turn, transports them to target organs and tissues, leading to the observed calciotropic properties of vitamin D hormone. Our objectives are to probe the vitamin D sterol-binding domain of DBP by our recently developed photoaffinity labelling method. Thus, we plan to photoaffinity label purified human serum DBP (hDBP) with 3H-25-ANE, a radiolabelled photoaffinity analog of 25-hydroxyvitamin D3, followed by proteolytic and chemical cleavages, and isolation of the labelled peptide/peptides. Mass spectrometric analysis of this peptide fragment/fragments will enable us to define the vitamin D-binding site in hDBP. We will also develop additional photoaffinity analogs of 25-OH-D3 containing (i) photoactive groups at various part of the parent molecule, (ii) spacer arm having different lengths, and (iii) photo probes with carbene precursors; and carry out studies similar to those described with 3H-25-ANE. This will lead to unequivocal identification of the vitamin D sterol-binding domain in hDBP as well as points of contact within the binding site.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK044337-01A2
Application #
3245892
Study Section
General Medicine B Study Section (GMB)
Project Start
1993-01-01
Project End
1995-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118

  Publications

Ray, Arjun; Swamy, Narasimha; Ray, Rahul (2008) Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D. Biochem Biophys Res Commun 365:746-50
Swamy, Narasimha; Ray, Rahul (2008) Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different. Bioorg Chem 36:165-8
Fernandez-Gacio, Ana; Fernandez-Marcos, Carlos; Swamy, Narasimha et al. (2003) Affinity labeling of the nuclear vitamin D receptor with nonsteroidal alkylating agents. Bioorg Med Chem Lett 13:213-6
Kisker, Oliver; Onizuka, Shinya; Becker, Christian M et al. (2003) Vitamin D binding protein-macrophage activating factor (DBP-maf) inhibits angiogenesis and tumor growth in mice. Neoplasia 5:32-40
Addo, James K; Swamy, Narasimha; Ray, Rahul (2002) The C(19) position of 25-hydroxyvitamin D(3) faces outward in the vitamin D sterol-binding pocket of vitamin D-binding protein. Bioorg Med Chem Lett 12:279-81
Swamy, Narasimha; Head, James F; Weitz, Daniel et al. (2002) Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein. Arch Biochem Biophys 402:14-23
Swamy, N; Ghosh, S; Schneider, G B et al. (2001) Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity. J Cell Biochem 81:535-46
Mohr, S C; Swamy, N; Xu, W et al. (2001) Why do we need a three-dimensional architecture of the ligand-binding domain of the nuclear 1alpha,25-dihydroxyvitamin D(3) receptor? Steroids 66:189-201
Swamy, N; Xu, W; Paz, N et al. (2000) Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3. Biochemistry 39:12162-71
Swamy, N; Addo, J; Vskokovic, M R et al. (2000) Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols. Arch Biochem Biophys 373:471-8

Showing the most recent 10 out of 23 publications