Vitamin D-binding protein (DBP) binds vitamin D and its metabolites as well as G-actin with high affinity and serves to transport the former to target tissues, and to scavenge the latter so as to prevent its polymerization during cell-injury. DBP also binds fatty acids, and enhances complement activation on neutrophil chemotaxis by binding to C5a des Arg. Additionally, DBP is known to be converted in vivo to a potent macrophage and osteoclast activating factor (DBP-MAF). The objectives of this proposal are to study various structure-functional aspects of DBP and DBP-MAF using a multiple-methods approach including chemical modification of the vitamin D sterol and fatty acid-binding domains of DBP by affinity/photoaffinity labeling, manipulations of the DBP-gene to express mutants and segments of DBP and their functional characterization, determination of the 3-D structures of DBP and DBP-actin complex by X-ray crystallography, and macrophage-superoxide production and bone resorption assays (for DBP-MAF activities). Multi-tasking nature of DBP is directly related to specific recognition/binding of various ligands by DBP. Such processes are strongly influenced by the structure of the protein, particularly the 3-D structures of the domains of the protein that are responsible for such recognition and binding. Hence, these studies will provide information about the importance of independent domains in the multi-domained structure of DBP, possible sharing of a common binding pocket by various endogenous ligands, degree of cross-talk among various domains, interdependence among various ligands, and influence of ligand-binding on DBP-MAF activities. The role of DBP is crucial in the metabolic activation of 1,25(OH)2D3, the calciotropic hormone, and its tissue-specific delivery, so that the latter can be responsible in calcium and phosphorus homeostasis, regulation of growth and maturity of cells, antiproliferation of malignant cells, and immunoregulation. Interaction of DBP with G-actin is important in the prevention of actin-polymerization, and clogging of arteries during cellular injury. On the other hand, macrophage-activation by DBP-MAF has stressed the possible immunoregulatory property of DBP. Furthermore, osteoclast-activation (by DBP-MAF) has raised the possibility that this cytokine may be involved in inflammatory joint diseases such as osteoarthritis, rheumatoid arthritis, periodontal diseases, etc. The proposed structure-function studies should be valuable in evaluating multiple functions and their possible physiological implications of DBP and DBP-MAF.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK044337-04A2
Application #
2016464
Study Section
Special Emphasis Panel (ZRG4-GMB (04))
Program Officer
Margolis, Ronald N
Project Start
1993-01-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Boston University
Department
Miscellaneous
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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
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; 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
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

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