STRUCTURE/FUNCTION STUDIES OF VITAMIN D BINDING PROTEIN

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DESCRIPTION (Adapted from the Applicant's Abstract): 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.