Lead toxicity remains a major health problem in the U.S. affecting 17% of all preschool children. Bone is its major reservoir. Although animal and bone cell studies have revealed significant insights into pb2+ toxicity, at the molecular level very little is known. Some of the properties of a noncollagenous protein secreted from the osteoblast, osteocalcin, have been found to be altered by Pb2+. Osteocalcin plays a role in bone mineralization as well as resorption. Structural features associated with its function are a Ca2+ dependent alpha-helical conformation in the region containing its 3 gamma- carboxyglutamic acid (Gla) residues which is necessary for enhanced binding to bone mineral, hydroxyapatite. An accessible COOH terminus is thought to be involved in promoting bone resorption. While Ca2+ promotes binding, Pb2+ has been found to inhibit the binding of osteocalcin to hydroxyapatite. The mechanism for this is unknown. Presently, no high resolution structures have been determined for Ca2+ or Pb2+ - osteocalcin complexes and very little in general is known about the effect of a toxic metal on structure and function relationships in proteins. The goal of this research is to further understand the mechanism of Pb2+ toxicity at the molecular level, in bone, by comparing precise structural and functional data of Ca2+ - and Pb2+ - osteocalcin.
The specific aims are to: 1) Determine whether Pb2+ effects the binding of osteocalcin to hydroxyapatite in-vitro and in mineralizing osteoblastic bone cells. 2) Elucidate a mechanism for the enhanced binding of Ca2+-osteocalcin to hydroxyapatite comparing the distances between the free side chain COOH groups, from the 2D NMR structure, to the known Ca2+ spacings in the hydroxyapatite crystal. 3) Elucidate a mechanism for the inhibitory effect of Pb2+ on osteocalcin binding to hydroxyapatite by contrasting the structure of Pb2+- osteocalcin to that of Ca2+-osteocalcin. 4) To determine and compare the structures of Ca2+- and Pb2+-osteocalcin using small amounts of human (2Gla) osteocalcin. 5) To investigate whether Pb2+ alters the role of osteocalcin as a regulator of mineralization. The results of this study should aid in elucidating a molecular mechanism for Pb2+ toxicity.
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