IN VIVO/IN VITRO STUDIES OF BONE ACIDIC GLYCOPROTEIN-75

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DESCRIPTION (Adapted from the Applicant's Abstract): Bone acid glycoprotein-75 (BAG-75) is a new Mr-75,000 phosphorylated acidic glycoprotein with 44 moles of organic phosphate per mole. Interest in rat BAG-75 derives from its tissue specificity: synthesis is restricted predominantly to bone and calcifying cartilage, whereas the protein was also only detected in extracts of these tissues. Recent studies of osteoclast ontogeny have demonstrated that assumption of "osteoclastic" functional properties closely parallels cell surface expression of a vitronectin receptor (previously denoted as osteoclast functional antigen). The identity of the matrix ligand for osteoclasts in calcified cartilage and bone is unknown, but bone acidic glycoprotein-75 is a prime candidate for this role. Second, a Mr+50 kDa presumed fragment of BAG-75 is co-localized with the Mr=75 kDa precursor in bone, but is additionally found in serum. Although both species react with anti-BAG-75 antibodies, Western blotting permits separate analysis of each antigenic form in blood from disease models. Third, its 29.3% ASP/GLU content, its presumed extracellular location, and the presence of an extended polyacidic amino acid stretch suggest that BAG-75 may be a member of a new class of metal-binding proteins devoid of "EF hand" motif consensus sequences which exhibit low affinity, but large capacity (i.e., sarcoplasmic reticulum calsequestrin). Since the current method for extraction of BAG-75 uses 4M guanidine-HCL, concern over irreversible effects upon conformation (and function) sites necessitates alternate means for extraction and preparation without exposure to denaturants. The rat was chosen for study because of its availability, the relative immaturity of its bone, the existence of normal and transformed bone cell systems, and the potential for in vivo studies. The principal goals of this pilot project are to: 1) determine whether the level of BAG-75 antigens (Mr+75 and/or 50 kDa) vary with onset of postmenopausal osteoporosis, hereditary osteopetrosis, and osteosarcoma in rat models; 2) purify "native" BAG-75 and the Mr=50 kDa presumed fragment without use of denaturants; 3) examine the effect of BAG-75 on osteoclast ontogeny, surface adhesion, and resorption activity; 4) determine the capacity of osteoclasts to produce partial degradation products of BAG-75 which may influence osteoblasts; and 5) determine the calcium-binding properties of BAG-75 and the Mr=50 kDa presumed fragment in solution and as bound to a substratum. Although the function of bone acidic glycoprotein-75 is presently unknown, these pilot projects are intended to provide fundamental biochemical, cell biological, and physiological data necessary to develop functional hypotheses and systems for further study of this interesting tissue-restricted protein with calcium-binding potential.