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

描述（改编自申请人的摘要）：骨酸 糖蛋白-75（BAG-75）是一种新的Mr-75，000磷酸化酸性糖蛋白， 糖蛋白，每摩尔含44摩尔有机磷酸盐。 利率利息 BAG-75来源于其组织特异性：合成受到限制 主要是骨骼和钙化软骨，而蛋白质 也仅在这些组织的提取物中检测到。 最近的研究 破骨细胞个体发生已经证明了“骨细胞”假设 功能特性与细胞表面表达 玻连蛋白受体（以前称为破骨细胞功能性抗原）。 钙化软骨中破骨细胞基质配体的鉴定 骨的情况尚不清楚，但骨酸性糖蛋白-75是一个主要的候选者。 为了这个角色。 第二，BAG-75的Mr+50 kDa推定片段是 与骨中的Mr=75 kDa前体共定位，但另外 在血清中发现。 虽然这两个物种都与抗BAG-75抗体反应， 蛋白质印迹法允许对血液中的每种抗原形式进行单独分析 疾病模型。 第三，其29.3%的ASP/GLU含量， 细胞外定位，以及存在延伸的多酸氨基酸 酸拉伸表明BAG-75可能是一类新的 缺乏“EF手”基序共有序列的金属结合蛋白， 表现出低亲和力，但大容量（即，肌浆网 钙螯合蛋白）。 由于BAG-75的当前提取方法使用4 M 盐酸胍，对构象不可逆影响的关注（和 功能）部位需要备用的提取和制备方法 而不暴露于变性剂。 选择大鼠进行研究是因为 它的可用性，它的骨骼相对不成熟， 正常和转化的骨细胞系统，以及体内的潜力 问题研究 该试点项目的主要目标是：1）确定 BAG-75抗原（Mr+75和/或50 kDa）的水平是否随发作而变化 绝经后骨质疏松症、遗传性骨硬化症和骨肉瘤 在大鼠模型中; 2）纯化“天然”BAG-75，并假定Mr=50 kDa 3）检查BAG-75对DNA片段的影响; 破骨细胞个体发育、表面粘附和吸收活性; 4） 确定破骨细胞产生部分降解的能力 可能影响成骨细胞的BAG-75的产物;和5）确定 BAG-75的钙结合特性和Mt =50 kDa的推测片段， 解决方案，并绑定到一个底层。 虽然骨骼的功能 酸性糖蛋白-75目前是未知的，这些试点项目是 旨在提供基本的生物化学，细胞生物学， 发展功能假说和系统所必需的生理数据 为了进一步研究这种有趣的组织限制性蛋白质， 钙结合电位

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.