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

描述(改编自申请者摘要)：骨酸 糖蛋白-75(BAG-75)是一种新的MR-75000磷酸化酸性蛋白 每摩尔含有44摩尔有机磷酸盐的糖蛋白。对老鼠的兴趣 BAG-75源于其组织特异性：合成受到限制 主要是骨骼和钙化的软骨，而蛋白质是 也只在这些组织的提取物中检测到。最新的研究 破骨细胞个体发育学证明了“破骨细胞”假设 功能特性与细胞表面的表达密切相关 Vitronectin受体(以前称为破骨细胞功能抗原)。 钙化软骨中破骨细胞基质配体的同源性 骨骼尚不清楚，但骨酸性糖蛋白-75是首选候选蛋白 为了这个角色。其次，BAG-75的MR+50 kDa推定片段是 与MR=75 kDa前体在骨骼中共定位，但另外 在血清中发现。尽管这两个物种都能与抗BAG-75抗体发生反应， Western blotting允许对血液中的每种抗原形式进行单独分析 来自疾病模型。第三，其ASP/GLU含量为29.3%，其推定 胞外位置，以及延伸的多酸氨基的存在 酸性拉伸提示BAG-75可能是一个新的类别 缺乏“EF Hand”基序的金属结合蛋白 亲和力低，但容量大(即肌浆网 钙调素)。由于目前提取BAG-75的方法采用4M 盐酸胍，关注对构象的不可逆影响(和 功能)部位需要替代的提取和制备手段 没有暴露在变性剂中。这只老鼠被选为研究对象是因为 它的可用性，它的骨骼的相对不成熟， 正常和转化的骨细胞系统，以及体内移植的可能性 学习。这一试点项目的主要目标是：1)确定 BAG-75抗原(MR+75和/或50 kDa)水平是否随发病而变化 绝经后骨质疏松症、遗传性骨质疏松和骨肉瘤 在大鼠模型中；2)纯化天然的BAG-75，推测其MR=50 kDa 不使用变性剂的碎片；3)检测BAG-75对 破骨细胞个体发育、表面黏附和吸收活性；4) 测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产物；以及5)测定 BAG-75和MR=50 kDa假定片段的钙结合特性 溶液和AS与底物结合。虽然骨骼的功能 酸性糖蛋白-75目前尚不清楚，这些试点项目是 旨在提供基本的生化、细胞生物学和 开发功能假说和系统所需的生理数据 为了进一步研究这种有趣的组织限制性蛋白， 钙结合潜力。

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.