Gorski J P, Griffin D, Dudley G, Stanford C, Thomas R, Huang C, Lai E, Karr B, Solursh M
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110.
J Biol Chem. 1990 Sep 5;265(25):14956-63.
Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.
制备了抗肽和抗蛋白质抗血清，它们都能识别骨酸性糖蛋白-75（分子量=75,000）以及钙化组织和/或血清中一种明显的片段或生物合成中间体（分子量=50,000）。从长期储存后纯化蛋白经蛋白水解可产生紧密间隔的分子量为50,000的双峰多肽这一事实来看，提示存在片段-前体关系。未检测到与骨桥蛋白、骨唾液蛋白或小骨蛋白聚糖的反应性。骨酸性糖蛋白-75占新生颅骨或生长板外植体培养物、颅骨外植体培养物以及大鼠骨肉瘤细胞合成的总放射性标记蛋白的0.5-1%。外植体培养物和颅骨外植体培养物产生的量与成骨细胞的主要产物骨桥蛋白的量相似。在骨肉瘤培养物中，80%的标记抗原与细胞层部分相关，其中特异性免疫沉淀沉淀出分子量为50,000和75,000大小的抗原。骨酸性糖蛋白-75（分子量=75,000）在新生大鼠骨骼和生长板组织的4M盐酸胍/0.5 EDTA提取物中富集，而在心脏、肺、脾、肝、脑和肾中基本不存在。这些非钙化组织的外植体培养物也合成骨酸性糖蛋白-75抗原，但产生的量仅为颅骨培养物的5%或更少。通过免疫组织化学，抗原性与长骨的骨干和钙化软骨相关，但在相关软组织中不存在。这些发现表明骨酸性糖蛋白-75在抗原性上是独特的，主要定位于钙化组织，是正常成骨细胞的主要产物，并且在体内和体外可能会经历特征性的片段化。
