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Analysis of osteoclast-specificmembrane-bound proteins that are expressed during osteoclast differentiation

破骨细胞分化过程中表达的破骨细胞特异性膜结合蛋白的分析

基本信息

批准号：
06671825
负责人：
HAKEDA Yoshiyuki
金额：
$ 1.41万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

It is postulated that osteoclast-specific functional proteins are expressed during the process of osteoclastic differentiation. Based on this hypothesis, we attempted the identification of an antigen (5H-antigen) for an osteoclast-specific antibody (Mab-5H) that had been established in our laboratory. Using a experimental system of osteoclastic cell formation in culture of rabbit unfractionated bone cells, we found that the 5H-antigen was expressed in the osteoclast percursors in parallel with the expression of tartrate-resistant acid phosphatase (TRAP) whichis a marker enzyme of osteoclast-diffeerntiation. When Mab-5H simultaneously with parathyroid hormone (PTH) was added into the cultures, the formation and the bone-resorbing activity of osteoclastic cells induced by PTH were abolished by the Mab-5H.These results suggest that the 5H-anitgen specifically produced by osteoclast precursors is involved in the differentiation and function of osteoclasts. In addition, 5H-antigen had two d … More ifferent forms in the osteoclasts, e.g., one was a cytosolic form and the other was a plasma membrane-bound form. Thus, we tried to purify these two distinct forms. However, it was difficult to purify the antigen from osteoclast extracts due to the small number of osteoclasts that we could harvest from the bones. Then, we sought other sources beside osteoclasts to purify the 5H-antigens, and found that intestine also expressed the 5H-antigens enough to purify them. The purified 5H-antigen in a membrane-bound form was a highly-glycosylated protein with molecular mass of 65 kDa in SDS-PAGE analysis, and was very unstable. The cytosolic antigen was also a glycoprotein with molecular mass of 600 kDa under a neutral condition and of 65 kDa in SDS-PAGE,and much more atabel the membrane-bound form. Then we sought the amino acid sequence of the cytosolic 5H-antigen. However, the amino acid analysis was unsuccessful because of a blockage of N-terminus of the antigen. We also tried to analyze the amino acid sequence of peptide fragments of the antigen generated by the limited hydrolysis, but we could not obtain the enough amount of the fragment ot be analyzed. In this study, we found the 5H-antigen was predominantly expressed in osteoclast precursors and playd an important role in osteoclast differentiation and function. However, we could not identify the 5H-antigens. Less

据推测，破骨细胞特异性功能蛋白在骨细胞分化过程中表达。基于这一假设，我们尝试鉴定本实验室已建立的破骨细胞特异性抗体（Mab-5 H）的抗原（5 H-抗原）。利用兔未分级骨细胞体外培养成骨细胞实验系统，发现破骨细胞前体中5 H抗原的表达与破骨细胞前体化标志酶抗酒石酸酸性磷酸酶（TRAP）的表达平行。当Mab-5 H与甲状旁腺激素（PTH）共同作用时，Mab-5 H可阻断PTH诱导的破骨细胞的形成和骨吸收活性，提示破骨细胞前体特异性产生的5 H抗原参与破骨细胞的分化和功能。此外，5 H抗原有两个阳性反应， ...更多信息破骨细胞中的不同形式，例如，一种是胞质形式，另一种是质膜结合形式。因此，我们试图净化这两种不同的形式。然而，由于我们可以从骨中收获的破骨细胞数量很少，因此很难从破骨细胞提取物中纯化抗原。然后，我们寻找破骨细胞以外的其他来源来纯化5 H-抗原，发现肠也表达足够的5 H-抗原来纯化它们。纯化的膜结合形式的5 H-抗原在SDS-PAGE分析中是高度糖基化的蛋白，分子量为65 kDa，并且非常不稳定。胞浆抗原也是糖蛋白，中性条件下分子量约为600 kDa，SDS-PAGE电泳显示分子量约为65 kDa，更接近膜结合形式。然后我们寻找胞质5 H抗原的氨基酸序列。然而，由于抗原的N-末端的封闭，氨基酸分析不成功。我们还尝试分析由有限水解产生的抗原的肽片段的氨基酸序列，但是我们不能获得足够量的待分析片段。本研究发现，5 H抗原主要表达于破骨细胞前体细胞中，在破骨细胞的分化和功能中起重要作用。然而，我们不能鉴定5 H抗原。少