Characterization and identification of the inhibitory domain of GDF-8 propeptide
Characterization and identification of the inhibitory domain of GDF-8 propeptide
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DOI：
10.1016/j.bbrc.2004.01.085
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发表时间：
2004-03-12
影响因子：
3.1
通讯作者：
Stotish, R
中科院分区：
文献类型：
--
作者：
Jiang, MS;Liang, LF;Stotish, R
GDF - 8是骨骼肌质量的一种负性调节因子。调节GDF - 8生物学活性的机制尚未阐明。与转化生长因子-β（TGF - β）系统类似，GDF - 8前肽与GDF - 8结合并抑制其活性。在这些研究中，我们确定了GDF - 8前肽抑制活性所必需的关键结构域。两分子的GDF - 8前肽单体可抑制一分子GDF - 8同型二聚体的生物学活性。尽管在哺乳动物细胞中合成时前肽含有N - 连接糖基化，但这种糖基化对于抑制GDF - 8并非必需。利用细菌表达系统，我们表达并纯化了保留完全抑制活性的GDF - 8前肽。为了确定前肽的功能区域，我们表达了一系列截短的谷胱甘肽S - 转移酶（GST） - 前肽融合蛋白，并检测了它们的抑制活性。我们观察到含有C - 末端区域（氨基酸残基99 - 266）的融合蛋白非常稳定，但不表现出抑制活性；而含有N - 末端区域（氨基酸残基42 - 115）的融合蛋白不稳定，但具有关键的抑制活性。数据表明，C - 末端区域可能在GDF - 8前肽的稳定性中起作用，并且抑制结构域位于氨基酸42和115之间的区域。（C）2004爱思唯尔公司。保留所有权利。
GDF-8 is a negative regulator of skeletal muscle mass. The mechanisms which regulate the biological activity of GDF-8 have not yet been elucidated. Analogous to the TGF-beta system, GDF-8 propeptide binds to and inhibits the activity of GDF-8. In these studies, we define the critical domain of the GDF-8 propeptide necessary for inhibitory activity. Two molecules of GDF-8 propeptide monomer inhibit the biological activity of one molecule of GDF-8 homodimer. Although the propeptide contains N-linked glycosylation when synthesized in mammalian cells, this glycosylation is not necessary for the inhibition of GDF-8. Taking advantage of the bacterial expression system, we express and purify GDF-8 propeptide which retains full inhibitory activity. To define the functional regions of the propeptide, we express a series of truncated GST-propeptide fusion proteins and examined their inhibitory activity. We observe that fusion proteins containing the C-terminal region (amino acid residues 99-266) are very stable, but do not exhibit inhibitory activity; while fusion proteins containing the N-terminal region (amino acid residues 42-115) are labile but contain essential inhibitory activity. The data suggest that the C-terminal region may play a role in the stability of the GDF-8 propeptide and that the inhibitory domain is located in the region between amino acids 42 and 115. (C) 2004 Elsevier Inc. All rights reserved.