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首页> 外文期刊>Journal of cellular physiology. >A missense mutation sheds light on a novel structure-function relationship of RANKL
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A missense mutation sheds light on a novel structure-function relationship of RANKL

机译：一个错义突变揭示了小说RANKL的结构关系

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摘要

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-kappaB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.
机译：肿瘤坏死因子(TNF)——核心领域核factor-kappaB受体激活配体(RANKL)是一个功能领域至关重要对破骨细胞分化。错义突变患者识别osteoclast-poor常染色体隐性骨硬化病(ARO)位于残留蛋氨酸199被替换为赖氨酸(M199K)在TNF-like核心域。这种突变的关系还不清楚。序列排列显示片段包含人类M199高度守恒和相当于M200老鼠。定点诱变,我们生成的三个突变细胞RANKL M200K by A / E (M200s)取代200年蛋氨酸和赖氨酸(M200K),丙氨酸(M200A)和谷氨酸(M200E),代表不同的物理性质。TRAcP染色和骨坑试验表明m200未能破骨细胞形成和支持骨吸收,伴随着受损osteoclast-related信号转导。没有发现在m200拮抗效应野生型老鼠RANKL。RANKL结构预测,这蛋氨酸残留坐落的疏水核心内的蛋白质,因此,可能是至关重要的蛋白质折叠和稳定性。差示扫描荧光分析建议m200不太稳定。污点分析分析进一步揭示了受损由m200 RANKL三聚。receptor-ligand绑定试验显示打断了交互m200的内在受体。分子基础的人类M199-induced ARO和阐明啮齿动物的不可或缺的作用残留M200(相当于人类M199)RANKL函数。

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