The Institute for Environmental and Gender-Specific Medicine, Urayasu, Chiba 279-0021, Japan.
Nitric Oxide. 2011 Aug 1;25(2):176-82. doi: 10.1016/j.niox.2011.05.005. Epub 2011 May 27.
Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels (p<0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC-ESI-MS-MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.
神经元生长因子（NGF）在 PC12 细胞中的信号转导，PC12 细胞源自大鼠肾上腺髓质嗜铬细胞瘤，可诱导神经元型一氧化氮合酶（nNOS）的表达和一氧化氮（NO）的产生。随后，NO 导致 PC12 细胞向具有形态变化的神经元细胞分化，如轴突延伸。在这项研究中，我们表明含 6-硝基色氨酸的蛋白质在 PC12 细胞（未分化的 PC12 细胞）和/或 NGF 诱导的 PC12 细胞（分化的 PC12 细胞）中产生。用抗 6-硝基色氨酸抗体对未分化的 PC12 细胞和分化的 PC12 细胞的蛋白质提取物进行 Western blot 分析，显示出几个免疫反应性条带，随后用胰蛋白酶消化并进行 LC-ESI-MS-MS 分析。来自五个核糖体蛋白的肽，即 60S 核糖体蛋白 L7（Trp154）、60S 酸性核糖体蛋白 P1（Trp43）、40S 核糖体蛋白 S2（Trp60）、40S 核糖体蛋白 S6（Trp45）和 40S 核糖体蛋白 S19（Trp52），被鉴定为含硝基色氨酸残基的蛋白质，具有显著的离子得分水平（p<0.05）。在这些蛋白质中，只有 S19 蛋白在分化的 PC12 细胞中观察到色氨酸硝化，而只有 L7 蛋白在未分化的 PC12 细胞中观察到色氨酸硝化。核糖体蛋白 P1、S2 和 S6 的色氨酸硝化在未分化和分化的 PC12 细胞中均有观察到。Western 印迹中含硝基色氨酸的蛋白质的阳性信号约为 16 kDa（Band 1），包括 40S 核糖体蛋白 S19，用 NOS 抑制剂 L-NAME 处理后被抑制。LC-ESI-MS-MS 分析该样品时未观察到 40S 核糖体蛋白的色氨酸硝化。这是首次不仅在存活的培养细胞中，而且在生理过程（细胞分化）中鉴定蛋白质上硝基色氨酸的几个特定位点的研究。
