一种用于蛋白质精氨酸特异性 ADP 核糖基化的新检测方法——抗 ADP 核糖基精氨酸抗体与 ADP 核糖基精氨酸水解酶的联合应用。

A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase.

作者信息

Osago Harumi, Terashima Masaharu, Hara Nobumasa, Yamada Kazuo, Tsuchiya Mikako

机构信息

Department of Biochemistry, Faculty of Medicine, Shimane University, Izumo 693-8501, Japan.

出版信息

J Biochem Biophys Methods. 2008 Apr 24;70(6):1014-9. doi: 10.1016/j.jprot.2007.11.008. Epub 2007 Nov 28.

DOI:10.1016/j.jprot.2007.11.008

PMID:18160133

Abstract

Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH). In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (alphaADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised alphaADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with alphaADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.

摘要

精氨酸特异性 ADP-核糖基化是蛋白质翻译后修饰之一，通过将 NAD 的一个 ADP-核糖部分转移到靶蛋白的精氨酸残基上实现。这种修饰由 ADP-核糖基转移酶（Art）催化，并由 ADP-核糖基精氨酸水解酶（AAH）逆转。在本研究中，我们描述了一种将抗 ADP-核糖基精氨酸抗体（αADP-R-Arg Ab）和 AAH 相结合用于检测 ADP-核糖基化靶蛋白的新方法。我们用 ADP-核糖基化组蛋白制备了 αADP-R-Arg Ab，并检测了该抗体与经 Art 和/或 AAH 处理的蛋白质以及小鼠 T 细胞原位 ADP-核糖基化系统中蛋白质的反应性。我们的结果表明，用 αADP-R-Arg Ab 和 AAH 检测 ADP-核糖基化蛋白是探索 ADP-核糖基化靶蛋白的有用工具。我们应用该方法在大鼠中寻找内源性 ADP-核糖基化蛋白，并在具有高 Art 活性的骨骼肌中检测到了可能的靶蛋白。