Generation of polyclonal antiserum for the detection of methylarginine proteins
Generation of polyclonal antiserum for the detection of methylarginine proteins
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DOI：
10.1016/j.jim.2007.01.006
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发表时间：
2007-03-30
影响因子：
2.2
通讯作者：
Aletta, John M.
中科院分区：
文献类型：
--
作者：
Duan, Peng;Xu, Ye;Aletta, John M.
这份报告描述了一种基于免疫试剂的研究甲基精氨酸蛋白生物学的方法，该试剂能够识别细胞蛋白的甲基精氨酸状态。基于在已验证的含有二甲基精氨酸的蛋白质中发现最普遍的氨基酸序列基序，制备了两种形式的免疫肽。其中一种形式的多肽由7个精氨酸残基和8个甘氨酸残基交替构成。合成中使用的精氨酸都没有甲基化。该肽的另一种形式是用相同的重复GRG序列合成的，但在每个精氨酸残基上带有不对称的二甲基精氨酸。利用后者制备了甲基精氨酸特异性抗血清。分别用或不用不对称二甲基精氨酸合成的富含甘氨酸精氨酸的多肽的酶联免疫吸附试验和免疫印迹试验证明了该抗血清的甲基特异性。甲基精氨酸特异性抗体与高度甲基化的天然核素蛋白共定位，明显集中在核仁中。仅当GRG肽或RBP16分别与重组蛋白精氨酸甲基转移酶1或细胞提取物孵育后，甲基精氨酸特异性抗血清才识别GRG肽并在细菌中表达RBP 16。从处于不同发育状态的细胞中分离出的蛋白质在蛋白质印迹中显示出不同的反应性模式。最后，甲基精氨酸特异性试剂与细胞hnRNPA1的甲基精氨酸和在培养的PC12细胞中表达的人类脆性X智力低下蛋白特异性地相互作用。一种能够检测细胞甲基蛋白质的甲基精氨酸状态的免疫试剂将有助于在广泛的研究和生物医学应用中对蛋白质精氨酸甲基化进行细胞和分子分析。(C)2007 Elsevier B.V.保留所有权利。
This report describes an approach for the study of the biology of methylarginine proteins based on the generation of immunological reagents capable of recognizing the methylarginine status of cellular proteins. Two forms of an immunizing peptide were prepared based upon an amino acid sequence motif found most prevalently among verified dimethylarginine-containing proteins. One form of the peptide was constructed with 7 arginine residues alternating with 8 glycine residues. None of the arginines used in the synthesis were methylated. The alternative form of the peptide was synthesized with the identical repeating GRG sequence, but with asymmetrical dimethylarginine at each arginine residue. A methylarginine-specific antiserum was generated using the latter peptide. ELISA and western blotting of glycine arginine-rich peptides, each synthesized with or without asymmetric dimethylarginine, demonstrate the methyl specificity of the antiserum. The methylarginine-specific antibody co-localizes with the highly methylated native nucleolin protein conspicuously concentrated in the nucleolus. The methylarginine-specific antiserum recognizes a GRG peptide and bacterially expressed RBP 16 only after incubation of the peptide or RBP16 with recombinant protein arginine methyltransferase 1, or cell extracts, respectively. Proteins isolated from cells in different developmental states exhibit different patterns of reactivity observed by western blots. Finally, the methylarginine-specific reagent interacts specifically with the methylarginine of cellular hnRNPA1 and human fragile X mental retardation protein expressed in cultured PC12 cells. An immunological reagent capable of detecting the methylarginine status of cellular methylproteins will facilitate the cellular and molecular analysis of protein arginine methylation in a wide variety of research and biomedical applications. (c) 2007 Elsevier B.V. All rights reserved.