Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins
Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins
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DOI：
10.1021/acs.biochem.7b00670
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发表时间：
2017-12-05
期刊：
影响因子：
2.9
通讯作者：
Kraus, W. Lee
中科院分区：
文献类型：
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作者：
Gibson, Bryan A.;Conrad, Lesley B.;Kraus, W. Lee
adp核糖基化是一种酶催化的蛋白质翻译后修饰，其中NAD+的adp核糖（ADPR）部分被转移到底物蛋白质中的特定氨基酸上。adp核糖基化的生物学功能是多种多样的，从正常生理状态到病理状态。对ADPR不同形态和功能的生物化学和细胞研究需要可用于检测和富集的免疫试剂。缺乏一套完整的工具来识别所有形式的ADPR[即单核苷酸、寡核苷酸和多核苷酸（adp -核糖）]阻碍了进展。在此，我们描述了一组重组抗体样adp核糖结合蛋白的产生和表征，其中天然存在的ADPR结合结构域，包括大结构域和WWE结构域，通过融合到兔免疫球蛋白的Fc区而功能化。这些试剂可识别具有不同特异性的所有形式的ADPR，可用于广泛的基于抗体的检测，如免疫印迹、细胞免疫荧光染色和免疫沉淀。这些实验的观察结果表明，ADPR的生物学比以前认为的更加多样化、丰富和复杂。本文描述的ARBD Fc融合蛋白将为未来探索adp核糖的化学、生物化学和生物学提供有用的工具。
ADP-ribosylation is an enzyme-catalyzed post-translational modification of proteins in which the ADP-ribose (ADPR) moiety of NAD+ is transferred to a specific amino acid in a substrate protein. The biological functions of ADP-ribosylation are numerous and diverse, ranging from normal physiology to pathological conditions. Biochemical and cellular studies of the diverse forms and functions of ADPR require immunological reagents that can be used for detection and enrichment. The lack of a complete set of tools that recognize all forms of ADPR [i.e., mono-, oligo-, and poly(ADP-ribose)] has hampered progress. Herein, we describe the generation and characterization of a set of recombinant antibody-like ADP-ribose binding proteins, in which naturally occurring ADPR binding domains, including macrodomains and WWE domains, have been functionalized by fusion to the Fc region of rabbit immunoglobulin. These reagents, which collectively recognize all forms of ADPR with different specificities, are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation. Observations from these assays suggest that the biology of ADPR is more diverse, rich, and complex than previously thought. The ARBD Fc fusion proteins described herein will be useful tools for future exploration of the chemistry, biochemistry, and biology of ADP-ribose.