Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins,Biochemistry

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Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins
Biochemistry ( IF 2.9 ) Pub Date : 2017-11-22 00:00:00 , DOI: 10.1021/acs.biochem.7b00670
Bryan A. Gibson _{1,

2} , Lesley B. Conrad _{1,

2,

3} , Dan Huang _{1,

2,

4} , W. Lee Kraus _{1,

2}

Affiliation

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.

中文翻译：

重组抗体样ADP核糖结合蛋白的产生与表征

ADP-核糖基化是酶催化的蛋白质翻译后修饰，其中NAD ⁺的ADP-核糖（ADPR）部分将其转移至底物蛋白中的特定氨基酸。ADP-核糖基化的生物学功能是多种多样的，范围从正常生理到病理状况。对ADPR多种形式和功能的生化和细胞研究需要可用于检测和富集的免疫试剂。缺乏能够识别所有形式的ADPR的完整工具集（即单，寡和聚（ADP-核糖））阻碍了进展。本文中，我们描述了一组重组抗体样ADP-核糖结合蛋白的生成和表征，其中天然存在的ADPR结合域（包括大域和WWE域）已通过与兔免疫球蛋白的Fc区融合而功能化。这些试剂，它们可以共同识别具有不同特异性的所有形式的ADPR，可用于各种基于抗体的测定，例如免疫印迹，细胞的免疫荧光染色和免疫沉淀。这些测定的观察结果表明，ADPR的生物学比以前认为的要多样化，丰富和复杂。本文所述的ARBD-Fc融合蛋白将成为未来探索ADP-核糖的化学，生物化学和生物学的有用工具。

更新日期：2017-11-22

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