TY - JOUR
T1 - Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins
AU - Gibson, Bryan A.
AU - Conrad, Lesley B.
AU - Huang, Dan
AU - Kraus, W. Lee
N1 - Funding Information:
*Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-8511. Phone: 214-648-2388. Fax: 214-648-0383. E-mail: Lee.kraus@utsouthwestern.edu. ORCID W. Lee Kraus: 0000-0002-8786-2986 Author Contributions W.L.K. conceived of the ARBD−Fc fusion proteins. B.A.G. designed and generated the fusion protein expression constructs, expressed and purified the fusion proteins, and conducted the biochemical assays. L.B.C. performed the ADPR detection assays (immunoblotting and immunofluorescence) in HeLa and MCF-7 cells. D.H. performed the ADP-ribose blocking and removal assays in Figure 5. B.A.G., L.B.C., and D.H. designed and executed the experiments and analyzed the data, with input from W.L.K. W.L.K. secured funding and provided intellectual support. B.A.G., L.B.C., and W.L.K. prepared the figures and wrote the paper. L.B.C. and D.H. contributed equally to this work. Funding This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK069710) and the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K. Notes The authors declare the following competing financial interest(s): W.L.K. is a founder and consultant for Ribon Therapeutics, Inc. W.L.K. and B.A.G. hold the patent on the ARBD−Fc fusion proteins described herein (U.S. Patent 9,599,606). The University of Texas Southwestern Medical Center has licensed the fusion proteins to EMD Millipore, which markets them for research purposes.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/12/5
Y1 - 2017/12/5
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.biochem.7b00670
DO - 10.1021/acs.biochem.7b00670
M3 - Article
C2 - 29053245
AN - SCOPUS:85038124025
VL - 56
SP - 6305
EP - 6316
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 48
ER -