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

Abstract

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.

Figure 1.. Design of antibody-like ADP-ribose binding reagents.

(A and B) The chemical structure of (A) a poly(ADP-ribosyl)ated amino acid or (B) a mono(ADP-ribosyl)ated amino acid, with the amino acids shown in heteroatomic colors and the ADP-ribose units colored in grey (proximal to the amino acid) and black. The blue box with a dashed line highlights the chemical moiety in the ADP-ribose modification recognized by the WWE domain (the iso-ADP-ribose linkage between two ADP-ribose monomers). The green boxes with dashed lines highlight the chemical moiety in the ADP-ribose modification recognized by the macrodomains. The WWE and macrodomains used in this study are indicated (species and protein). (C) Ribbon diagram depicting the X-ray crystal structure of a monoclonal IgG antibody (PDBID:1IGY), with the variable fragment (Fv) in green and the homodimerized Fc fragment in purple and pink. (D) Schematic diagram of the plasmid constructs used to express the ADP-ribose binding domain-Fc (ARBD-Fc) fusion proteins in bacteria. The constructs contain DNA segments encoding: (1) 10xHis and/or Strep[II] tags, (2) an ADP-ribose binding domain (green), (3) a flexible glycine and serine linker, and (4) rabbit IgG constant fragment (Fc) (pink).

Figure 2.. Expression and purification of ADP-ribose binding domain-Fc (ARBD-Fc) fusion proteins.

ARBD-Fc fusion proteins were expressed in E. coli and purified using Ni-NTA affinity chromatography. (Top) The purified proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows indicate protein bands with the expected molecular weight of the ARBD-Fc fusion proteins. The asterisks indicate breakdown products or contaminating E. coli proteins, which do not alter the functionality of the ARBD-Fc fusion protein. (Bottom) List of four ARBD-Fc fusion proteins and their expected molecular weights in kilodaltons (kDa).

Figure 3.. Immunoblot and dot blot analyses of mono-, oligo-, and poly(ADP-ribosyl)ated PARP proteins using ARBD-Fc fusion proteins.

(A) Purified recombinant PARP-1 and PARP-3 were incubated with or without NAD⁺ under different reaction conditions to promote auto(ADP-ribosyl)ation. The yield was mono(ADP-ribosyl)ated PARP-3 (red), oligo(ADP-ribosyl)ated PARP-1 (green), or poly(ADP-ribosyl)ated PARP-1 (blue). (B through F) Immunoblot analyses. The mono, oligo, and poly(ADP-ribosyl)ated PARP proteins were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the 10H anti-PAR monoclonal antibody, as well as four ARBD-Fc fusion proteins, as labeled. Each lane contained approximately the same number of terminal ADP-ribose units, to our best approximation. The molecular weights in kilodaltons (kDa) are indicated. (G) Dot blot analyses. Purified recombinant PARP-1 and PARP-3 were incubated with or without NAD⁺ under different reaction conditions to promote auto(ADP-ribosyl)ation. The yield was mono(ADP-ribosyl)ated PARP-3 (red), oligo(ADP-ribosyl)ated PARP-1 (green), or poly(ADP-ribosyl)ated PARP-1 (blue). Serial dilutions of the auto(ADP-ribosyl)ated PARP proteins were applied to nitrocellulose membranes for dot blotting using the 10H anti-PAR monoclonal antibody, as well as four ARBD-Fc fusion proteins, as labeled. Each spot contained approximately the same number of terminal ADP-ribose units, to our best approximation.

Figure 4.. Immunoblot blot analysis of ADP-ribosylation in HeLa and MCF-7 cells using ARBD-Fc fusion proteins.

(A) Nuclear extracts were prepared from HeLa cells (left) and MCF-7 cells (right) maintained under standard culture conditions. Aliquots of the nuclear extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated above each lane. (B) Whole cell extracts were prepared from HeLa cells grown in culture following treatment with or without 2 mM H₂O₂ and 20 μM PARP inhibitor PJ34 (PARPi), as indicated. Aliquots of the whole cell extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated.

Figure 5.. Blocking or removing ADP-ribose eliminates detection by ARBD-Fc fusion proteins in immunoblotting assays using cell extracts.

Whole cell extracts were prepared from HeLa cells treated with 2 mM H₂O₂. Aliquots of the extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated above each lane. (A) Immunoblotting after blocking the ADPR-binding reagents with 10 mM free ADPR prior to incubation with the membrane (B) Immunoblotting after treating the membranes with 1 M hydroxylamine, which cleaves ADPR from glutamate and aspartate residues, prior to immunoblotting. (C) Immunoblotting after incubating the extracts with 1 μM purified ARH3, a glycohydrolase that cleaves PAR chains as well as the proximal ADPR units from proteins^–, prior to immunoblotting.

Figure 6.. Immunofluorescent staining of ADP-ribosylation in HeLa cells using ARBD-Fc fusion proteins.

HeLa cells grown in culture on glass cover slips were treated with vehicle, H₂O₂, or PJ34 (PARPi), as indicated. Following treatment, the cells were fixed with methanol, stained with TO-PRO-3 (a DNA stain), and immunostained for ADP-ribose using the ARBD-Fc fusion proteins, as indicated. The cover slips were affixed to glass slides, and the cells were imaged for fluorescence by laser scanning confocal microscopy. (A) Fluorescence images for all conditions and all ADPR-Fc fusion proteins, as indicated. The ADPR (green), DNA (red), or merged images are indicated. Scale bar = 50 μm (same for all images). (B) Nine-fold magnification of selected conditions from (A). Exclusion of staining from the nucleoli is indicated by the yellow arrows. Scale bar = 10 μm (same for all images).

Figure 7.. Immunoprecipitation of ADP-ribosylated PARP-1 from 293T cells using ARBDFc fusion proteins.

(A) Protein A-agarose binding assay with ADPR-Fc fusion proteins. The ADPR-Fc fusion proteins indicated were incubated with protein A-agarose. The efficiency of binding was assessed by SDS-PAGE analyses of the eluted material, with subsequent staining using Coomassie brilliant blue. (B) 293T cells were treated with vehicle or H₂O₂, as indicated. Nuclear extracts prepared from the treated cells were subjected to immunoprecipitation using the indicated ADPR-Fc fusion proteins (the lane numbers above the gel correspond to the number labels for the ADPR-Fc fusion proteins shown in panel A). The immunoprecipitated material was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using an anti-PARP-1 antibody. The molecular weights in kilodaltons (kDa) are indicated.