ADP Ribosylation

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What Is Adenosine Diphosphate-Ribosylation?

Adenosine diphosphate-ribosylation (ADPr) is a reversible form of post-translational modification (PTM) and involves the addition of ADP-ribose to specific amino acids on target proteins. This process is controlled by transferases, including poly (ADP-ribose) polymerases (PARPs), which add either a single ADP-ribose to their targets (mono-ADPr) or create branching ADPr chains (poly-ADPr) (Qi et al. 2020). PARP1 is an example of a transferase which is able to create branching ADPr chains and is important within the DNA damage response, rapidly producing ADPr chains on damaged chromatin.

ADPr Function and Therapeutic Potential

In addition to the DNA damage response, ADPr controls multiple fundamental biological processes including cell proliferation and differentiation, metabolism, stress, and the immune response (Palazzo et al. 2019). Dysregulation of ADPr has been implicated in inherited and acquired diseases including neurological diseases and cancer. For this reason, control of ADPr is of therapeutic interest. PARP inhibitors, which suppress DNA damage repair and induce apoptosis in tumor cells with defective repair, are used for the treatment of multiple cancer types (Kassab et al. 2020).

ADPr Research

ADPr is understudied relative to other forms of PTM such as phosphorylation and methylation. This is partly explained by the lack of robust tools for studying this process; it has remained a challenge to generate antibodies that specifically recognize ADPr events. However, novel methods for the generation of ADP-ribosylated peptides using recombinant antibody technology have recently resulted in the successful generation of five antibodies (Bonfiglio et al. 2020). The recombinant antibodies were generated using Human Combinatorial Antibody Libraries (HuCAL®) and a proprietary method of phage display.

HuCAL technology is proven and well published and has been used by the custom antibody team at Bio-Rad, to generate antibodies for research and diagnostic applications since 2004. Read HuCAL explained to discover more about this recombinant technology and watch our “Designed Just for You” video to see inside Bio-Rad’s custom antibody facility, and learn how your requirements for specialized custom antibodies can become a reality through the use of HuCAL technology.

ADPr Antibodies

The ADPr antibodies, shown in Table 1, provide different options for the detection of ADPr, including antibodies that are pan and site specific. The site-specific antibodies are particularly important as reagents capable of unambiguously detecting cellular ADPr by PARP1 when its active site is completed by HPF1. The mono-specific antibodies have enabled specific immunoaffinity purification of mono-ADP-ribosylated substrates, with the identification of 272 mono-ADP-ribosylated sites on 151 primary PARP1 targets. There are also 6 Fab formats of these full-length antibodies for the detection of ADPr. Tagged with a SpyTag2 at the C-terminus of the Fab heavy chain, it enables the user to couple these antibodies to a SpyCatcher reagent for conversion to alternative formats in less than an hour.

The antibodies have been characterized by ELISA, immunofluorescence, and western blotting. They are valuable tools for further investigation of ADP-ribosylation in a variety of biological processes, opening previously inaccessible avenues of research. Bonfiglio et al. (2020) have published details about the generation, characterization, and use of these antibodies.

Table 1. HuCAL generated antibodies for the detection of ADPr.

Target	Antibody Specification	Antibody Clone	Applications	Image	Catalog #	Fab format Catalog #
Pan-ADP-ribose	Recognizes both mono- and poly- ADPr	AbD33641	WB, ELISA		HCA353	(Pan-ADP-ribose): TZA024
Mono-ADP-ribose	Recognizes mono-ADPr Binding preference for ubiquitin Arg-ADPr and mono-ADPr catalyzed by other PARPs	AbD33204	ELISA, IF, WB		HCA354	(Mono-ADP-ribose): TZA019
Mono-ADP-ribose	Recognizes mono-ADPr Binding preference for Ser-mono-ADP-riboses	AbD33205	IF, WB		HCA355	(Mono-ADP-ribose): TZA021
PARP1-S499-ADP-ribose	Recognizes PARP1, ADP-ribosylated at serine 499 Does not detect poly-ADP-ribose in a site-specific manner	AbD34251	ELISA, WB		HCA356	(PARP1-S499-ADP-ribose): TZA022
H3-S10-ADP-ribose	Recognizes histone H3, ADP-ribosylated at serine 10 Also recognizes the less abundant H3 site H3-S28-ADP-ribose, with a lower affinity Does not detect poly-ADP-ribose in a site-specific manner	AbD33644	ELISA, WB		HCA357	(H3-S10-ADP-ribose): TZA023
Mono-ADP-ribose	Recognizes mono-ADPr Binding preference for mono serine-ADPr Fab format	AbD43647	ELISA, IF, WB		N/A	TZA020

Abbreviations: IF, immunofluorescence; WB, western blotting.

Additional Antibodies

To further facilitate research into ADPr and PARPs, Bio-Rad also offers:

Table 2. Antibodies for the detection of ADPr.

Abbreviations: IF, immunofluorescence; IHC-F, immunohistology – frozen sections; IHC-P, immunohistology – paraffin sections; IP, immunoprecipitation; WB, western blotting.

*A PrecisionAb Antibody that has enhanced western blotting validation.

References

• Bonfiglio J et al. (2020). An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation. Cell 183, 1,086–1,102.
• Kassab MA et al. (2020). Targeting dePARylation for cancer therapy. Cell Biosci 10. Accessed September 16, 2021.
• Palazzo L et al. (2019). ADP-ribosylation signalling and human disease. Open Biol 9; 190041
• Qi H et al. (2020). Multiple roles for mono- and poly(ADP-ribose) in regulating stress responses. Trends Genet 35, 159–172.