Identification and Characterization of a Mammalian 39-kDa Poly(ADP-ribose) Glycohydrolase

Shunya Oka,Jiro Kato,Joel Moss

doi:10.1074/jbc.m510290200

Abstract

ADP-ribosylation is a post-translational modification resulting from transfer of the ADP-ribose moiety of NAD to protein. Mammalian cells contain mono-ADP-ribosyltransferases that catalyze the formation of ADP-ribose-(arginine) protein, which can be cleaved by a 39-kDa ADP-ribose-(arginine) protein hydrolase (ARH1), resulting in release of free ADP-ribose and regeneration of unmodified protein. Enzymes involved in poly(ADP-ribosylation) participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. We report here the identification of an ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, which exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG.

Highlights

Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to specific amino acid in proteins, altering their structure and function [1]
Materials—[adenine-U-14C]NAD (252 mCi/mmol) was purchased from Amersham Biosciences; [U-14C]L-arginine (50 mCi/mmol) and [adenylate-32P]NAD were from PerkinElmer Life Sciences; ␤-NAD was from Sigma; Affi-Gel boronate was from Bio-Rad; the plasmid DNA isolation QIAprep Spin Miniprep Kit was from Qiagen (Valencia, CA); the ABI PRISM Big Dye Cycle Sequencing Ready Reaction kit was from Applied Biosystems (Foster City, CA); glutathione-Sepharose 4B was from Amersham Biosciences; and cholera toxin A subunit, pertussis toxin, Pseudomonas exotoxin A, and Botulinum C3 enzyme were from List Biological Laboratories (Campbell, CA)
ARH3 is more identical to the catalytic region of the 111-kDa poly(ADP-ribose) glycohydrolase (PARG) (19%) than to ARH1 (10%) or ARH2 (13%)

Summary

EXPERIMENTAL PROCEDURES

Materials—[adenine-U-14C]NAD (252 mCi/mmol) was purchased from Amersham Biosciences; [U-14C]L-arginine (50 mCi/mmol) and [adenylate-32P]NAD were from PerkinElmer Life Sciences; ␤-NAD was from Sigma; Affi-Gel boronate was from Bio-Rad; the plasmid DNA isolation QIAprep Spin Miniprep Kit was from Qiagen (Valencia, CA); the ABI PRISM Big Dye Cycle Sequencing Ready Reaction kit was from Applied Biosystems (Foster City, CA); glutathione-Sepharose 4B was from Amersham Biosciences; and cholera toxin A subunit, pertussis toxin, Pseudomonas exotoxin A, and Botulinum C3 enzyme were from List Biological Laboratories (Campbell, CA). For Western blotting, HepG2 human liver carcinoma cells were purchased from the American Type Culture Collection (Manassas, VA); goat antirabbit IgG conjugated to horseradish peroxidase was from Southern Biotech (Birmingham, AL); SuperSignal chemiluminescent substrate was from Pierce; and the QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Mouse brain membrane fraction (1 mg of protein) as ADP-ribose acceptor plus activated cholera toxin and 10 ␮M [32P]NAD (10 ␮Ci/reaction), 10 mM MgCl2, and 5 mM DTT in 50 mM potassium phosphate, pH 7.5 (total volume, 1 ml), were incubated at 37 °C for 2 h. Extracted G-protein (1 mg) as ADP-ribose acceptor plus activated pertussis toxin (50 ␮g), 10 ␮M [32P]NAD (10 ␮Ci/reaction), 10 mM MgCl2, and 5 mM DTT in 50

PCR primers

RESULTS

DISCUSSION