Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

Atsushi Kasamatsu,Motoyuki Nakao,Brian C Smith,Lindsay R Comstock,Tohru Ono,Jiro Kato,John M Denu,Joel Moss

doi:10.1074/jbc.m111.237636

Abstract

O-acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD(+)-dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2″ and C-3″ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in α-anomeric linkage to C-1″ of ADP-ribose. Because both ARH3- and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1″ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2″- or 3″-position, and we postulated the existence of a third isomer, 1″-OAADPr, in equilibrium with 2″- and 3″-isomers. A third isomer, consistent with 1″-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3″-OAADPr predominated. Consistent with our hypothesis, IC(50) values for ARH3 inhibition by 2″- and 3″-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H(2)(18)O resulted in incorporation of one (18)O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1″ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1″-O linkage in their structurally diverse substrates.

Highlights

Mono-ADP-ribosylation is a post-translational modification, in which the ADP-ribose (ADPr)5 moiety of NAD is transferred to an acceptor protein [1]
These studies demonstrate that ARH1 and ADP-ribosylhydrolase 3 (ARH3) show a similar preference for substrates and that hydrolysis proceeds with attack at the C-1Љ position
Prior studies that showed the presence of C-2Љ and C-3Љ O-acetyl-ADP-ribose are in agreement with our current findings that at neutral pH, the equilibrium favors these two isomers and not C-1Љ O-acetyl-ADP-ribose

Summary

EXPERIMENTAL PROCEDURES

Materials—␤-NAD, ADPr, DTT, TFA, and acetonitrile were purchased from Sigma. Adenine [U-14C]NAD (252 mCi (1 Ci ϭ 37 GBq)) was from GE Healthcare. Identification of Isomers of O-Acetyl-ADP-ribose—For identification of OAADPr isomers, 1 ␮M OAADPr in 50 mM potassium phosphate (pH 9.0), 10 mM MgCl2, and 5 mM DTT (total volume, 200 ␮l) were incubated (5 min at room temperature), before separation of isomers using RP-HPLC and analysis by MALDI-TOF mass spectrometry as described previously [11]. Reaction mixtures (200 ␮l) containing 50 mM potassium phosphate (pH 7.0), 10 mM MgCl2, 5 mM DTT, 16.7 pmol of purified human ARH3, 12.5 ␮M [3H]OAADPr, and either 2Љ- or 3Љ-N-acetyl-ADPr, as indicated, were incubated for 30 min at 30 °C. Assays containing 25 ␮M ␣-ADP-ribosylarginine and purified recombinant ARH1 (3 pmol) in 50 mM potassium phosphate (pH 5.0, 7.0, or 9.0), 10 mM MgCl2, and 5 mM DTT (total volume, 200 ␮l) were incubated for the indicated time at 30 °C.

AB C

RESULTS

HO O

DISCUSSION