Characterization of mouse Rt6.1 NAD:arginine ADP-ribosyltransferase

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.