Mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase

Mouse T-cell antigens Rt6.1 and Rt6.2 are glycosylphosphatidylinositol-anchored arginine-specific adenosine diphosphate (ADP)-ribosyltransferases. In the present study, we obtained evidence that an arginine-specific ADP-ribosyltransferase activity liberated from BALB/c mouse splenocytes by phosphatidylinositol-specific phospholipase C increased fivefold in the presence of dithiothreitol and that the activity was immunoprecipitated by polyclonal antibodies generated against recombinant rat RT6.1. When mouse Rt6.1 was expressed as a recombinant protein, the transferase activity of Rt6.1 was stimulated by dithiothreitol, and inhibited by N-ethylmaleimide, while activities of recombinant mouse Rt6.2 and the Glu-207 mutant of rat RT6.1 [Hara, N., Tsuchiya, M., and Shimoyama, M. (1996) J. Biol. Chem. 271, 29552-29555] were unaffected by either agent. In addition to four cysteine residues conserved among mouse Rt6 and rat RT6 antigens, Rt6.1 has two extra cysteine residues at positions 80 and 201. To investigate a contribution of these extra cysteines in mouse Rt6.1 to thiol dependency of Rt6.1 transferase activity, Cys-80 and Cys-201 of Rt6.1 were replaced with serine and phenylalanine, respectively, the corresponding residues of mouse Rt6. 2 and rat RT6.1. Transferase activity of the Phe-201 mutant of Rt6.1 lost thiol dependency while that of the Ser-80 mutant remained thiol-dependent. Thus, we conclude that mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase, and that Cys-201 confers thiol dependency on Rt6.1 transferase. Our study indicates that arginine-specific ADP-ribosyltransferase activity detected on BALB/c mouse splenocytes is attributed to Rt6.1 and that Rt6.1 differs from Rt6.2 in enzymatic property of the transferase and perhaps in immunoregulatory functions.