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Hara, N, Terashima, M, Shimoyama, M and Tsuchiya, M (2000) Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity. J. Biochem. 128:601-7

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.

PubMed

ADP Ribose Transferases; Adenosine Diphosphate Ribose/metabolism; Animals; Antigens, Differentiation, T-Lymphocyte; COS Cells; Cysteine/genetics; Cysteine/metabolism; Dithiothreitol/pharmacology; Escherichia coli; Ethylmaleimide/pharmacology; Histocompatibility Antigens/chemistry; Histocompatibility Antigens/genetics; Histocompatibility Antigens/immunology; Histocompatibility Antigens/metabolism; Hydrolysis/drug effects; Kinetics; Macrophages, Peritoneal/metabolism; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mutation; NAD/metabolism; NAD+ Nucleosidase/chemistry; NAD+ Nucleosidase/genetics; NAD+ Nucleosidase/immunology; NAD+ Nucleosidase/metabolism; Poly(ADP-ribose) Polymerases/metabolism; Recombinant Fusion Proteins/metabolism; Sulfhydryl Compounds/metabolism; Sulfhydryl Compounds/pharmacology; T-Lymphocytes/drug effects; T-Lymphocytes/enzymology; T-Lymphocytes/immunology; Transfection