Glutamic acid 207 in rodent T-cell RT6 antigens is essential for arginine-specific ADP-ribosylation.
A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that kcat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.
Hara
N
N
Department of Biochemistry, Shimane Medical University, 89-1, Izumo, Shimane 693, Japan.
Tsuchiya
M
M
Shimoyama
M
M
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D001120:Arginine / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006649:Histocompatibility Antigens / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology*
D000945:Antigens, Differentiation, T-Lymphocyte; D006649:Histocompatibility Antigens; D008562:Membrane Glycoproteins; D000246:Adenosine Diphosphate Ribose; D018698:Glutamic Acid; D001120:Arginine; D036002:ADP Ribose Transferases; C449322:Art2a protein, mouse; C090163:Art2b protein, rat