Regulatory role of arginine 204 in the catalytic activity of rat alloantigens ART2a and ART2b.
ART2a (RT6.1) and ART2b (RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), NH2-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited enhanced NADase activity. Incubation with NAD and auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b (R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine. Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.
Stevens
Linda A
LA
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590, USA. stevensl@nhlbi.nih.gov
Bourgeois
Christelle
C
Bortell
Rita
R
Moss
Joel
J
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D017931:DNA Primers; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000945:Antigens, Differentiation, T-Lymphocyte; D017931:DNA Primers; D008562:Membrane Glycoproteins; D001120:Arginine; D036002:ADP Ribose Transferases; C090163:Art2b protein, rat