Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase.
Mono-ADP-ribosylation of arginine is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing reactions in the cycle. ADP-ribosylarginine hydrolases differ in their dithiothreitol (DTT) requirements. Rat and mouse hydrolases require DTT for maximal activity, but calf, guinea pig, and human hydrolases are DTT-independent. To define the molecular basis for these differences, brain ADP-ribosylarginine hydrolases were cloned. Deduced amino acid sequences of mouse and rat hydrolases were 94% identical with 5 conserved cysteines. The human hydrolase sequence was 83% identical to that of rat but contained only 4 cysteines with cysteine 108 in rat corresponding to serine 103 in human. To investigate the role of rat cysteine 108, human and rat wild-type hydrolases and mutants, in which serine 103 in human was replaced by cysteine (S103C) and cysteine 108 in rat was replaced by serine (C108S), were expressed in Escherichia coli. Affinity-purified anti-rat brain hydrolase antibodies reacted with recombinant wild-type rat hydrolase, but only weakly with the C108S mutant. They did not react with human wild-type or the S103C mutant. Human hydrolase and rat C108S were DTT-independent; human S103C was, however, DTT-dependent. These data clearly show that cysteine 108 in rat hydrolase plays a critical role in DTT dependence and may be important in immunoreactivity.
Takada
T
T
Laboratory of Cellular Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Iida
K
K
Moss
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004229:Dithiothreitol / Q000494:pharmacology; D006026:Glycoside Hydrolases; D006168:Guinea Pigs; D006801:Humans; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D006867:Hydrolases; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase; D004229:Dithiothreitol