NAD(+)-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes.
Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase. Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows. When intact rat lymphocytes expressing RT6.1 mRNA were incubated with [alpha-32P]NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000. The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum. The radioactivity incorporated into the 31-kDa protein was recovered as 5'-[32P]AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment. These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue. When intact lymphocytes, in which RT6.1 had been once modified by [32P]ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the [32P]ADP-ribosylated RT6.1. The reduced radioactivity was recovered from the incubation medium as [32P]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture. Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes.
Maehama
T
T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Nishina
H
H
Hoshino
S
S
Kanaho
Y
Y
Katada
T
T
D036002:ADP Ribose Transferases; D018528:ATP-Binding Cassette Transporters; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D001120:Arginine; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006649:Histocompatibility Antigens / Q000096:biosynthesis / Q000378:metabolism*; D007519:Isoantigens / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D009243:NAD / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012910:Snake Venoms; D013601:T-Lymphocytes / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000945:Antigens, Differentiation, T-Lymphocyte; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D017261:Glycosylphosphatidylinositols; D006649:Histocompatibility Antigens; D007519:Isoantigens; D058619:Maltose-Binding Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012910:Snake Venoms; C461599:maltose transport system, E coli; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D000249:Adenosine Monophosphate; D008320:Maltose; D001120:Arginine; D036002:ADP Ribose Transferases; C090163:Art2b protein, rat; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase