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.