Abstract
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.
Highlights
ADP-ribosylation is one of the post-translational modifications of cellular proteins, in which the ADP-ribose moiety of NADϩ is transferred to specific amino acid residues of mostly GTP-binding proteins
An ecto-enzyme activity of NADϩ:arginine ADP-ribosyltransferase was found in myogenically differentiated C2C12 cells, and its substrate was identified as a cell surface adhesion molecule, integrin ␣7 [7]
RT6 alloantigen is expressed in the cell surface of T lymphocytes [11], it is not detected in thymocytes, bone marrow cells, or B lymphocytes [11], suggesting that its expression is restricted to the final stages of post-thymic T lymphocyte development
Summary
ADP-ribosylation is one of the post-translational modifications of cellular proteins, in which the ADP-ribose moiety of NADϩ is transferred to specific amino acid residues of mostly GTP-binding proteins. For ADP-ribosylation of intact RT6 protein present in cell surface, lymphocytes (2– 8 ϫ 106 cells/ml) were incubated with [␣-32P]NADϩ (0.5–10 TBq/mmol) at 37 °C in HBSS containing 2 mM ATP, 2 mM ADP-ribose, 1 mM FAD, 12.5 M NADPϩ, 1 mM MgCl2, 0.1 mM MnCl2, and 10 M CaCl2. When the purified MBP-trRT6.1 having the Mr of 66,000 (Fig. 1, lane 1) was incubated with [␣-32P]NADϩ, the radiolabeled nucleotide was hydrolyzed into [32P]ADP-ribose and nicotinamide (data not shown), as had been observed with RT6.2 exogenously expressed in rat adenocarcinoma cells [9].
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