CHARACTERIZATION OF MAMMALIAN ADP-RIBOSYLTRANSFERASES

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Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Similar to cholera toxin, some mammalian ADP- ribosyltransferases specifically use the guanidino group of arginine as an ADP-ribose acceptor. Five mammalian NAD: arginine ADP- ribosyltransferases (ART) were cloned from various tissues. ART1 was shown to be a cell surface protein, linked through a glycosylphosphatidylinositol (GPI) anchor. The transferases appear to be selectively expressed in mammalian tissues. ART-1 is found in skeletal and cardiac muscle and lymphoid cells, ART-2 in lymphocytes, ART-4 in spleen andART-5 in testis. ART-5 is primarily an NAD glycohydrolase, with significantly less NAD: arginine ADP- ribosyltransferase. Upon incubation with NAD under standard assay conditions, in the presence or absence of agmatine, an arginine analog, the NAD glycohydrolase activity gradually declines; in contrast, the ADP-ribosyltransferase, activity after a brief delay, increases in velocity, and then, after about one hour, appears to be constant for several hours. The loss of NAD glycohydrolase activity and the increase in ADP-ribosyltransferase activity is associated with the appearance on sodium dodecyl sulfate-polyacrylamide gels of an auto-ADP-ribosylated form of ART-5. One ADP-ribosylation appears to be sufficient for the dramatic changes in NAD glycohydrolase and auto-ADP-ribosyltransferase activities. Incubation for greater than one hour, however, results in the further addition of ADP-ribose moieties. To verify that the auto- ADP-ribosylated ART-5 exhibited differences in enzymatic activities, the transferase was incubated with NAD for one or eight hours, then purified by sequential chromatography over two PD-10 gel permeation columns. The auto-ADP-ribosylated enzyme exhibited an increase in ADP- ribosyltransferase activity and decrease in NAD glycohydrolase when compared to control. The fact that ART-5 incubated with NAD was modified with ADP-ribose was verified by mass spectrometry, which demonstrated an increase of 541 mass units. These data support the hypothesis that the auto-ADP-ribosylation can serve a regulatory role and determine the relative levels of NAD glycohydrolase and ADP- ribosyltransferase activities. - Mono-ADP-ribosylation, ADP- ribosyltransferases, bacterial toxins