Increase in ADP-ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini, Although the first Glu in the common motif is replaced by Gin (Q(207)EEVLIP) in rat T lymphocyte alloantigens RT6.1 anti RT6.2, the two RT6s appear to have both activities of NAD(+) glycohydrolase and ADP-ribosyltransferase to a lesser extent, To investigate the significance of the Glu-rich motif in the two enzyme activities, we produced a mutant RT6.1 (Q207E), in which Gln(207) was replaced by Gin, together with wild-type RT6s, in Escherichia coli, Kinetic analysis revealed that there were no marked differences in the V-max, and K-m values of NAD(+) glycohydrolases among the three recombinant proteins, The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, although the latter modification was somewhat higher than the former, In contrast, much greater auto-modification was observed for the Q207E mutant, Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate, Thus, the single amino acid mutation of RT6.1 caused a marked increase in its ADP-ribosylate transferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.