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

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Abstract
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 Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and 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 Gln207 was replaced by Glu, 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‐ribosyltransferase activity, indicating that the Glu‐rich motif near the carboxy terminus plays an important role in the enzyme activity.

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