MOUSE T-CELL MEMBRANE-PROTEINS RT6-1 AND RT6-2 ARE ARGININE PROTEIN MONO(ADPRIBOSYL)TRANSFERASES AND SHARE SECONDARY STRUCTURE MOTIFS WITH ADP-RIBOSYLATING BACTERIAL TOXINS

Mono ADP-ribosylation is a posttranslational protein modification that has been implicated in the regulation of key biological functions in bacteria as well as in animals, Recently, the first cDNAs for eucaryot ic mono(ADPribosyl)transferases were cloned and found to exhibit signi ficant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6, In this paper we describe secondary stru cture analyses of Rt6 and related proteins and show conserved structur e motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreo ver, we have expressed soluble mouse Rt6-1 and Rt6-2 gene products in which C-terminal tags (FLAG-His(6)) replace the native glycosylphospha tidylinositol anchor signal sequences. Purified recombinant Rt6-2, but not Rt6-1, shows NAD(+) glycohydrolase activity, which is inhibited b y the arginine analogue agmatine. Immunoprecipitation of recombinant R t6-1 and Rt6-2 with anti-FLAG M2 antibody followed by incubation with [P-32]NAD(+) leads to rapid and covalent incorporation of radioactivit y into the light chain of the M2 antibody, The bound label is resistan t to treatment with HgCl2 but sensitive to NH2OH, characteristic of ar ginine-linked ADP-ribosylation. These results demonstrate that Rt6-1 a nd Rt6-2 possess the enzymatic activities typical for NAD(+)-dependent arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31), They are the first such enzymes to be molecularly characterized in the immune system.