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

Friedrich Koch-Nolte,David Petersen,Sriram Balasubramanian,Friedrich Haag,Dominik Kahlke,Thomas Willer,Robert Kastelein,Fernando Bazan,Heinz-Günter Thiele

doi:10.1074/jbc.271.13.7686

Friedrich Koch-Nolte, David Petersen + Show 7 more

Open Access

https://doi.org/10.1074/jbc.271.13.7686

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Abstract

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 eucaryotic mono(ADPribosyl)transferases were cloned and found to exhibit significant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6. In this paper we describe secondary structure analyses of Rt6 and related proteins and show conserved structure motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreover, we have expressed soluble mouse Rt6-1 and Rt6-2 gene products in which C-terminal tags (FLAG-His6) replace the native glycosylphosphatidylinositol anchor signal sequences. Purified recombinant Rt6-2, but not Rt6-1, shows NAD+ glycohydrolase activity, which is inhibited by the arginine analogue agmatine. Immunoprecipitation of recombinant Rt6-1 and Rt6-2 with anti-FLAG M2 antibody followed by incubation with [32P]NAD+ leads to rapid and covalent incorporation of radioactivity into the light chain of the M2 antibody. The bound label is resistant to treatment with HgCl2 but sensitive to NH2OH, characteristic of arginine-linked ADP-ribosylation. These results demonstrate that Rt6-1 and 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.

Highlights

Mono ADP-ribosylation is a posttranslational protein modification in which the ADP-ribose (ADPR)1 moiety of NADϩ is transferred from NADϩ to a specific amino acid residue in a target protein, while the nicotinamide moiety is released [1, 2]
Many of the bacterial toxins can modify proteins other than their physiologic targets and, in the absence of target protein, some toxins can use water as an alternative acceptor resulting in the hydrolysis of NADϩ to nicotinamide and ADPR, which can be measured as NADϩ glycohydrolase activity
The results show that these proteins, should be classified as arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31)

Summary

Introduction

Mono ADP-ribosylation is a posttranslational protein modification in which the ADP-ribose (ADPR)1 moiety of NADϩ is transferred from NADϩ to a specific amino acid residue in a target protein, while the nicotinamide moiety is released [1, 2]. For analysis of ADP-ribosyltransferase activity, purified Rt6 or Rt6 precipitated from Sf9 cell supernatants with M2-antibody affinity matrix was suspended in 50 ␮l of PBS (0.2 ␮g Rt6/ml) containing 2 ␮Ci of [32P]NADϩ (5000 Ci/mol, Amersham Corp.) or 2 ␮Ci of [32P]ADPR.

Results

Conclusion