Glutamic Acid 207 in Rodent T-cell RT6 Antigens Is Essential for Arginine-specific ADP-ribosylation

Abstract

A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that k cat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.Arginine-specific ADP-ribosyltransferase catalyzes transfer of the ADP-ribose moiety of NAD to simple guanidino compounds such as arginine or an arginine residue of a target protein, forming ADP-ribose-acceptor adducts (1, 2). Molecular cloning has revealed the primary structures of ADP-ribosyltransferases in eucaryotes, including rabbit (3) and human skeletal muscles (4) and chicken bone marrow cells (5). Homology searches revealed highly conserved regions in the deduced amino acid sequences of chicken and skeletal muscle transferases (3-5). A gene with overall sequence similarity to these transferases was cloned from chicken erythroblasts; the functional expression was not described (6).The only known protein to which these arginine-specific ADP-ribosyltransferases show significant homology is the rat (7,8) and mouse (9) T-cell antigenic system RT6 (3, 5). It has been reported that RT6-specific antisera activate T-cells (10) and that defects in RT6 expression are associated with the pathogenesis of autoimmune insulin-dependent diabetes in diabetes-prone BB rats (11). Based on sequence similarity, Takada et al. (12) examined enzyme activities of the rat RT6.2 expressed in mammary adenocarcinoma cells and found that cells transfected with the RT6.2 gene exhibited NAD glycohydrolase (NADase), but not ADP-ribosyltransferase, activity. In contrast, Haag et al. (13) detected an arginine-specific auto-ADP-ribosylation of RT6.2 but no modification on RT6.1. More recently, Maehama et al. (14) have reported that RT6.1 was auto-ADP-ribosylated at arginine residues. Thus, whether rat RT6 antigens are indeed arginine-specific ADP-ribosyltransferases has remained controversial.We report here that the mutant RT6.1 in which Gln-207 was replaced with glutamic acid exhibited arginine-specific ADPribosyltransferase activity, while wild-type RT6.1 exhibited only NADase activity. Furthermore, the mouse homologue of rat RT6 (MRT6H), a recently characterized arginine-specific ADP-ribo...

Modification of the ADP-ribosyltransferase and NAD Glycohydrolase Activities of a Mammalian Transferase (ADP-ribosyltransferase 5) by Auto-ADP-ribosylation

“…Unlike the rat RT6 proteins, mouse Rt6 -1 is primarily a transferase, with a relatively low level of NADase activity (25). The differences between RT6 and Rt6 appear to result from the presence of glutamine or glutamate, respectively, at the active site (26,27).…”

“…Unlike the rat RT6 proteins, mouse Rt6 -1 is primarily a transferase, with a relatively low level of NADase activity (25). The differences between RT6 and Rt6 appear to result from the presence of glutamine or glutamate, respectively, at the active site (26,27).Two lymphocyte ADP-ribosyltransferases, termed Yac-1 (12) and Yac-2 (13), were cloned from mouse lymphoma (Yac-1) cells. Yac-1, a GPI-linked exoenzyme, is the murine equivalent of ART1 and exhibits 75 and 77% similarity of amino acid sequence to the rabbit and human muscle enzymes, respectively.…”

Elevation of Cellular NAD Levels by Nicotinic Acid and Involvement of Nicotinic Acid Phosphoribosyltransferase in Human Cells

“…NAPRT was immunodetected with antihuman NAPRT and peroxidase-conjugated anti-mouse IgG (MBL, Nagoya, Japan) antibodies, as described previously (27). Protein loading was assessed using rabbit anti-actin (Sigma) and anti-rabbit IgG (MBL) antibodies.…”

In silicoinvestigation of PARP-1 catalytic domains inholoandapostates for the design of high-affinity PARP-1 inhibitors

“…This computational result is in a good agreement with the experimental finding which pointed out that His862 and Glu988 have significant contributions to the interactions at the active site 35,36 . Among the PARP-1 active site amino acids, Glu988 is important for NAD + catalysis 37,38 , Lys903 is critical for the DNA polymerization process 39 . The second part of the inhibitor molecule forms strong hydrophobic interaction with His862 of beta sheet-like Glu988, it also participates in NAD + binding 40 .…”