Characterization of Mouse Rt6.1 NAD:Arginine ADP-ribosyltransferase

Joel Moss,Linda A Stevens,Eleanor Cavanaugh,Ian J Okazaki,Rita Bortell,Toshihiro Kanaitsuka,John P Mordes,Dale L Greiner,Aldo A Rossini

doi:10.1074/jbc.272.7.4342

Abstract

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.

Highlights

Chloride-induced autoimmune glomerulonephritis [10]
Expression of mouse Rt6 protein by immunoblotting and RT6 gene expression appear to be restricted to T cells [18, 21]
It was suggested that cytotoxic T cell (CTL) activity in mice may be regulated by GPI-linked ADP-ribosyltransferases that have many of the characteristics of the rat RT6 proteins [22, 23] and catalyze the modification of surface proteins [24]

Summary

EXPERIMENTAL PROCEDURES

Materials pAcSG2 vector was purchased from PharMingen (San Diego, CA); pMAMneo expression vector and ligation express kit from Clontech (Palo Alto, CA); gene Amp PCR reagent kit and DNA thermal cycler from Perkin-Elmer; ABI 394 DNA/RNA synthesizer from Applied Biosystems, Inc. (Foster City, CA); NuSieve and GTG-agarose from FMC Corp. (Rockland, ME); QIAEX gel extraction kit and QIAGEN plasmid purification kit from QIAGEN (Chatsworth, CA); Tris acetate-EDTA buffer, LB broth, Eagle’s minimal essential medium with Earle’s balanced salt solution containing L-glutamine, Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum, heat-inactivated, from BioWhittaker, Inc. (Walkersville, MD); XhoI and ampicillin, sodium salt from Boehringer Mannheim; NheI, DH5␣-competent cells, G418, buffer-saturated phenol, and phenol/chloroform/isoamyl alcohol (25:24:1) from Life Technologies, Inc.; 3 M sodium acetate from Quality Biological Inc. (Gaithersburg, MD); ethanol from the Warner Graham Co. (Cockeyesville, MD); rat mammary adenocarcinoma cells (NMU cells) from American Type Culture Collection (Rockville, MD); dexamethasone sodium phosphate from MG Scientific (Buffalo Grove, IL); trypsin and 7-deaza-dGTP sequencing kit from U. Materials pAcSG2 vector was purchased from PharMingen (San Diego, CA); pMAMneo expression vector and ligation express kit from Clontech (Palo Alto, CA); gene Amp PCR reagent kit and DNA thermal cycler from Perkin-Elmer; ABI 394 DNA/RNA synthesizer from Applied Biosystems, Inc. (Rockland, ME); QIAEX gel extraction kit and QIAGEN plasmid purification kit from QIAGEN (Chatsworth, CA); Tris acetate-EDTA buffer, LB broth, Eagle’s minimal essential medium with Earle’s balanced salt solution containing L-glutamine, Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum, heat-inactivated, from BioWhittaker, Inc. (San Francisco, CA); ␤-NAD, phosphatidylinositol-specific phospholipase C (PI-PLC), and agmatine sulfate from Sigma; [adenine-U-14C]NAD (243 mCi/mmol), [carbonyl14C]nicotinamide adenine dinucleotide (53 mCi/mmol), and L-[U14C]arginine monohydrochloride (297 mCi/mmol) from Amersham Corp.; Dowex AG1-X2 resin, 200 – 400 mesh, chloride form, from BioRad; Long Ranger gel solution from J.

Methods

AND DISCUSSION

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