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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP Ribose Transferases*
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Adenosine Diphosphate Ribose / metabolism
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Amino Acid Sequence
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Animals
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Antigens, Differentiation, T-Lymphocyte
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Arginine
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Bacterial Toxins / chemistry*
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Base Sequence
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Cell Membrane / enzymology
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Chickens
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Chromatography, Affinity
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Cloning, Molecular
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Conserved Sequence
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DNA Primers
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Histocompatibility Antigens / biosynthesis
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Histocompatibility Antigens / chemistry*
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Histocompatibility Antigens / metabolism*
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Membrane Glycoproteins*
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Mice
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Mice, Inbred BALB C
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Mice, Inbred C57BL
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Mice, Inbred Strains
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Molecular Sequence Data
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NAD / metabolism
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Poly(ADP-ribose) Polymerases / biosynthesis
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Poly(ADP-ribose) Polymerases / chemistry*
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Poly(ADP-ribose) Polymerases / metabolism*
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Polymerase Chain Reaction
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Protein Structure, Secondary*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Sequence Tagged Sites
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Spleen / enzymology
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T-Lymphocytes / enzymology*
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Transfection
Substances
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Antigens, Differentiation, T-Lymphocyte
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Bacterial Toxins
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DNA Primers
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Histocompatibility Antigens
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Membrane Glycoproteins
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Recombinant Proteins
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NAD
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Adenosine Diphosphate Ribose
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Arginine
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ADP Ribose Transferases
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Poly(ADP-ribose) Polymerases
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Art2a protein, mouse
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Art2b protein, mouse
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Art2b protein, rat
Associated data
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GENBANK/K01995
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GENBANK/L27629
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GENBANK/M87319
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GENBANK/P00588
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GENBANK/P01555
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GENBANK/P04977
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GENBANK/P06717
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GENBANK/P14299
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GENBANK/P15879
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GENBANK/P24121
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GENBANK/Q00901
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GENBANK/S27514
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GENBANK/X15811
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GENBANK/X69893
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GENBANK/X71131