ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.Keywords: ADP-ribosylation; recombinant proteins; PSI-BLAST; orthologous genes; paralogous gene; cross-species PCR; database searches The purpose of this study was to identify all recognizable human and mouse members of the family of toxin-related mono-ADP-ribosyltransferases; to clone, sequence, and chromosomally map their genes and cDNAs; to express the gene products as recombinant proteins and assay their enzyme activities; and to assess structural and sequence similarities between bacterial and mammalian ADP-ribosyltransferases.ADP-ribosylation is an enzyme-catalyzed post-translational protein modification in which the ADP-ribose moiety is transferred from NAD+ to a specific amino acid in a Reprint requests to: Friedrich Koch-Nolte, Institute for Immunology, University Hospital, Martinistr. 52, D-20246 Hamburg, Germany, e-mail: nolte@uke.uni-hamburg.de; fax: 49-40-42803-4243.Abbreviations: ARH, ADP-ribosylhydrolase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; HPRT, hypoxanthine phosphoribosyltransferase; mART, mono(ADP-ribosyl)transferase; NAD+, nicotine adenine dinucleotide; PBS, phosphate-buffered saline; pART, poly...

We searched the database of expressed sequence tags (dbEST) for relatives of the known human and murine mono(ADP-ribosyl)transferases (mADPRT), poly(ADP-ribosyl)polymerases (PARP), ADP-ribosyl cyclases, and ADP-ribosylarginine hydro lases (ARH). By May 31, 1996, all of the known enzymes except for RT6 were represented in dbEST by exact sequence matches from mouse and/or human tissues. Several ESTs show significant sequence similarity but not identity to known mADPRTs. We isolated, cloned, and sequenced the corresponding genes. Our results show that seven human ESTs stem from a novel gene, provisionally designated LART, which is specifically expressed in lymphatic tissues. Five human ESTs stem from a novel gene, here designated TARTJ, which is specifically expressed in testis. This gene is also represented by a single mouse EST. One other mouse EST stems from a distinct gene, here designated TART2, which is also expressed in testis. These genes have similar exon/intron structures. The predicted LART and TARTI gene products contain hydrophobic N-and C-terminal signal peptides characteristic for GPI-anchored surface proteins, TART2 lacks the GPI-anchor signal peptide. The predicted native proteins show 28-42% sequence identity to one another. They each contain four cysteine residues that probably form conserved disulfide bonds. They each also contain a conserved glutamic acid residue within the proposed active site motif. LART and TARTI show interesting deviations from the surrounding consensus sequence.