RESUMEN
Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome 7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six exons spanning 8 kb of genomic DNA. The 5' end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfecting an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.
Asunto(s)
ADP Ribosa Transferasas/genética , Genes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Mapeo Cromosómico , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Exones , Femenino , Expresión Génica , Intrones , Isoenzimas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Muridae , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
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 hydrolases (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 TART1, 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 TART1 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 TART1 show interesting deviations from the surrounding consensus sequence.