Purification, characterization and molecular cloning of glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferases from chicken

Abstract

Mono-ADP-ribosylation is a post-translational modification that regulates the functions of target proteins or peptides by attaching an ADP-ribose moiety. Here we report the purification, molecular cloning, characterization and tissue-specific distribution of novel arginine-specific Arts (ADP-ribosyltransferases) from chicken. Arts were detected in various chicken tissues as GPI (glycosylphosphatidylinositol)-anchored forms, and purified from the lung membrane fraction. By molecular cloning based on the partial amino acid sequence using 5′- and 3′-RACE (rapid amplification of cDNA ends), two full-length cDNAs of chicken GPI-anchored Arts, cgArt1 (chicken GPI-anchored Art1) and cgArt2, were obtained. The cDNA of cgArt1 encoded a novel polypeptide of 298 amino acids which shows a high degree of identity with cgArt2 (82.9%), Art6.1 (50.2%) and rabbit Art1 (42.1%). In contrast, the nucleotide sequence of cgArt2 was identical with that of Art7 cloned previously from chicken erythroblasts. cgArt1 and cgArt2 proteins expressed in DT40 cells were shown to be GPI-anchored Arts with a molecular mass of 45 kDa, and these Arts showed different enzymatic properties from the soluble chicken Art, Art6.1. RNase protection assays and real-time quantitative PCR revealed distinct expression patterns of the two Arts; cgArt1 was expressed predominantly in the lung, spleen and bone marrow, followed by the heart, kidney and muscle, while cgArt2 was expressed only in the heart and skeletal muscle. Thus GPI-anchored Arts encoded by the genes cgArt1 and cgArt2 are expressed extensively in chicken tissues. It may be worthwhile determining the functional roles of ADP-ribosylation in each tissue.