title: Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells.
abstract: The mammalian mono-ADP-ribosyltransferases are a family of enzymes related to bacterial toxins that can catalyse both intracellular and extracellular mono-ADP-ribosylation of target proteins involved in different cellular processes, such as cell migration, signalling and inflammation. Here, we report the molecular cloning and functional characterisation of a novel glycosylphosphatidylinositol (GPI)-anchored mono-ADP-ribosyltransferase isoform from Chinese hamster ovary (CHO) cells (cARTC2.1) that has both NAD-glycohydrolase and arginine-specific ADP-ribosyltransferase activities. cARTC2.1 has the R-S-EXE active-site motif that is typical of arginine-specific ADP-ribosyltransferases, with Glu209 as the predicted catalytic amino acid. When over-expressed in CHO cells, the E209G single point mutant of cARTC2.1 cannot hydrolyse NAD(+), although it retains low arginine-specific ADP-ribosyltransferase activity. This ADP-ribosyltransferase activity was abolished only with an additional mutation in the R-S-EXE active-site motif, with both of the glutamate residues of the EKE sequence of cARTC2.1 mutated to glycine (E207/209G). These glutamate-mutated proteins localise to the plasma membrane, as does wild-type cARTC2.1. Thus, the partial or total loss of enzymatic activity of cARTC2.1 that arises from these mutations does not affect its cellular localisation. Importantly, an endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells, while a similar activity cannot be detected in ovarian cancer cells. With respect to this endogenous ecto-ART activity, we have identified two cell populations: ART-positive and ART-negative CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population.
authors: Consorzio Mario Negri Sud, Via Nazionale, 8/A 66030 Santa Maria Imbaro, Chieti, Italy.
authors: Annalisa
authors: A
authors: Stilla
authors: Simone
authors: S
authors: Di Paola
authors: Nadia
authors: N
authors: Dani
authors: Christian
authors: C
authors: Krebs
authors: Antonella
authors: A
authors: Arrizza
authors: Daniela
authors: D
authors: Corda
authors: Friedrich
authors: F
authors: Haag
authors: Friederich
authors: F
authors: Koch-Nolte
authors: Maria
authors: M
authors: Di Girolamo