Journal
EUROPEAN JOURNAL OF CELL BIOLOGY
Volume 90, Issue 8, Pages 665-677Publisher
ELSEVIER GMBH, URBAN & FISCHER VERLAG
DOI: 10.1016/j.ejcb.2011.03.007
Keywords
ADP-ribosyltransferase; ART; ARTC; Post-translational modification; Mono-ADP-ribosylation; NAD-glycohydrolase; NAD; CHO; SKOV-3
Categories
Funding
- Ministero dell'Istruzione, Universita e Ricerca (MIUR) [RBIP06LSS2_005, DM26545]
- Fondazione Negri Sud
- DFG [310/6]
- Italian Association for Cancer Research (AIRC)
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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. (C) 2011 Elsevier GmbH. All rights reserved.
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