Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells.
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Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells. / Stilla, Annalisa; Simone, Di Paola; Dani, Nadia; Krebs, Christian; Arrizza, Antonella; Corda, Daniela; Haag, Friedrich; Koch Nolte, Friedrich; Maria, Di Girolamo.
in: EUR J CELL BIOL, Jahrgang 90, Nr. 8, 8, 2011, S. 665-677.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells.
AU - Stilla, Annalisa
AU - Simone, Di Paola
AU - Dani, Nadia
AU - Krebs, Christian
AU - Arrizza, Antonella
AU - Corda, Daniela
AU - Haag, Friedrich
AU - Koch Nolte, Friedrich
AU - Maria, Di Girolamo
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
KW - Animals
KW - Female
KW - CHO Cells
KW - Cricetinae
KW - Cricetulus
KW - Mutation
KW - Amino Acid Sequence
KW - Base Sequence
KW - Sequence Analysis, DNA
KW - Polymerase Chain Reaction
KW - Glycosylphosphatidylinositols/metabolism
KW - Amino Acid Substitution/genetics
KW - ADP Ribose Transferases/chemistry/genetics/isolation & purification/metabolism
KW - Cell Membrane/enzymology
KW - Isoenzymes/chemistry/genetics/isolation & purification/metabolism
KW - NAD+ Nucleosidase
KW - Animals
KW - Female
KW - CHO Cells
KW - Cricetinae
KW - Cricetulus
KW - Mutation
KW - Amino Acid Sequence
KW - Base Sequence
KW - Sequence Analysis, DNA
KW - Polymerase Chain Reaction
KW - Glycosylphosphatidylinositols/metabolism
KW - Amino Acid Substitution/genetics
KW - ADP Ribose Transferases/chemistry/genetics/isolation & purification/metabolism
KW - Cell Membrane/enzymology
KW - Isoenzymes/chemistry/genetics/isolation & purification/metabolism
KW - NAD+ Nucleosidase
M3 - SCORING: Journal article
VL - 90
SP - 665
EP - 677
JO - EUR J CELL BIOL
JF - EUR J CELL BIOL
SN - 0171-9335
IS - 8
M1 - 8
ER -
