title: Targeting of the dual oxidase 2 N-terminal region to the plasma membrane.
abstract: Dual oxidase 2 (Duox2) is a cell surface glycoprotein that probably provides thyroperoxidase with the H2O2 required to catalyze thyroid hormone synthesis. No functional H2O2-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the N-terminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596-685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation.
authors.affiliation: Unité 486 INSERM, Université Paris 11, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296 Chātenay-Malabry, France. stanislas.morand@cep.u-psud.fr
authors.forename: Stanislas
authors.initials: S
authors.lastname: Morand
authors.forename: Diane
authors.initials: D
authors.lastname: Agnandji
authors.forename: Marie-Sophie
authors.initials: MS
authors.lastname: Noel-Hudson
authors.forename: Valérie
authors.initials: V
authors.lastname: Nicolas
authors.forename: Sandrine
authors.initials: S
authors.lastname: Buisson
authors.forename: Laetitia
authors.initials: L
authors.lastname: Macon-Lemaitre
authors.forename: Sédami
authors.initials: S
authors.lastname: Gnidehou
authors.forename: Jacques
authors.initials: J
authors.lastname: Kaniewski
authors.forename: Renée
authors.initials: R
authors.lastname: Ohayon
authors.forename: Alain
authors.initials: A
authors.lastname: Virion
authors.forename: Corinne
authors.initials: C
authors.lastname: Dupuy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D000074623:Dual Oxidases; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D019255:NADPH Oxidases; D017434:Protein Structure, Tertiary; D051381:Rats; D013552:Swine; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D005420:Flavoproteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human; C486611:Duox2 protein, rat; D003545:Cysteine