title: N-Linked glycosylation of the superoxide-producing NADPH oxidase Nox1.
abstract: Nox1 is a membrane-integrated protein that belongs to the Nox family of superoxide-producing NADPH oxidases. Here we show that human Nox1 undergoes glycosylation at Asn-162 and Asn-236 in the second and third extracellular loops, respectively. Simultaneous threonine substitution for these residues completely abrogates the glycosylation, but does not prevent Nox1 from forming a heterodimer with p22(phox), trafficking to the cell surface, or producing superoxide. In the absence of p22(phox), Nox1 is transported to the plasma membrane mainly as a form with high mannose N-glycans, although their conversion into complex N-glycans is induced by expression of p22(phox). These findings indicate that glycosylation and subsequent N-glycan maturation of Nox1 are both dispensable for its cell surface recruitment. Superoxide production by unglycosylated Nox1 is largely dependent on p22(phox), which is abrogated by glutamine substitution for Pro-156 in p22(phox), a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22(phox) directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.
authors.affiliation: Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Miyano
authors.affiliation: Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: hsumi@med.kyushu-u.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sumimoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D013481:Superoxides / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011134:Polysaccharides; D013481:Superoxides; D001216:Asparagine; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C431570:Nox3 protein, human; C086986:CYBA protein, human
keywords: Duox; ER; Endo H; Glycosylation; NADPH oxidase; Nox; Nox1; PNGase F; ROS; Superoxide; dual oxidase; endoglycosidase H; endoplasmic reticulum; p22(phox); peptide: N-glycosidase F; reactive oxygen species