N-linked glycosylation of the superoxide-producing NADPH oxidase Nox1

•Human Nox1 undergoes glycosylation at Asn-162 and Asn-236.•Nox1 can be transported to the cell surface as no or high mannose N-glycan form.•Nox1 N-glycosylation is dispensable for its catalytic function.•p22phox allows maturation of Nox1 N-glycans into more complex oligosaccharides.•p22phox directly contributes to Nox1 activation independently of Nox1 glycosylation. 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 p22phox, trafficking to the cell surface, or producing superoxide. In the absence of p22phox, 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 p22phox. 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 p22phox, which is abrogated by glutamine substitution for Pro-156 in p22phox, a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22phox directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.