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Free Radic Biol Med. 2014 Mar;68:196-204. doi: 10.1016/j.freeradbiomed.2013.12.013. Epub 2013 Dec 19.

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase.

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Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Electronic address:
Radioisotope Center, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Inflammation Program and Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Coralville, IA 52241, USA; Veterans Administration Medical Center, Iowa City, IA 52240, USA.


Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60kDa accompanied by a broad band of 60-90kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn(161) and Asn(241) on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.


Antibodies; N-glycosylation; NADPH oxidase; Reactive oxygen species; Site-directed mutagenesis

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