Abstract
Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the 'exon skipping' mode of alternative splicing, lacking exons 4-10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein - herein termed Nox2β - that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Atherosclerosis / enzymology
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Atherosclerosis / genetics
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Base Sequence
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Blood Vessels / metabolism
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Gene Expression Regulation, Enzymologic
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Gene Knockdown Techniques
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Humans
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Macrophages / enzymology*
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Macrophages / metabolism
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Male
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / deficiency
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Mice
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Models, Molecular
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Molecular Sequence Data
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NADPH Oxidase 2
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NADPH Oxidases / chemistry*
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NADPH Oxidases / deficiency
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NADPH Oxidases / genetics
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NADPH Oxidases / metabolism*
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Protein Isoforms / chemistry
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Protein Isoforms / deficiency
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Structure, Tertiary
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RNA, Small Interfering / genetics
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Sequence Deletion
Substances
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Membrane Glycoproteins
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Protein Isoforms
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RNA, Small Interfering
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CYBB protein, human
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Cybb protein, mouse
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NADPH Oxidase 2
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NADPH Oxidases
Grants and funding
Sources of funding included: National Health and Medical Research Council of Australia (
www.nhmrc.gov.au/) for Fellowship and Project Grant Support (I.D. 606472, 1006017, 545942, 570861, 606488 and 1010984), the Australian Research Council (
www.arc.gov.au) for fellowship support (FT120100876), the National Heart Foundation of Australia (
www.heartfoundation.org.au/) for Grant-in-Aid support (G 09M 4398 and G 10M 5218) and the Cancer Council of Victoria (
www.cancervic.org.au) for Project Grant support (606674). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.