NOX family NADPH oxidases in mammals: Evolutionary conservation and isoform-defining sequences

SUMMARY

NADPH oxidases are superoxide-producing enzymes that play a role in host defense, biosynthetic pathways, as well as cellular signaling. Humans have 7 NOX isoforms (NOX1-5, DUOX1,2), while mice and rats lack NOX5 and therefore have only 6 NOX isoforms. Whether all human NOX isoforms or their subunits (CYBA, NCF1, 2, 4, NOXO1, NOXA1, DUOXA1, 2) are present and conserved in other mammalian species is unknown. In this study, the authors led by GCIR member Professor Karl-Heinz Krause have analyzed the conservation of the NOX family during mammalian evolution using an in-silico approach. Complete genomic sequences of 164 mammalian species were available. The possible absence of genes coding for NOX isoforms was investigated using the NCBI orthologs database followed by manual curation. Conservation of a given NOX isoform during mammalian evolution was evaluated by multiple alignment and identification of highly conserved sequences. There was no convincing evidence for the absence of NOX2, 3, 4, and DUOX1, 2 in all the available mammalian genome. However, NOX5 was absent in 27 of 31 rodent, in 2 of 3 lagomorph and in 2 out of 18 bat species. NOX1 was absent in all sequenced Afrotheria and Monotremata species, as well as in 3 of 18 bat species. NOXA1 was absent in all Afrotheria and in 3 out of 4 Eulipotyphla species. The authors also investigated amino acid sequence conservation among given NOX isoforms. Highly conserved sequences were observed for most isoforms except for NOX5. Interestingly, the highly conserved region of NOX2 sequence was relatively small (11 amino acids), as compared to NOX1, 3, 4. The highly conserved domains are different from one NOX isoform to the other, raising the possibility of distinct evolutionary conserved functional domains. These results published in Redox Biology shed a new light on the essentiality of different NOX isoforms. They also identified isoform-defining sequences, i.e., hitherto undescribed conserved domains within specific NOX isoforms.

Full article: https://doi.org/10.1016/j.redox.2023.102851

This study was granted by Swiss National Science Foundation.

Why is it important?

Humans have seven enzymes (NOX1-5, and DUOX 1-2) whose main activity on a molecular level is the production of reactive oxygen species (ROS). ROS, also referred to as oxygen radicals, are biologically active derivatives of oxygen, including superoxide and hydrogen peroxide.  ROS-producing enzymes are called NADPH oxidases or NOX enzymes. Their biological function is best illustrated by what happens if you don’t have one of them. In humans, genetic loss of NOX2 leads to severe immune deficiency, loss of DUOX2 leads to severe hypothyroidism (fatal if not corrected rapidly after birth), and loss of function mutations in NOX1 and DUOX1 appear to be involved in inflammatory bowel disease. In mice, genetic loss of NOX3 leads to absence of otoconia and therefore to severe equilibrium problems.

Before this study, it was generally assumed that most mammals have – similar to humans - seven NOX isoforms, with the notable exception of a loss of NOX5 in mice and rats. The question whether there are sequences within different NOX isoforms that are highly conserved during mammalian evolution (and thus have a particular functional or structural importance) has never been addressed.

This study was an exciting expedition into mammalian zoology and allowed to shed new light on ROS-producing enzymes and their role in mammalian biology. The human situation with 7 NOX enzymes is commonly found in many mammals, but only 5 NOX enzymes (NOX2, NOX3, NOX4, DUOX1, and DUOX2) are found in all mammalian species. NOX5 got lost during evolution in most (but not all) rodents, as well as in some lagomorphs and some bats. This fits well with the observation that conservation of NOX5 sequences is quite low. Hence NOX5 is probably the most dispensable among the ROS-producing enzymes. NOX1 is completely lacking in two mammalian clades: Monotremata (egg-laying mammals such as platypus) and Afrotheria (mammals that developed in isolation when Africa was still an island without connection to other continents). The best-known examples of afrotheria are elephants and manatees. Based on the phylogenetic analysis, it is likely that NOX5 and NOX1 got lost several times during evolution. Relatively small, but highly conserved isoform-specific sequences are observed in NOX1, NOX2, NOX4, and DUOX1. In contrast, NOX3 and DUOX2 have much larger areas of highly conserved sequences.

The identification of these highly conserved sequences is entirely novel and provides new elements for the understanding and further studies of the biology of ROS-producing enzymes.

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