Karen Bedard and Karl-Heinz Krause

Within the spleen, NOX5 shows a distinct localization
within the mantle zone, which is rich in mature B cells,
and in the periarterial lymphoid sheath area, which is
enriched with T lymphocytes (56). Interestingly, NOX5
could not be detected within circulating lymphocytes
(56). These data are based on mRNA expression; no data
on the tissue distribution or subcellular distribution of the
NOX5 protein are published. Presently there is also no
information on the NOX5 promoters or on factors controlling
gene expression of the EF-hand expressing NOX5
isoforms (�-�). However, a recent study shed first light on
mechanisms regulating expression of the NOX5� isoform:
acid induces NOX5� expression in Barrett’s esophageal
adenocarcinoma cells through mechanisms involving the
cAMP response element binding protein CREB (274).
Nothing is known about the activation of the EF handdeficient
NOX5�; thus the activation mechanisms summarized
below are based on studies using EF hand-containing
NOX5 isoforms. NOX5 does not require p22 phox for activity,
as demonstrated by siRNA suppression of p22 phox leading to
a decrease in the activity of NOX1 to NOX4, but not of NOX5
(446). NOX5 does not require cytosolic organizer or activator
subunits (56) and has been shown to function in a
cell-free system without the requirements of any cytosolic
proteins (58). As predicted by the presence of EF hands,
activation of NOX5 is mediated by an increase in the cytoplasmic
Ca 2� concentration (58). The Ca 2� -binding domain
of NOX5 behaves as an independent folding unit and undergoes
conformational changes in response to Ca 2� elevations
(58). This is thought to activate the enyzme through an
intramolecular protein-protein interaction between the
Ca 2� -binding region and the catalytic COOH terminus of the
enzyme (56, 58).
6. DUOX1 and DUOX2
For several novel NOX isoforms, the identification of
the protein preceded the definition of its function. In the
case of DUOX1 and DUOX2, the situation was reversed. It
had been known for a long time that thyroid epithelial
cells produce H 2O 2 at the apical plasma membrane in a
Ca 2� - and NADPH-dependent manner (88). Researchers
in the thyroid field were actively looking for an NADPH
oxidase. It took 15 years from the discovery of this function
to the identification of DUOX proteins (originally
called thyroid oxidase). They were identified from thyroid
gland by two groups using different methods: purification
and partial sequencing of the DUOX2 enzyme followed by
rapid amplification of cDNA ends polymerase chain reaction
(RACE PCR) (228) and low-temperature hybridization
of a thyroid cDNA phage library with a NOX2 probe
(189, 228). The genes for both human DUOX isoforms are
located on chromosome 15. The two DUOX genes are
somewhat unusual in their arrangement. They are arranged
in a head-to-head configuration, separated by a
THE NOX FAMILY OF ROS-GENERATING NADPH OXIDASES 255
Physiol Rev VOL 87 JANUARY 2007 www.prv.org
relatively short (16 kb) region with the direction of transcription
away from one another (675).
In addition to a NOX1–4 homology domain and an
EF-hand region, DUOX proteins have a seventh transmembrane
domain at the NH 2 terminus with an ectofacing
peroxidase like domain. Within the NOX backbone,
DUOX isoforms share �50% identity with NOX2 (189). An
NH 2-terminally truncated form of DUOX2 mRNA has been
found in rat thyroid cell lines (625).
DUOX enzymes are glycosylated. Both DUOX1 and
DUOX2 have two N-glycosylation states: the high mannose
glycosylated form found in the ER, which runs by gel
electrophoresis at 180 kDa, and a fully glycosylated form
found at the plasma membrane that runs at 190 kDa (188,
624). Carbohydrate content analysis of plasma membrane
DUOX revealed specific oligosaccharides indicative of
Golgi apparatus processing (623). When totally deglycosylated,
the molecular mass of both DUOX1 and DUOX2
drops to 160 kDa (188).
It is not clear whether the peroxidase homology domain
of DUOX enzymes functions as a peroxidase. One
study suggests that DUOX peroxidase homology domains,
when expressed as recombinant proteins, have a peroxidase
function (239). However, from a structural point of
view, this is surprising. Indeed, the DUOX peroxidase
homology domains lack many amino acid residues identified
as essential for peroxidase function (168, 181, 653).
The fact that a peroxidase is usually coexpressed in
DUOX expressing systems, e.g., thyroid peroxidase in the
thyroid gland and lactoperoxidase in salivary glands, also
questions the peroxidase function of DUOX. This is particularly
well documented for the thyroid, where thyroid
peroxidase deficiency leads to severe hypothyroidism,
due to a lack of peroxidase-dependent hormone synthesis
(690). Still, the peroxidase homology region of DUOX2
seems to be of functional importance, as hypothyroidism
in patients with mutations in the extracellular domain has
been reported (918).
Based on its homology with NOX2 and the fact that
heme enzymes are monoelectron transporters, DUOX enzymes
should generate superoxide. However, a generation
of hydrogen peroxide by thyrocytes has been detected
in many studies. This led to a heated debate over
the question of whether the thyroid oxidase directly generates
hydrogen peroxide or whether the hydrogen peroxide
generation occurs via a superoxide intermediate
(231, 527, 645, 646). In a recent study, the immature,
partially glycosylated form of DUOX2 generated superoxide,
while the mature form generated hydrogen peroxide
(27). The authors speculate that posttranslational modifications
favor intramolecular dismutation of superoxide to
hydrogen peroxide. Taken together, it is likely that the
primary product of DUOX enzymes is superoxide and that
a rapid dismutation precludes in many instances the detection
of a superoxide intermediate. The substrate selec-
Downloaded from
physrev.physiology.org on February 2, 2010