The topology model shown in
is proposed for Duox based on its primary structure and on analogy with known features of gp91phox
. In activated phagocytes, gp91phox
is integrally associated with the plasma membrane. The COOH-terminal half of gp91phox
is homologous to known FAD proteins (Rotrosen et al., 1992
; Segal et al., 1992
; Sumimoto et al., 1992
) and contains a predicted NADPH-binding site. This region is thus likely to fold into a discrete intracellular flavoprotein domain. Gp91phox
also contains an NH2
-terminal hydrophobic domain, comprising nearly half of the molecule. The corresponding region in Duox is predicted to cross the membrane six times ( A), placing both the NH2
terminus and the COOH terminus (attached to the flavoprotein domain) on the cytosolic side. Several features of the model have been verified for gp91phox
. For example, the model places known glycosylation sites on the cell exterior (Wallach and Segal, 1997
) and a binding loop for the cytosolic regulatory protein p47phox
(Biberstine-Kinkade et al., 1999
) on the cytosolic side.
Proposed topology model for Duox. See text for details.
The gp91phox homology domains in h-Duox1/2 and Ce-Duox1/2 show the same hydropathy profile and predicted transmembrane α helices as gp91phox (, hashed bars within the gp91phox homology region). We therefore assume that the gp91phox transmembrane model will also apply to the COOH-terminal portion of the Duox proteins. Such a model predicts that the domain containing the EF-hands is on the interior of the cell. The presence of a secretion export signal peptide sequence at the extreme NH2 terminus ( A) and the presence of an additional predicted transmembrane hydrophobic sequence intervening between the EF-hand domain and the peroxidase homology domain predicts that the peroxidase domain will reside on the exterior of the cell (). Although features of this model will need to be tested directly, this structure is attractive as it is consistent with the genetic and biochemical data, implicating Ce-Duox1 in the generation of extracellular tyrosine cross-links in cuticle proteins.
The phagocyte NADPH-oxidase serves as a model for the function of the gp91phox
homology domain of Duox. The gp91phox
component of the phagocyte oxidase generates reactive oxygen outside of the cell or in the phagosome (which is topologically extracellular). NADPH reduces the FAD within the flavoprotein domain, and the FAD then passes electrons through the two heme groups located within the transmembrane NH2
terminus of gp91phox
, reducing oxygen to form superoxide outside of the cell with secondary production of hydrogen peroxide by dismutation. Such a function has been demonstrated for p138Tox
(Duox2), which was purified as the hydrogen peroxide–generating NADPH oxidase from thyroid (Dupuy et al., 1999
). These authors proposed that p138Tox
functions to provide H2
to thyroid peroxidase, which is known to iodinate the thyroid hormone precursor. A recent study (De Deken et al., 2000
) identified a peroxidase homology domain in Duox1 and Duox2 (ThOX1 and ThOX2), but the authors suggested that this domain was inactive based on an absence of putative catalytically important residues.
Oxidative reactions are generally thought to be deleterious to the cell, but the results of the current study suggest that protein oxidation by peroxidases plays a critical role in normal physiology. Insights into the function of the Duox peroxidase domain come from the phagocyte system in which cell activation is accompanied by both activation of the phagocyte NADPH-oxidase and secretion of MPO. Hydrogen peroxide generated indirectly by the phagocyte NADPH-oxidase combines with chloride in an oxidation catalyzed by MPO to form hypochlorous acid, a species which functions in bactericidal reactions. In the case of Duox enzymes, both the NADPH-oxidase moiety and the peroxidase moiety are integrated into a single molecule. The hydrogen peroxide generated by the gp91phox
-homology domain in Duox should then serve as a substrate for the peroxidase domain. For Ce-Duox1, the cosubstrate is protein tyrosine residues, which are converted to di- and trityrosine presumably via a reaction involving the tyrosyl radical based on mechanisms established for other well-studied peroxidases; tyrosyl radical recombination would generate di- and trityrosine, resulting in protein cross-linking to stabilize the nematode cuticle. Peroxidases including human MPO (Heinecke et al., 1993
) and the sea urchin ovoperoxidase (Deits et al., 1984
) catalyze this tyrosine cross-linking, albeit in the former case somewhat inefficiently. Thus, the proposed topological structure of Duox is well suited to support transmembrane peroxidative reaction using intracellular reducing equivalents from NADPH.
Precedent for the involvement of a peroxidase in extracellular matrix structure comes from the fertilization reaction in sea urchin oocytes. Fertilization results in activation of tyrosine cross-linking of extracellular proteins in the oocyte, forming a protective fertilization envelope (Deits et al. 1984
). This reaction is catalyzed by ovoperoxidase, a peroxidase secreted by the oocyte. In this system, the hydrogen peroxide is generated by an unknown NADPH-oxidase. Thus, in the case of the sea urchin oocyte individual proteins carry out the peroxide generation and the peroxidative functions respectively. Yeast spore coats also contain dityrosine cross-links that are formed by a heme protein (Briza et al. 1996
The closest known structural homologues of h-Duox1/2 are Ce-Duox1 and Ce-Duox2. Both h-Duox1/2 and Ce-Duox1 show the same domain structure, including the gp91phox
homology domain, the EF-hand domain, and the peroxidase domain. In the case of h-Duox1/2, the critical calcium-binding residues in the EF-hand domain are well conserved, suggesting a role for calcium in the regulation of the enzyme activity. This has been proposed to account for the calcium dependence for the NADPH-oxidase activity of p138Tox
(Duox2), (Dupuy et al., 1999
). In contrast, the calcium-binding ligands in the EF-hand regions in Ce-Duox1/2 are poorly conserved, suggesting that calcium may not be involved at this site. Calcium-binding regions within the peroxidase domain are well conserved in both Ce-Duox1/2 and h-Duox, suggesting a distinct role for calcium as has been noted for other peroxidases.
The similarity between the peroxidase domain of Ce-Duox1 and h-Duox1/2 raises the possibility that their function will be similar. The peroxidase domains of h-Duox1/2 and Ce-Duox1 are 37% identical to one another, whereas the peroxidase domains of Duox proteins are only 19–20% identical with known mammalian peroxidases. Thus, among known peroxidases or peroxidase domains the h-Duox is most similar to Ce-Duox1, and this may imply similar catalytic specificities. Supporting this idea, biochemical data show that the recombinant-expressed peroxidase domains from human and C. elegans
Duox are both capable of generating di- and trityrosine cross-links. This conservation of domain structure, sequence, and biochemical activity is suggestive of a similar function for Ce-Duox1 and h-Duox1/2. In addition, our data showing that Duox1 and Duox2 expression is not restricted to thyroid suggests that their function is not limited to a thyroid-specific function. In thyroid, the peroxidase domains of h-Duox1 and h-Duox2 may participate in iodination of thyroglobulin, the precursor of thyroxine. If this is the case, then the function is redundant to that of the well-characterized thyroid peroxidase (Taurog, 1999
). However, because other tissues in which Duox1/2 are expressed lack iodine uptake systems such a function in these tissues seems unlikely. In addition to iodination, mammalian Duox might also function in forming the ether bridge of thyroid hormone, a reaction that is analogous to pulcherosine synthesis.
Is mammalian Duox involved in generation of tyrosine cross-links? Dityrosine cross-links stabilize several types of extracellular matrices including not only the cuticle of nematodes (above), but also insect elastomer resilin (Anderson, 1963
), insect cuticle (Locke, 1969
; Hall, 1978
; Klebanoff et al., 1979
; Deits et al., 1984
), the chorion envelope of Drosophila
eggs (Georgi and Deri, 1976
; Mindrinos et al., 1980
), and the yeast acrospore wall (Briza et al., 1996
). The occurrence of dityrosine and a possible role in extracellular matrix in mammalian system is less well studied. In mammals, dityrosine is a marker of inflammation as occurs in low density lipoprotein isolated from atherosclerotic plaques (Leeuwenburgh et al., 1997
). Dityrosine is proposed to be formed during inflammation through the action of MPO secreted by inflammatory cells (Zaitsu et al., 1981
) or in the mouth by salivary lactoperoxidase (Tenovuo and Paunio, 1979
), but Duox-dependent mechanisms are not ruled out. A major constituent of extracellular matrix and basement membrane is collagen, and mammalian collagen has been reported previously to contain very low concentrations of dityrosine linkages (~1 in 100,000 tyrosines) (Keeley et al., 1969
; Waykole and Heidemann, 1976
), in addition to the predominant cross-links involving lysines and histidines. However, such cross-linking may be nonspecifically associated with aging or artifactually induced during sample preparation. Dityrosine linkages have been isolated from other structural proteins and hard tissues such as elastin (LaBella et al., 1967
), fibrin and keratin (Raven et al., 1971
), and cataractous human lens protein (Garcia-Castineiras et al., 1978
). In elastin, although the predominant cross-link involves lysine it has been speculated that the tyrosine cross-links may be critical at early stages of elastin biogenesis and that this is followed later by extensive cross-linking at lysines (LaBella et al., 1967
). Low concentrations of tyrosine cross-links might help order and align elastin and/or collagen fibrils, aligning further high abundance cross-linking at lysines. Such a role for human Duox is attractive and is consistent with its expression in lung, which possesses a high content of elastin. To our knowledge, dityrosine linkages in mammalian basement membrane or extracellular matrix other than collagen and elastin have not been described, perhaps owing to a lack of sufficient quantities of material for analysis. In addition, another peroxidase-catalyzed cross-link is formed from the deamination of protein lysyl ε-amino groups to form lysyl aldehydes, which then react with amino acid residues of adjacent molecules (Stahmann et al., 1977
; Clark et al., 1986
; Hazen et al., 1997
). Thus, it is also possible that h-Duox generates this type of extracellular matrix cross-link. Its transmembrane nature and results from RNAi studies in C. elegans
support the hypothesis that this enzyme participates in the formation or modification of extracellular protein matrix.