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Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, Edens HA, Tang X, Sullards C, Flaherty DB, Benian GM, Lambeth JD - J. Cell Biol. (2001)

Bottom Line: In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals.The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester.Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University Medical School, Atlanta, GA 30322, USA.

ABSTRACT
High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

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Proposed topology model for Duox. See text for details.
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fig9: Proposed topology model for Duox. See text for details.

Mentions: The topology model shown in Fig. 9 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 (Fig. 1 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.


Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, Edens HA, Tang X, Sullards C, Flaherty DB, Benian GM, Lambeth JD - J. Cell Biol. (2001)

Proposed topology model for Duox. See text for details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196470&req=5

fig9: Proposed topology model for Duox. See text for details.
Mentions: The topology model shown in Fig. 9 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 (Fig. 1 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.

Bottom Line: In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals.The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester.Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University Medical School, Atlanta, GA 30322, USA.

ABSTRACT
High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Show MeSH
Related in: MedlinePlus