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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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EM of Ce-Duox RNAi animals. (A) Cross-sectional view of wild-type animals showing normal cuticle structure. Arrows indicate the location of struts. (B) Cross-sectional view of an RNAi animal showing separation of the cuticle into two layers. Solid arrows indicate broken struts attached to the basal layer, and the open arrow indicates the cortical layer that has detached from the basal layer. (C) Cross-sectional view of a RNAi animal showing the full view of a blister. The animals shown are representative of 10 in each group. Magnification was 8,155× (C) and 12,575× (A and B).
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fig6: EM of Ce-Duox RNAi animals. (A) Cross-sectional view of wild-type animals showing normal cuticle structure. Arrows indicate the location of struts. (B) Cross-sectional view of an RNAi animal showing separation of the cuticle into two layers. Solid arrows indicate broken struts attached to the basal layer, and the open arrow indicates the cortical layer that has detached from the basal layer. (C) Cross-sectional view of a RNAi animal showing the full view of a blister. The animals shown are representative of 10 in each group. Magnification was 8,155× (C) and 12,575× (A and B).

Mentions: The similarity in phenotypes among animals defective in collagen and cuticle biosynthesis compared with the RNAi Duox animals suggested that Duox participates in cuticle biogenesis. To confirm this hypothesis, EM was carried out on wild-type and RNAi animals. As shown in Fig. 6 , cuticle of RNAi Duox animals was grossly abnormal. In normal animals (Fig. 6 A), three cuticle layers are seen clearly: the cortical (outer), median, and basal (inner) layer as described previously (Cox et al., 1981). The median layer is composed of struts (Fig. 6 A, arrows) connecting the cortical and basal layers with a fluid-filled space between these layers. The RNAi animals (Fig. 6, B and C) frequently showed separation between the cortical and the basal layers with marked expansion of the fluid cavity and broken and distended struts that are still visible on these layers (Fig. 6 B, arrows). These separations occurred mainly over bundles of muscle fiber (Fig. 6, B and C) and are likely to account for the formation of the blisters seen by light microscopy. Thus, the cuticle structure was severely affected in RNAi Duox animals.


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)

EM of Ce-Duox RNAi animals. (A) Cross-sectional view of wild-type animals showing normal cuticle structure. Arrows indicate the location of struts. (B) Cross-sectional view of an RNAi animal showing separation of the cuticle into two layers. Solid arrows indicate broken struts attached to the basal layer, and the open arrow indicates the cortical layer that has detached from the basal layer. (C) Cross-sectional view of a RNAi animal showing the full view of a blister. The animals shown are representative of 10 in each group. Magnification was 8,155× (C) and 12,575× (A and B).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: EM of Ce-Duox RNAi animals. (A) Cross-sectional view of wild-type animals showing normal cuticle structure. Arrows indicate the location of struts. (B) Cross-sectional view of an RNAi animal showing separation of the cuticle into two layers. Solid arrows indicate broken struts attached to the basal layer, and the open arrow indicates the cortical layer that has detached from the basal layer. (C) Cross-sectional view of a RNAi animal showing the full view of a blister. The animals shown are representative of 10 in each group. Magnification was 8,155× (C) and 12,575× (A and B).
Mentions: The similarity in phenotypes among animals defective in collagen and cuticle biosynthesis compared with the RNAi Duox animals suggested that Duox participates in cuticle biogenesis. To confirm this hypothesis, EM was carried out on wild-type and RNAi animals. As shown in Fig. 6 , cuticle of RNAi Duox animals was grossly abnormal. In normal animals (Fig. 6 A), three cuticle layers are seen clearly: the cortical (outer), median, and basal (inner) layer as described previously (Cox et al., 1981). The median layer is composed of struts (Fig. 6 A, arrows) connecting the cortical and basal layers with a fluid-filled space between these layers. The RNAi animals (Fig. 6, B and C) frequently showed separation between the cortical and the basal layers with marked expansion of the fluid cavity and broken and distended struts that are still visible on these layers (Fig. 6 B, arrows). These separations occurred mainly over bundles of muscle fiber (Fig. 6, B and C) and are likely to account for the formation of the blisters seen by light microscopy. Thus, the cuticle structure was severely affected in RNAi Duox animals.

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