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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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Loss of function phenotypes of Ce-Duox1 resulting from RNAi. (A) Wild-type animals showing normal morphology and sigmoidal shape. (B) Mutant animal showing a large blister and defect in movement evidenced by local expulsion of eggs and local clearing of bacteria near the anterior of the worm. (C) Mutant worm showing a “dumpy”-like phenotype and local clearing of the bacterial lawn. (D) Mutant worm on left demonstrating translucent appearance compared with wild-type worm on right. Duox1 dsRNA was injected into N2 hermaphrodites in six independent experiments. In each, although the resulting phenotypes showed a range of severities the phenotypes were identical.
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fig5: Loss of function phenotypes of Ce-Duox1 resulting from RNAi. (A) Wild-type animals showing normal morphology and sigmoidal shape. (B) Mutant animal showing a large blister and defect in movement evidenced by local expulsion of eggs and local clearing of bacteria near the anterior of the worm. (C) Mutant worm showing a “dumpy”-like phenotype and local clearing of the bacterial lawn. (D) Mutant worm on left demonstrating translucent appearance compared with wild-type worm on right. Duox1 dsRNA was injected into N2 hermaphrodites in six independent experiments. In each, although the resulting phenotypes showed a range of severities the phenotypes were identical.

Mentions: To gain insights regarding the biological function of Duox enzymes, we used the reverse genetic tool, RNAi, to “knock out” Duox in C. elegans (Fire et al., 1998). This technique involves injection of double-stranded RNA (dsRNA), encoding a segment of Ce-Duox1 or Ce-Duox2 into gonads of C. elegans wild-type hermaphrodites. Injected animals were then allowed to lay eggs, the harvested eggs were allowed to develop, and the progeny were observed for phenotypes. This procedure specifically diminishes or eliminates the expression of the gene of interest. dsRNA corresponding to three distinct regions of Ce-Duox1 and Duox2 were used in separate experiments. The first two correspond to regions of identity between Ce-Duox1 and Ce-Duox2 and are predicted to block the expression of both forms of Duox. The third dsRNA corresponds to the extreme COOH terminus of Ce-Duox1, which does not have a counterpart in Ce-Duox2, and therefore blocks only the expression of Ce-Duox1. All three dsRNA forms resulted in the same range of phenotypes. In replicate experiments, the percentage of animals exhibiting any given phenotype was somewhat variable, probably due to differences in amount of RNAi or site of injection. However, in a typical experiment >90% of the animals were affected by one or more phenotypes. In a typical experiment, phenotypes included the presence of large superficial blisters (Fig. 5 B, ∼50% of animals) and short or “dumpy” animals (Fig. 5 C, ∼35% of animals) and animals with retained eggs or larvae (unpublished data). In addition, whereas wild-type animals showed a dark appearance, >80% of RNAi animals were translucent (compare wild-type, Fig. 5, A and D, animal on right with affected animals in D, animal on left, and B and C). Around half of RNAi animals showed an inability to move on plates in a normal serpentine manner: affected animals were either completely paralyzed or moved only the anterior region, clearing a localized swath of Escherichia coli in the vicinity of the head (Fig. 5, B and C).


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)

Loss of function phenotypes of Ce-Duox1 resulting from RNAi. (A) Wild-type animals showing normal morphology and sigmoidal shape. (B) Mutant animal showing a large blister and defect in movement evidenced by local expulsion of eggs and local clearing of bacteria near the anterior of the worm. (C) Mutant worm showing a “dumpy”-like phenotype and local clearing of the bacterial lawn. (D) Mutant worm on left demonstrating translucent appearance compared with wild-type worm on right. Duox1 dsRNA was injected into N2 hermaphrodites in six independent experiments. In each, although the resulting phenotypes showed a range of severities the phenotypes were identical.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196470&req=5

fig5: Loss of function phenotypes of Ce-Duox1 resulting from RNAi. (A) Wild-type animals showing normal morphology and sigmoidal shape. (B) Mutant animal showing a large blister and defect in movement evidenced by local expulsion of eggs and local clearing of bacteria near the anterior of the worm. (C) Mutant worm showing a “dumpy”-like phenotype and local clearing of the bacterial lawn. (D) Mutant worm on left demonstrating translucent appearance compared with wild-type worm on right. Duox1 dsRNA was injected into N2 hermaphrodites in six independent experiments. In each, although the resulting phenotypes showed a range of severities the phenotypes were identical.
Mentions: To gain insights regarding the biological function of Duox enzymes, we used the reverse genetic tool, RNAi, to “knock out” Duox in C. elegans (Fire et al., 1998). This technique involves injection of double-stranded RNA (dsRNA), encoding a segment of Ce-Duox1 or Ce-Duox2 into gonads of C. elegans wild-type hermaphrodites. Injected animals were then allowed to lay eggs, the harvested eggs were allowed to develop, and the progeny were observed for phenotypes. This procedure specifically diminishes or eliminates the expression of the gene of interest. dsRNA corresponding to three distinct regions of Ce-Duox1 and Duox2 were used in separate experiments. The first two correspond to regions of identity between Ce-Duox1 and Ce-Duox2 and are predicted to block the expression of both forms of Duox. The third dsRNA corresponds to the extreme COOH terminus of Ce-Duox1, which does not have a counterpart in Ce-Duox2, and therefore blocks only the expression of Ce-Duox1. All three dsRNA forms resulted in the same range of phenotypes. In replicate experiments, the percentage of animals exhibiting any given phenotype was somewhat variable, probably due to differences in amount of RNAi or site of injection. However, in a typical experiment >90% of the animals were affected by one or more phenotypes. In a typical experiment, phenotypes included the presence of large superficial blisters (Fig. 5 B, ∼50% of animals) and short or “dumpy” animals (Fig. 5 C, ∼35% of animals) and animals with retained eggs or larvae (unpublished data). In addition, whereas wild-type animals showed a dark appearance, >80% of RNAi animals were translucent (compare wild-type, Fig. 5, A and D, animal on right with affected animals in D, animal on left, and B and C). Around half of RNAi animals showed an inability to move on plates in a normal serpentine manner: affected animals were either completely paralyzed or moved only the anterior region, clearing a localized swath of Escherichia coli in the vicinity of the head (Fig. 5, B and C).

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