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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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Biochemical activities of Duox peroxidase domains. Peroxidase domains from h-Duox1 and Ce-Duox1 were expressed in E. coli as described in Materials and methods. (A) Lysates (100 μg protein) from vector control E. coli or E. coli expressing Ce-Duox or h-Duox were added to an assay mixture containing tetramethylbenzidine and hydrogen peroxide, the mixture was incubated at 25°C for 2 min, and the optical density was read at 655 nm. Some incubations contained 30 μM aminobenzohydrazide. (B) Lysates (100 μg protein) from E. coli from vector control cells (c) or cells expressing Ce-Duox1 (a) or h-Duox1 (b) were incubated at 25°C for 60 min with tyrosine ethyl ester. The reaction was quenched, and the ester was hydrolyzed as described in Materials and methods. The product was analyzed by HPLC, monitoring fluorescence, and the results are representative of three experiments. Peak 1 is dityrosine, and peak 2 is trityrosine as in Fig. 7.
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fig8: Biochemical activities of Duox peroxidase domains. Peroxidase domains from h-Duox1 and Ce-Duox1 were expressed in E. coli as described in Materials and methods. (A) Lysates (100 μg protein) from vector control E. coli or E. coli expressing Ce-Duox or h-Duox were added to an assay mixture containing tetramethylbenzidine and hydrogen peroxide, the mixture was incubated at 25°C for 2 min, and the optical density was read at 655 nm. Some incubations contained 30 μM aminobenzohydrazide. (B) Lysates (100 μg protein) from E. coli from vector control cells (c) or cells expressing Ce-Duox1 (a) or h-Duox1 (b) were incubated at 25°C for 60 min with tyrosine ethyl ester. The reaction was quenched, and the ester was hydrolyzed as described in Materials and methods. The product was analyzed by HPLC, monitoring fluorescence, and the results are representative of three experiments. Peak 1 is dityrosine, and peak 2 is trityrosine as in Fig. 7.

Mentions: The peroxidase domains of Ce-Duox (residues 1–590) and h-Duox1 (residues 1–593) were expressed in E. coli as described in Materials and methods. A lysate from these cells was analyzed for peroxidase activity, and the results are summarized in Fig. 8 A. As shown, the lysates from E. coli expressing both the human and the C. elegans peroxidase homology domains from Duox demonstrated peroxidase activity towards 3,3′,5,5′-tetramethylbenzidine (TMB), a well-characterized peroxidase substrate. The activity was inhibited by the peroxidase inhibitor aminobenzohydrazide. Lysates from E. coli expressing the peroxidase domains of h-Duox and Ce-Duox but not those from vector control cells also catalyzed the cross-linking of tyrosine ethyl ester (Fig. 8 B). Two major fluorescent products were seen; peak 1 was identified by cochromatography with authentic material and mass spectral analysis as dityrosine, whereas peak 2 was identified as trityrosine by mass spectral analysis as above.


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)

Biochemical activities of Duox peroxidase domains. Peroxidase domains from h-Duox1 and Ce-Duox1 were expressed in E. coli as described in Materials and methods. (A) Lysates (100 μg protein) from vector control E. coli or E. coli expressing Ce-Duox or h-Duox were added to an assay mixture containing tetramethylbenzidine and hydrogen peroxide, the mixture was incubated at 25°C for 2 min, and the optical density was read at 655 nm. Some incubations contained 30 μM aminobenzohydrazide. (B) Lysates (100 μg protein) from E. coli from vector control cells (c) or cells expressing Ce-Duox1 (a) or h-Duox1 (b) were incubated at 25°C for 60 min with tyrosine ethyl ester. The reaction was quenched, and the ester was hydrolyzed as described in Materials and methods. The product was analyzed by HPLC, monitoring fluorescence, and the results are representative of three experiments. Peak 1 is dityrosine, and peak 2 is trityrosine as in Fig. 7.
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Related In: Results  -  Collection

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

fig8: Biochemical activities of Duox peroxidase domains. Peroxidase domains from h-Duox1 and Ce-Duox1 were expressed in E. coli as described in Materials and methods. (A) Lysates (100 μg protein) from vector control E. coli or E. coli expressing Ce-Duox or h-Duox were added to an assay mixture containing tetramethylbenzidine and hydrogen peroxide, the mixture was incubated at 25°C for 2 min, and the optical density was read at 655 nm. Some incubations contained 30 μM aminobenzohydrazide. (B) Lysates (100 μg protein) from E. coli from vector control cells (c) or cells expressing Ce-Duox1 (a) or h-Duox1 (b) were incubated at 25°C for 60 min with tyrosine ethyl ester. The reaction was quenched, and the ester was hydrolyzed as described in Materials and methods. The product was analyzed by HPLC, monitoring fluorescence, and the results are representative of three experiments. Peak 1 is dityrosine, and peak 2 is trityrosine as in Fig. 7.
Mentions: The peroxidase domains of Ce-Duox (residues 1–590) and h-Duox1 (residues 1–593) were expressed in E. coli as described in Materials and methods. A lysate from these cells was analyzed for peroxidase activity, and the results are summarized in Fig. 8 A. As shown, the lysates from E. coli expressing both the human and the C. elegans peroxidase homology domains from Duox demonstrated peroxidase activity towards 3,3′,5,5′-tetramethylbenzidine (TMB), a well-characterized peroxidase substrate. The activity was inhibited by the peroxidase inhibitor aminobenzohydrazide. Lysates from E. coli expressing the peroxidase domains of h-Duox and Ce-Duox but not those from vector control cells also catalyzed the cross-linking of tyrosine ethyl ester (Fig. 8 B). Two major fluorescent products were seen; peak 1 was identified by cochromatography with authentic material and mass spectral analysis as dityrosine, whereas peak 2 was identified as trityrosine by mass spectral analysis as above.

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