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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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Absence of di- and trityrosine linkages resulting from RNAi. Total protein from wild-type (A) and RNAi (B) animals was extracted, hydrolyzed, and analyzed by HPLC monitoring fluorescence. Peak 1 was identified as dityrosine, and peak 2 was identified as trityrosine as described. The experiment is representative of three.
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fig7: Absence of di- and trityrosine linkages resulting from RNAi. Total protein from wild-type (A) and RNAi (B) animals was extracted, hydrolyzed, and analyzed by HPLC monitoring fluorescence. Peak 1 was identified as dityrosine, and peak 2 was identified as trityrosine as described. The experiment is representative of three.

Mentions: Cross-linking of collagen and other cuticle proteins in nematodes occurs through di- and trityrosine linkages, which bridge and stabilize the proteinaceous structure (Fetterer and Rhoads, 1990; Fetterer et al., 1993). Because peroxidases such as sea urchin ovoperoxidase and human MPO carry out this reaction (LaBella et al., 1968; Malanik and Ledvina, 1979; Deits et al., 1984), we hypothesized that the function of Ce-Duox1 (and possibly Ce-Duox2) is to generate tyrosine cross-links and that the defective cuticle in the Ce-Duox RNAi animals is due to an inability to form tyrosine cross-links. A role for an unknown peroxidase in tyrosine cross-linking in Ascaris was suggested previously based on studies in which tyrosine cross-linking activity was inhibited using the peroxidase inhibitors 4-amino-2,3,4 aminotriazole, phenylhydrazine, and N-acetyl tyrosine (Fetterer et al., 1993). We therefore examined the wild-type and Ce-Duox1/2 RNAi knockout animals for di- and trityrosine linkages. An HPLC profile of an acid hydrolysate of the wild-type C. elegans is shown in Fig. 7 , trace A. The first large peak was identified as dityrosine based on comparison with authentic standard and mass spectral analysis, and the second peak is identified as trityrosine based on its migration on HPLC relative to dityrosine and mass spectral analysis. Based on peak areas and assuming equivalent ionization, dityrosine and tyrosine were present in a ratio of 1:200 in adult wild-type animals. In addition, the fluorescence excitation/emission maxima were determined at alkaline and acidic pH and were in good agreement with previously reported values (Jacob et al., 1996). Mass spectral analysis of purified C. elegans cuticle and noncuticular material determined that >99.99% of dityrosine and trityrosine are located in cuticle material, since dityrosine was undetectable in the noncuticle fraction (unpublished data). Dityrosine and trityrosine peaks were absent in hydrolysates of Ce-Duox RNAi nematodes (Fig. 7, trace B). Thus, interference with the expression of Ce-Duox1 eliminates formation of di- and trityrosine linkages.


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)

Absence of di- and trityrosine linkages resulting from RNAi. Total protein from wild-type (A) and RNAi (B) animals was extracted, hydrolyzed, and analyzed by HPLC monitoring fluorescence. Peak 1 was identified as dityrosine, and peak 2 was identified as trityrosine as described. The experiment is representative of three.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Absence of di- and trityrosine linkages resulting from RNAi. Total protein from wild-type (A) and RNAi (B) animals was extracted, hydrolyzed, and analyzed by HPLC monitoring fluorescence. Peak 1 was identified as dityrosine, and peak 2 was identified as trityrosine as described. The experiment is representative of three.
Mentions: Cross-linking of collagen and other cuticle proteins in nematodes occurs through di- and trityrosine linkages, which bridge and stabilize the proteinaceous structure (Fetterer and Rhoads, 1990; Fetterer et al., 1993). Because peroxidases such as sea urchin ovoperoxidase and human MPO carry out this reaction (LaBella et al., 1968; Malanik and Ledvina, 1979; Deits et al., 1984), we hypothesized that the function of Ce-Duox1 (and possibly Ce-Duox2) is to generate tyrosine cross-links and that the defective cuticle in the Ce-Duox RNAi animals is due to an inability to form tyrosine cross-links. A role for an unknown peroxidase in tyrosine cross-linking in Ascaris was suggested previously based on studies in which tyrosine cross-linking activity was inhibited using the peroxidase inhibitors 4-amino-2,3,4 aminotriazole, phenylhydrazine, and N-acetyl tyrosine (Fetterer et al., 1993). We therefore examined the wild-type and Ce-Duox1/2 RNAi knockout animals for di- and trityrosine linkages. An HPLC profile of an acid hydrolysate of the wild-type C. elegans is shown in Fig. 7 , trace A. The first large peak was identified as dityrosine based on comparison with authentic standard and mass spectral analysis, and the second peak is identified as trityrosine based on its migration on HPLC relative to dityrosine and mass spectral analysis. Based on peak areas and assuming equivalent ionization, dityrosine and tyrosine were present in a ratio of 1:200 in adult wild-type animals. In addition, the fluorescence excitation/emission maxima were determined at alkaline and acidic pH and were in good agreement with previously reported values (Jacob et al., 1996). Mass spectral analysis of purified C. elegans cuticle and noncuticular material determined that >99.99% of dityrosine and trityrosine are located in cuticle material, since dityrosine was undetectable in the noncuticle fraction (unpublished data). Dityrosine and trityrosine peaks were absent in hydrolysates of Ce-Duox RNAi nematodes (Fig. 7, trace B). Thus, interference with the expression of Ce-Duox1 eliminates formation of di- and trityrosine linkages.

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