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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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Comparison of the peroxidase domains of h-Duox, Ce-Duox1, and some known peroxidases. (A) Sequence alignments. TPO, thyroid peroxidase; EPO, eosinophil peroxidase; LPO, lactoperoxidase, Pxsn.dros, Drosophila peroxidasin. Residues which are conserved among all seven proteins are shown with black boxes, whereas those matching a derived consensus sequence are shown in line boxes. Filled circles indicate residues which are proposed to provide contacts with the heme based on the crystal structure of canine MPO (Zeng and Fenna, 1992). The superscripted double bar indicates residues comprising a calcium-binding region, and filled triangles indicate residues which appear in the crystal structure to bind directly to the calcium ion. (B) Phylogenetic relationships. The sequences shown in A and additional sequences are shown. OPO, ovoperoxidase; str.purp, Strongylocentrotus purpuratus; ly.var, Lytechinus variegatus; hemi.pulch; Hemicentrotus pulcherrimus.
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fig2: Comparison of the peroxidase domains of h-Duox, Ce-Duox1, and some known peroxidases. (A) Sequence alignments. TPO, thyroid peroxidase; EPO, eosinophil peroxidase; LPO, lactoperoxidase, Pxsn.dros, Drosophila peroxidasin. Residues which are conserved among all seven proteins are shown with black boxes, whereas those matching a derived consensus sequence are shown in line boxes. Filled circles indicate residues which are proposed to provide contacts with the heme based on the crystal structure of canine MPO (Zeng and Fenna, 1992). The superscripted double bar indicates residues comprising a calcium-binding region, and filled triangles indicate residues which appear in the crystal structure to bind directly to the calcium ion. (B) Phylogenetic relationships. The sequences shown in A and additional sequences are shown. OPO, ovoperoxidase; str.purp, Strongylocentrotus purpuratus; ly.var, Lytechinus variegatus; hemi.pulch; Hemicentrotus pulcherrimus.

Mentions: A BLAST search of the C. elegans genomic database using as a query the protein sequence of gp91phox identified two homologous genes contained in cosmids F56C11 and F53G12. The Ce-Duox1 conceptual transcript (sequence data available from GenBank/EMBL/DDBJ under accession no. AF043697) is predicted to be 8,197 bp before splicing, to contain 19 exons, and to encode a protein of 1,506 amino acids. Cloning of the cDNA for Ce-Duox1 (sequence data available from GenBank/EMBL/DDBJ under accession no. AF229855) revealed a cDNA of 4,491 bp (1,497 amino acids), which differed somewhat from the conceptual cDNA obtained from the gene structure due to inaccuracies in the predicted intron-exon junctions. The second transcript, Ce-Duox2 (sequence data available from GenBank/EMBL/DDBJ under accession no. AF043697), is predicted to be 5,308 bp before splicing, to contain 16 exons, and to encode a 1,313 amino acid protein. Alignment by homology of the genomic sequences of Ce-Duox1 and Ce-Duox2 identified two new exons 5′ of the first predicted exon of Duox2 that were highly homologous to the second and third exons of Duox1 (predicted cDNA structure available upon request), but an exon of Duox2 homologous to exon1 of Duox1 could not be identified by homology. The predicted amino acid sequences of both Ce-Duox1 and Ce-Duox2 show ∼30% identity with h-Duox1 and h-Duox2 (Fig. 1 and Fig. 2 A). Ce-Duox1 also contains the same domains as h-Duox1/2 (see below) and is roughly the same size. However, Ce-Duox2 contains a stop codon, which should eliminate the extreme COOH-terminal portion of the protein, which includes a segment of the pyridine nucleotide-binding site. Thus, whereas Ce-Duox2 should contain intact peroxidase and calmodulin-like domains, it is not predicted to encode a functioning NADPH-oxidase domain (Fig. 1). Except for this COOH-terminal region, Ce-Duox2 is 94% identical to Ce-Duox1 at the amino acid level. Both Ce-Duox1 and Ce-Duox2 are located near the end of chromosome I, separated by only 6 kb and in opposite orientations. The high degree of sequence identity and retention of intron structure (unpublished data) and the location of both near the end of a chromosome are consistent with a recent gene duplication.


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)

Comparison of the peroxidase domains of h-Duox, Ce-Duox1, and some known peroxidases. (A) Sequence alignments. TPO, thyroid peroxidase; EPO, eosinophil peroxidase; LPO, lactoperoxidase, Pxsn.dros, Drosophila peroxidasin. Residues which are conserved among all seven proteins are shown with black boxes, whereas those matching a derived consensus sequence are shown in line boxes. Filled circles indicate residues which are proposed to provide contacts with the heme based on the crystal structure of canine MPO (Zeng and Fenna, 1992). The superscripted double bar indicates residues comprising a calcium-binding region, and filled triangles indicate residues which appear in the crystal structure to bind directly to the calcium ion. (B) Phylogenetic relationships. The sequences shown in A and additional sequences are shown. OPO, ovoperoxidase; str.purp, Strongylocentrotus purpuratus; ly.var, Lytechinus variegatus; hemi.pulch; Hemicentrotus pulcherrimus.
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Related In: Results  -  Collection

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fig2: Comparison of the peroxidase domains of h-Duox, Ce-Duox1, and some known peroxidases. (A) Sequence alignments. TPO, thyroid peroxidase; EPO, eosinophil peroxidase; LPO, lactoperoxidase, Pxsn.dros, Drosophila peroxidasin. Residues which are conserved among all seven proteins are shown with black boxes, whereas those matching a derived consensus sequence are shown in line boxes. Filled circles indicate residues which are proposed to provide contacts with the heme based on the crystal structure of canine MPO (Zeng and Fenna, 1992). The superscripted double bar indicates residues comprising a calcium-binding region, and filled triangles indicate residues which appear in the crystal structure to bind directly to the calcium ion. (B) Phylogenetic relationships. The sequences shown in A and additional sequences are shown. OPO, ovoperoxidase; str.purp, Strongylocentrotus purpuratus; ly.var, Lytechinus variegatus; hemi.pulch; Hemicentrotus pulcherrimus.
Mentions: A BLAST search of the C. elegans genomic database using as a query the protein sequence of gp91phox identified two homologous genes contained in cosmids F56C11 and F53G12. The Ce-Duox1 conceptual transcript (sequence data available from GenBank/EMBL/DDBJ under accession no. AF043697) is predicted to be 8,197 bp before splicing, to contain 19 exons, and to encode a protein of 1,506 amino acids. Cloning of the cDNA for Ce-Duox1 (sequence data available from GenBank/EMBL/DDBJ under accession no. AF229855) revealed a cDNA of 4,491 bp (1,497 amino acids), which differed somewhat from the conceptual cDNA obtained from the gene structure due to inaccuracies in the predicted intron-exon junctions. The second transcript, Ce-Duox2 (sequence data available from GenBank/EMBL/DDBJ under accession no. AF043697), is predicted to be 5,308 bp before splicing, to contain 16 exons, and to encode a 1,313 amino acid protein. Alignment by homology of the genomic sequences of Ce-Duox1 and Ce-Duox2 identified two new exons 5′ of the first predicted exon of Duox2 that were highly homologous to the second and third exons of Duox1 (predicted cDNA structure available upon request), but an exon of Duox2 homologous to exon1 of Duox1 could not be identified by homology. The predicted amino acid sequences of both Ce-Duox1 and Ce-Duox2 show ∼30% identity with h-Duox1 and h-Duox2 (Fig. 1 and Fig. 2 A). Ce-Duox1 also contains the same domains as h-Duox1/2 (see below) and is roughly the same size. However, Ce-Duox2 contains a stop codon, which should eliminate the extreme COOH-terminal portion of the protein, which includes a segment of the pyridine nucleotide-binding site. Thus, whereas Ce-Duox2 should contain intact peroxidase and calmodulin-like domains, it is not predicted to encode a functioning NADPH-oxidase domain (Fig. 1). Except for this COOH-terminal region, Ce-Duox2 is 94% identical to Ce-Duox1 at the amino acid level. Both Ce-Duox1 and Ce-Duox2 are located near the end of chromosome I, separated by only 6 kb and in opposite orientations. The high degree of sequence identity and retention of intron structure (unpublished data) and the location of both near the end of a chromosome are consistent with a recent gene duplication.

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