Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox - PubMed (original) (raw)

. 2001 Aug 20;154(4):879-91.

doi: 10.1083/jcb.200103132.

L Sharling, G Cheng, R Shapira, J M Kinkade, T Lee, H A Edens, X Tang, C Sullards, D B Flaherty, G M Benian, J D Lambeth

Affiliations

Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox

W A Edens et al. J Cell Biol. 2001.

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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Figures

Figure 1.

Figure 1.

Structure of large homologues of gp91_phox_. Domain structure of Duox proteins. Secretory signal peptide sequences are indicated by a gray triangle, whereas predicted transmembrane α helices are indicated by hashed rectangles. White ovals indicate regions showing homology with EF-hand calcium-binding sites.

Figure 2.

Figure 2.

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.

Figure 2.

Figure 2.

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.

Figure 3.

Figure 3.

Tissue expression of mRNA for h-Duox. mRNA for h-Duox1, h-Duox2, and glyceraldehyde 3-phosphate dehydrogenase were detected by reverse transcriptase PCR as described in Materials and methods.

Figure 4.

Figure 4.

Cellular expression of Ce-Duox1. Animals were immunostained with antibodies to Ce-Duox1 (A–C, E, F, and H, green), myosin A (A and B, red), and MH4 (D, E, G, and H, red), and fluorescence was visualized using confocal microscopy. Merged images of Ce-Duox1 and myosin A staining (A and B) show that the Ce-Duox1 is expressed in a layer of cells just outside the muscle bundles. Longitudinal images (C and D) and cross section images (F and G) show the individual staining patters of Ce-Duox1 and the hypodermal cell marker MH4, respectively. Merged images (E and H) show areas of overlapping staining of Ce-Duox1 and MH4 in yellow, confirming the expression of Ce-Duox1 in the hypodermal layer of cells. The use of antibody to Ce-Duox1 that had been preincubated with Ce-Duox1(340–355) peptide eliminated green channel antibody staining (unpublished data). Photos are representative of >100 animals observed.

Figure 5.

Figure 5.

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.

Figure 6.

Figure 6.

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).

Figure 7.

Figure 7.

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.

Figure 8.

Figure 8.

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.

Figure 8.

Figure 8.

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.

Figure 9.

Figure 9.

Proposed topology model for Duox. See text for details.

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