Article |
Address correspondence to J. David Lambeth, Department of Biochemistry, Emory University Medical School, Atlanta, GA 30322. Tel.: (404) 727-5875. Fax: (404) 727-2738. E-mail: dlambe{at}bimcore.emory.edu
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Abstract |
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Key Words: NADPH-oxidase; peroxidase; extracellular matrix; cuticle; dityrosine
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Introduction |
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The phagocyte NADPH-oxidase consists of multiple subunits including gp91phox, the catalytic moiety (Babior, 1995; Yu et al., 1998). In activated cells, this subunit is associated with the plasma membrane (or with the phagosomal membrane, which is derived from the plasma membrane) and consists of a COOH-terminal flavoprotein domain containing the NADPH-binding site (Rotrosen et al., 1992; Segal et al., 1992; Taylor et al., 1993; Takeshige and Sumimoto, 1994) and an NH2-terminal hydrophobic region comprised of five to six transmembrane helices and harboring the two heme groups (Cross et al., 1995; Nisimoto et al., 1995; Yu et al., 1998). The structure of the enzyme permits the coupling of the oxidation of intracellular NADPH to the reduction of molecular oxygen to generate extracellular or phagosomal superoxide. Myeloperoxidase (MPO) is secreted extracellularly or into the phagosome, permitting reactive oxygen generated by the phagocyte oxidase to support hypochlorous acid generation in the extracellular/phagosomal compartment (Hampton et al., 1998; Nauseef, 1998).
Based on the hypothesis that reactive oxygen generation in nonphagocytic cells originates in part from homologues of the phagocyte oxidase, we have searched for and molecularly cloned homologues of gp91phox. The first of these, Nox1 (also termed Mox1, NOH-1), is expressed in nonphagocytic cells, including colonic epithelia and vascular smooth muscle (Suh et al., 1999), and functions in regulating cell growth and cell transformation. Alternative splicing of Nox1 to generate a portion of the membrane domain produces a proton channel (Banfi et al., 2000) with properties similar to voltage-gated channels. Additional homologues of gp91phox that are similar in size to gp91phox (65 kD) have been reported recently (Lambeth et al., 2000; Cheng et al., 2001).
In the present study, we describe large molecular weight homologues of gp91phox termed Duox, which are present in human and C. elegans. The term Duox, referring to dual oxidase, has been accepted by the Human Genome Organisation International Committee on Gene Nomenclature. A partial sequence of Duox2 was reported recently and termed p138Tox, referring to thyroid oxidase. Surprisingly, these homologues contain not only the gp91phox homology region at the COOH terminus but also an NH2-terminal region that is homologous to peroxidases including MPO. Using RNA interference (RNAi) in C. elegans to knock out the expression of the nematode Duox1, we find that Duox participates through tyrosine cross-linking in the biogenesis of cuticle, the collagenous extracellular matrix that forms the outer covering of nematodes. The tyrosine cross-linking reaction has also been reconstituted biochemically using the expressed peroxidase domains of human (h)-Duox and Caenorhabditis elegans (Ce)-Duox1.
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Results |
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Duox proteins have additional regions that are not present in gp91phox. A central region contains two EF-hand calcium-binding sequences as indicated in Fig. 1. The canonical residues involved in calcium ligation are well conserved in h-Duox1 and h-Duox2 but are poorly conserved in Ce-Duox1 and Ce-Duox2, suggesting that the function of this region may have evolved away from calcium binding in nematodes.
Surprisingly, the NH2-terminal third of Duox proteins is homologous to peroxidases including MPO, eosinophil peroxidase, thyroid peroxidase, lactoperoxidase, and sea urchin ovoperoxidases (Fig. 2, A and B). Overall, the identity with peroxidases within the entire region is 20%, but subregions show considerably higher homology. The Duox enzymes represent a distinct group within the peroxidase family (Fig. 2 B), and phylogenetically this group is marginally more closely related to sea urchin ovoperoxidases. Within the peroxidase homology region, only 2 of the 12 cysteine residues involved in the six intrachain disulfide bonds, which are conserved in the four homologous mammalian peroxidases, are present in Duox proteins (Fig. 2 A). In addition, the asparagine-linked glycosylation sites found in MPO are not present in Ce-Duox1 or Ce-Duox2. A calcium-binding site in MPO (aspartate 263 and residues 33534; Fig. 2 A, superior double bar) (Zeng and Fenna, 1992) is well conserved in the Duox family proteins, including three of the four candidate calcium liganding residues (Fig. 2 A,
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The extreme NH2-terminal 21 amino acids of Ce-Duox1 contain a secretory signal peptide sequence (Fig. 1), implying that the NH2-terminal peroxidase domain is in a compartment that is transmembrane to the cytosol (for example, extracellular or within a secretory vesicle). In addition, hydropathy plots reveal that the proteins contain a highly hydrophobic region corresponding to the NH2-terminal third of gp91phox. This region can be modeled as a cluster of six transmembrane helices as indicated in Fig. 1. An additional transmembrane helical region is present between the peroxidase homology domain and the calmodulin-like domain.
Tissue distribution of h-Duox mRNA
As shown in Fig. 3, h
-Duox1 mRNA was distributed among a variety of adult tissues with highest expression in lung and thyroid but with significant expression also seen in placenta, testis, and prostate, and with detectable expression in pancreas and heart. h-Duox1 mRNA was also widely expressed in fetal tissues where it was abundant in lung. As reported previously (Dupuy et al., 1999), Duox2 (p138Tox) is present in thyroid. In addition, we observed significant expression in a variety of fetal tissues and in adult colon with detectable expression in kidney, liver, lung, pancreas, prostate, and testis.
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The similarity in phenotypes among animals defective in collagen and cuticle biosynthesis compared with the RNAi Duox animals suggested that Duox participates in cuticle biogenesis. To confirm this hypothesis, EM was carried out on wild-type and RNAi animals. As shown in Fig. 6 , cuticle of RNAi Duox animals was grossly abnormal. In normal animals (Fig. 6 A), three cuticle layers are seen clearly: the cortical (outer), median, and basal (inner) layer as described previously (Cox et al., 1981). The median layer is composed of struts (Fig. 6 A, arrows) connecting the cortical and basal layers with a fluid-filled space between these layers. The RNAi animals (Fig. 6, B and C) frequently showed separation between the cortical and the basal layers with marked expansion of the fluid cavity and broken and distended struts that are still visible on these layers (Fig. 6 B, arrows). These separations occurred mainly over bundles of muscle fiber (Fig. 6, B and C) and are likely to account for the formation of the blisters seen by light microscopy. Thus, the cuticle structure was severely affected in RNAi Duox animals.
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Discussion |
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The phagocyte NADPH-oxidase serves as a model for the function of the gp91phox homology domain of Duox. The gp91phox component of the phagocyte oxidase generates reactive oxygen outside of the cell or in the phagosome (which is topologically extracellular). NADPH reduces the FAD within the flavoprotein domain, and the FAD then passes electrons through the two heme groups located within the transmembrane NH2 terminus of gp91phox, reducing oxygen to form superoxide outside of the cell with secondary production of hydrogen peroxide by dismutation. Such a function has been demonstrated for p138Tox (Duox2), which was purified as the hydrogen peroxidegenerating NADPH oxidase from thyroid (Dupuy et al., 1999). These authors proposed that p138Tox functions to provide H2O2 to thyroid peroxidase, which is known to iodinate the thyroid hormone precursor. A recent study (De Deken et al., 2000) identified a peroxidase homology domain in Duox1 and Duox2 (ThOX1 and ThOX2), but the authors suggested that this domain was inactive based on an absence of putative catalytically important residues.
Oxidative reactions are generally thought to be deleterious to the cell, but the results of the current study suggest that protein oxidation by peroxidases plays a critical role in normal physiology. Insights into the function of the Duox peroxidase domain come from the phagocyte system in which cell activation is accompanied by both activation of the phagocyte NADPH-oxidase and secretion of MPO. Hydrogen peroxide generated indirectly by the phagocyte NADPH-oxidase combines with chloride in an oxidation catalyzed by MPO to form hypochlorous acid, a species which functions in bactericidal reactions. In the case of Duox enzymes, both the NADPH-oxidase moiety and the peroxidase moiety are integrated into a single molecule. The hydrogen peroxide generated by the gp91phox-homology domain in Duox should then serve as a substrate for the peroxidase domain. For Ce-Duox1, the cosubstrate is protein tyrosine residues, which are converted to di- and trityrosine presumably via a reaction involving the tyrosyl radical based on mechanisms established for other well-studied peroxidases; tyrosyl radical recombination would generate di- and trityrosine, resulting in protein cross-linking to stabilize the nematode cuticle. Peroxidases including human MPO (Heinecke et al., 1993) and the sea urchin ovoperoxidase (Deits et al., 1984) catalyze this tyrosine cross-linking, albeit in the former case somewhat inefficiently. Thus, the proposed topological structure of Duox is well suited to support transmembrane peroxidative reaction using intracellular reducing equivalents from NADPH.
Precedent for the involvement of a peroxidase in extracellular matrix structure comes from the fertilization reaction in sea urchin oocytes. Fertilization results in activation of tyrosine cross-linking of extracellular proteins in the oocyte, forming a protective fertilization envelope (Deits et al. 1984). This reaction is catalyzed by ovoperoxidase, a peroxidase secreted by the oocyte. In this system, the hydrogen peroxide is generated by an unknown NADPH-oxidase. Thus, in the case of the sea urchin oocyte individual proteins carry out the peroxide generation and the peroxidative functions respectively. Yeast spore coats also contain dityrosine cross-links that are formed by a heme protein (Briza et al. 1996).
The closest known structural homologues of h-Duox1/2 are Ce-Duox1 and Ce-Duox2. Both h-Duox1/2 and Ce-Duox1 show the same domain structure, including the gp91phox homology domain, the EF-hand domain, and the peroxidase domain. In the case of h-Duox1/2, the critical calcium-binding residues in the EF-hand domain are well conserved, suggesting a role for calcium in the regulation of the enzyme activity. This has been proposed to account for the calcium dependence for the NADPH-oxidase activity of p138Tox (Duox2), (Dupuy et al., 1999). In contrast, the calcium-binding ligands in the EF-hand regions in Ce-Duox1/2 are poorly conserved, suggesting that calcium may not be involved at this site. Calcium-binding regions within the peroxidase domain are well conserved in both Ce-Duox1/2 and h-Duox, suggesting a distinct role for calcium as has been noted for other peroxidases.
The similarity between the peroxidase domain of Ce-Duox1 and h-Duox1/2 raises the possibility that their function will be similar. The peroxidase domains of h-Duox1/2 and Ce-Duox1 are 37% identical to one another, whereas the peroxidase domains of Duox proteins are only 1920% identical with known mammalian peroxidases. Thus, among known peroxidases or peroxidase domains the h-Duox is most similar to Ce-Duox1, and this may imply similar catalytic specificities. Supporting this idea, biochemical data show that the recombinant-expressed peroxidase domains from human and C. elegans Duox are both capable of generating di- and trityrosine cross-links. This conservation of domain structure, sequence, and biochemical activity is suggestive of a similar function for Ce-Duox1 and h-Duox1/2. In addition, our data showing that Duox1 and Duox2 expression is not restricted to thyroid suggests that their function is not limited to a thyroid-specific function. In thyroid, the peroxidase domains of h-Duox1 and h-Duox2 may participate in iodination of thyroglobulin, the precursor of thyroxine. If this is the case, then the function is redundant to that of the well-characterized thyroid peroxidase (Taurog, 1999). However, because other tissues in which Duox1/2 are expressed lack iodine uptake systems such a function in these tissues seems unlikely. In addition to iodination, mammalian Duox might also function in forming the ether bridge of thyroid hormone, a reaction that is analogous to pulcherosine synthesis.
Is mammalian Duox involved in generation of tyrosine cross-links? Dityrosine cross-links stabilize several types of extracellular matrices including not only the cuticle of nematodes (above), but also insect elastomer resilin (Anderson, 1963), insect cuticle (Locke, 1969; Hall, 1978; Klebanoff et al., 1979; Deits et al., 1984), the chorion envelope of Drosophila eggs (Georgi and Deri, 1976; Mindrinos et al., 1980), and the yeast acrospore wall (Briza et al., 1996). The occurrence of dityrosine and a possible role in extracellular matrix in mammalian system is less well studied. In mammals, dityrosine is a marker of inflammation as occurs in low density lipoprotein isolated from atherosclerotic plaques (Leeuwenburgh et al., 1997). Dityrosine is proposed to be formed during inflammation through the action of MPO secreted by inflammatory cells (Zaitsu et al., 1981) or in the mouth by salivary lactoperoxidase (Tenovuo and Paunio, 1979), but Duox-dependent mechanisms are not ruled out. A major constituent of extracellular matrix and basement membrane is collagen, and mammalian collagen has been reported previously to contain very low concentrations of dityrosine linkages (1 in 100,000 tyrosines) (Keeley et al., 1969; Waykole and Heidemann, 1976), in addition to the predominant cross-links involving lysines and histidines. However, such cross-linking may be nonspecifically associated with aging or artifactually induced during sample preparation. Dityrosine linkages have been isolated from other structural proteins and hard tissues such as elastin (LaBella et al., 1967), fibrin and keratin (Raven et al., 1971), and cataractous human lens protein (Garcia-Castineiras et al., 1978). In elastin, although the predominant cross-link involves lysine it has been speculated that the tyrosine cross-links may be critical at early stages of elastin biogenesis and that this is followed later by extensive cross-linking at lysines (LaBella et al., 1967). Low concentrations of tyrosine cross-links might help order and align elastin and/or collagen fibrils, aligning further high abundance cross-linking at lysines. Such a role for human Duox is attractive and is consistent with its expression in lung, which possesses a high content of elastin. To our knowledge, dityrosine linkages in mammalian basement membrane or extracellular matrix other than collagen and elastin have not been described, perhaps owing to a lack of sufficient quantities of material for analysis. In addition, another peroxidase-catalyzed cross-link is formed from the deamination of protein lysyl
-amino groups to form lysyl aldehydes, which then react with amino acid residues of adjacent molecules (Stahmann et al., 1977; Clark et al., 1986; Hazen et al., 1997). Thus, it is also possible that h-Duox generates this type of extracellular matrix cross-link. Its transmembrane nature and results from RNAi studies in C. elegans support the hypothesis that this enzyme participates in the formation or modification of extracellular protein matrix.
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Materials and methods |
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Cloning of the cDNA for human Duox2
A 535 base portion of an EST (zc92h03.rl; sequence data available from GenBank/EMBL/DDBJ under accession no. W52750) from human pancreatic islet was identified using the amino acid sequence of human gp91phox as a query in a BLAST search. The bacterial strain no. 595758 containing the EST sequence zc92h03.rl in the pBluescript SK-vector was purchased from American Type Culture Collection. The DNA was sequenced using primers to T7 and T3 vector promoters and sequence-specific internal primers. The EST encoded a 440 amino acid partial cDNA exhibiting 24.4% identity to gp91phox, including a stop codon corresponding to the COOH terminus of gp91phox. 5' and 3' RACE were carried out using human adult pancreas mRNA (CLONTECH) with the 5' RACE kit for Rapid Amplification of cDNA ends version 2.0 (GIBCO BRL). PCR was done with specific primers: 5' RACE: primer 1, 5'-GAAGTGGTGGGAGGCGAAGACATA-3'; primer 2, 5'-CCTGTCATACCTGGGACGGTCTGG-3'; primer 3, 5'-GAGCACAGTGAGATGCCTGTTCAG-3'; primer 4, 5'-GGAAGGCAGCAGAGAGCAATGATG-3'; primer 5, 5'-AGGTGGGATGCGGATGTTGAG-3' (for nested PCR); 3' RACE primer 6, 5'-ACATCTGCGAGCGGCACTTCCAGA-3'; primer 7, 5'-AGCTCGTCAACAGGCAGGACCGAGC-3'; primer 8, 5'-TCTCCATCAGAATCCACCTTAGGC-3' (for nested PCR). To complete the sequence, 5' RACE was carried out using human thyroid marathon-ready cDNA (CLONTECH) with primer 3 and adapter primer AP1 and primer 5 and adapter primer AP2. These procedures resulted in an additional 3.7-kb 5' region and a 1.5-kb 3' region.
Identification of genes for Ce-Duox1 and Ce-Duox2
A BLAST search using the cDNA sequence of human gp91phox identified two putative homologues (sequence data available from GenBank/EMBL/DDBJ under accession nos. AF043697 and AF003130) in the genomic sequence of C. elegans, both near the end of chromosome I and separated by 6 Kb.
Cloning of the cDNA for Ce-Duox1
Based on the gene sequence, PCR primers were designed to amplify two overlapping portions of the Ce-Duox1 gene: one extending from the 5' end and one extending from the 3' end. Primers were 5'-ATTCGTCGACAAATGCGCTCAAAACATGTGCTGT-3' and 5'-AACTTTGTGGATCAAAGTTAGCG-3' for the 5' region, and 5'-TTGGATTAGCATTTTGCTATGGAA-3' and 5'-GAGCGGCCGCGAACGTTTCAAAGCGATGTGCA-3' for the 3' region. PCR was carried out using a random primed C. elegans cDNA library in ACT (obtained from R. Barstead, Oklahoma Medical Research Foundation, Oklahoma City, OK) under the following conditions: denaturation at 95°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 1 min. The 5' piece and the 3' piece were digested with Dra III and ligated to produce the full-length Ce-Duox1 cDNA. The full-length Ce-Duox1 cDNA was inserted into the pBluescript SK-vector and was sequenced using T7 and T3 vector primers and sequence-specific primers.
Analysis of primary structure
Export signal sequences were predicted according to Nielsen et al. (1997). Transmembrane helices were predicted according to Sonnhammer et al. (1998). Both methods are available on the internet at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services). Multiple sequence alignments phylogenetic analysis were carried out using the clustal method using Megalign software (DNASTAR).
PCR detection of mRNA for human Duox
Based on the cloned h-Duox1 and hDuox2 cDNA sequence, we designed specific primers (Duox1: 5'-GCAGGACATCAACCCTGCACTCTC-3' and 5'-CTGCCATCTACCACACGGATCTGC-3'; Duox2: 5'-GCCCTCAACCTAAGCAGCTCACAACTG-3' and 5'-GAGCACAGTGAGATGCCTGTTCAG-3'), which were used to determine the tissue expression patterns of Duox1 and Duox2 using human multiple tissue PCR panels and human thyroid gland marathon-ready cDNA (CLONTECH). PCR conditions were as follows: 95°C for 30 s, 65°C for 20 s, and 72°C for 30 s, for a total of 35 cycles.
RNAi in C. elegans
The procedure was taken from Fire et al. (1998). RNA was transcribed from either pBluescript.Duox2, pBluescript.E17Duox1, or pBluescript.E18 + 19Duox1. For pBluescript.Duox2, exon 10 of Ce-Duox2 was amplified by PCR from genomic DNA using the forward primer 5'-GCTAGAGCTCTTCAGTTTGCTATGGAATTGGC-3' and reverse primer 5'-CATAAAGGATGAGGAGAATTCTGTG-3'. The 457-bp fragment generated was digested with SstI and EcoRI and subcloned into pBluescript. For pBluescript.E17Duox1, exon 17 of Duox1 was amplified by PCR from genomic DNA using the forward primer 5'-GCTAGAGCTCGGCTACTACTACGTTGTTGGACC-3' and the reverse primer 5'-GACTGAAGGACTTGTGGAACGTCTGAGTGAC-3'. The 659-bp fragment generated was digested with SstI and EcoRI and subcloned into pBluescript. For pBluescript.E18 + 19Duox1, exons 18 and 19 of Ce-Duox1 were amplified by PCR from a randomly primed C. elegans cDNA library (obtained from R. Barstead, Oklahoma Medical Research Foundation) using the forward primer 5'-GCTAGAGCTCACATTTGCGAGAAGCACTTCCG-3' and the reverse primer 5'-GTGTGAATTCAGCGATGTGCAAATGAAGGAGC-3'. The 266-bp fragment generated was digested with SstI and EcoRI and subcloned into pBluescript. Plasmids were linearized with either Sst1 or EcoR1, and transcription was carried out using T3 and T7 RNA polymerase (Promega) in separate reactions. Sense and antisense single-stranded RNAs were combined in equal concentrations and incubated for 10 min at 68°C followed by a 30 min incubation at 37°C to form dsRNA. dsRNAs were injected into the gonads of N2 hermaphrodite C. elegans. Injected animals were allowed to recover and lay eggs for 20 h after injection, transferred to individual plates, and allowed to lay eggs for a second 24-h period. The F1 progeny resulting from this second period of egg laying were evaluated. Phenotypes were observed in >90% of F1 animals.
Antibody production and purification
A 16 amino acid peptide corresponding to residues 340355 of Ce-Duox1 was synthesized by the Emory Microchemical Facility and coupled using gluteraldehyde to keyhole limpet hemocyanin. Rabbit antibody was prepared against keyhole limpet hemocyaninconjugated peptide by Lampire Biological Laboratories using standard protocols. Peptide (30 mg) was coupled to 1 ml of Affi-Gel 10 (Bio-Rad Laboratories) for antibody immunopurification; 2 ml of serum was dialyzed against PBS and was loaded onto the Affi-Gel column preequilibrated with PBS. The column was washed with 10 ml PBS containing 1 M NaCl. 0.5 ml-factions of antibody were eluted with 0.1 M glycine-HCl (pH 2.4) and were immediately neutralized with Tris, pH 9. Fractions containing the highest concentration of protein were used in immunofluorescence experiments.
Western blot
Nematodes were washed with M9 buffer, suspended in 0.5 ml sonication buffer (10 mM Tris HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride) and sonicated four times for 20 s. Protein was determined with the Bradford assay using BSA as a standard. 10 µg of whole animal extract was loaded onto a 10% SDS-page gel, which was then transferred to Immobilon-P membrane (Millipore). The blot was blocked for 1 h in a solution of 5% nonfat powdered milk and 0.05% Tween in PBS. The antibody to Ce-Duox1 was added in a 1 to 2,000 dilution, incubated overnight, and the membrane was washed three times for 15 min with blocking solution. The blot was then developed using the SuperSignal chemiluminescent kit from Pierce Chemical Co. A Western blot of C. elegans protein extract showed a single band with a molecular weight of 180,000 (unpublished data).
Indirect immunofluorescence
Immunofluorescence staining of C. elegans was carried out as in Benian et al. (1996). Goat antimouse rhodamine-conjugated antibody and goat antirabbit FITC-conjugated antibody were used as secondary antibodies for the detection of Ce-Duox1 antibody, myosin A antibody, and MH4 antibody. Mouse antibody to myosin A was a gift from D. Miller (Vanderbilt University, Nashville, TN) (Miller et al., 1983). The MH4 monoclonal antibody developed by G.R. Francis and R.H. Waterston (Washington University, St. Louis, MO) (Francis and Waterston, 1991) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences. To determine nonspecific binding of the Ce-Duox1 antibody, a 10-fold molar excess of Ce-Duox1(340355) peptide was added to neutralize the antibody. Microscopy was carried out using ZEISS 510 laser scanning confocal microscope.
Preparation of dityrosine standard
Dityrosine standard was synthesized and purified as in Abdelrahim et al. (1997) with minor modifications. Reaction products were dissolved in acidified methanol, filtered, and directly applied to the CP-11 cellulose phosphate, eliminating the rotary evaporation step. Samples with absorption properties characteristic of dityrosine were pooled and freeze dried. For mass spectrometry, the 1 ml of dityrosine standard (0.77 mg/ml) was added to 1 ml of methanolwater (1:1) in 0.1% acetic acid.
Analysis of dityrosine and trityrosine
Nematodes were washed with M9 buffer, suspended in 0.5 ml sonication buffer (10 mM Tris HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride), and sonicated four times for 20 s. Protein was determined with the Bradford assay using BSA as a standard. Whole worm extracts were lyophilized and resuspended in 6 N HCl. Samples were hydrolyzed for 24 h at 110°C under vacuum, dried under vacuum, and resuspended in the mobile phase for analysis by HPLC on a C18 column (0.46 x 26 cm; Fisher Scientific) using a Dionex AGP-1 HPLC instrument. The mobile phase consisted of 0.1 M KH2PO4 adjusted to pH 3.8 with 0.1 M phosphoric acid at a flow rate of 1 ml/min. The column eluent was monitored by fluorescence with an excitation 305395-nm bandpass filter and an emission filter at 450 nm with a bandpass of 40 nm. To verify the identity of dityrosine, authentic dityrosine standard was added to some samples and an increase in the intensity of the putative dityrosine band was observed (unpublished data).
Spectroscopic properties of di- and trityrosine
HPLC-purified samples of dityrosine and trityrosine from both C. elegans extracts and peroxidase domain cross-linking reactions were lyophilized and resolubilized in either 0.1 M HCl (3 ml) or 0.1 M NaOH (3 ml). Fluorescence excitation and emission spectra were obtained with a PerkinElmer LS-5B luminescence spectrometer.
Mass spectrometry
Mass spectrometry was preformed on a PerkinElmer sciex API 3000 triple quadruple mass spectrometer equipped with a turboionspray source. Dried dityrosine standard (20 mg) was reconstituted in 200 µl of H2O. A 50-µl aliquot of this was diluted to a final volume of 1 ml with 950 µl of 5 mM ammonium acetate in MeOH and 1% acetic acid. This solution infused at a flow rate of 5 µl min-1. The ionspray needle was held at +550 and -4500 V for positive and negative ion analysis, respectively. These experiments identified the singly protonated (positive ion mode) and deprotonated (negative ion mode) species of the standard to be m/z 361.3 and 359.3, respectively, corresponding to prediction.
Total protein, purified cuticle, and total protein minus purified cuticle acid hydrolysates from C. elegans, and standard were analyzed by reverse phase LC-MS/MS. A 50-µl volume of sample was injected onto a 15 cm x 2.1 mm Supelco Discovery C18 column at a flow rate of 300 µl min-1. Solvent A was 99:1 H2O to acetic acid and solvent B was 99:1 MeOHto acetic acid, both containing 5 mM ammonium acetate. The column was infused directly into the ion source of the mass spectrometer operating in positive ion mode. The column was preequilibrated with 100% solvent A for 6 min followed by sample injection. The column was then washed with 100% solvent A for 4 min and eluted with a 1 min linear gradient to 100% solvent B followed by a 4 min wash with 100% solvent B. For these experiments, both the precursor ions (as above for dityrosine; m/z 540.4/538.4 for trityrosine) and structurally distinctive breakdown ions were monitored. The transitions monitored for dityrosine were the neutral loss of both COOH groups, the neutral loss of both COOH groups and one NH2 group, and the neutral loss of both COOH groups and both NH2 groups (m/z 269.4, 252.2, and 235.0, respectively). For trityrosine, the transitions monitored were the neutral loss of a COOH group, the neutral loss of a COOH group and one NH2 group, the neutral loss of two COOH-termini, and the neutral loss of two COOH groups and two NH2 groups (m/z 494.3, 477.2, 448.2, and 431.2, respectively). C. elegans cuticle was purified according to methods developed previously by Cox et al. (1981).
Transmission electron microscopy
Approximately 120 wild-type or RNAi-blistered adult C. elegans were collected and washed first with M9 buffer and then with 0.1 M cacodylate buffer (pH 7.4). Animals were pelleted, added to 1 ml of 0.8% glutaraldeyde, 0.7% osmium tetroxide, 0.1 M cacodylate, pH 7.4, and incubated on ice for 1.5 h with occasional mixing. The animals were washed with 0.1 M cacodylate buffer, transferred to a glass depression slide, and cut in half with a 23-gauge needle. Bisected animals were transferred into a tube containing 1 ml of fresh fixative (0.8% glutaraldehyde, 0.7% osmium tetroxide, 0.1 M cacodylate, pH 7.4) and incubated on ice for 2 h. After washing with 0.1 M cacodylate buffer, the bisected animals were fixed overnight on ice in 1% osmium tetroxide in 0.1 M cacodylate buffer. Animals were washed several times in 0.1 M cacodylate buffer, dehydrated using graded alcohols through propylene oxide, infiltrated, and embedded in Embed-812 (Electron Microscopy Sciences). The animals were teased into parallel arrangement with an eyelash probe before polymerization at 60°C for 16 h. Sections (0.5 mm) were evaluated for orientation and ultrasections (800-Å thick) were collected on 200 mesh copper grids, stained with uranyl acetate and lead citrate, and cross sections were examined with a Philips EM201 electron microscope.
Construction of Duox peroxidase domain expression plasmids
The PCR was used to amplify the peroxidase domains of h-Duox (amino acid residues 1593) and Ce-Duox (amino acid residues 1590) from the cloned full-length sequences. The primers were designed to introduce an NH2-terminal BamH I site and a COOH-terminal Not I site. PCR productsLL were digested with BamH I and Not I and ligated into the pET-32a(+) vector from Novogen. Plasmids were transformed into BL21(DE3) cells containing the chloramphenicol-resistant plasmid pT-groE (Yasukawa et al., 1995), which expresses the chaperonins groES and groEL from the T7 promoter. The pT-groE expression vector in BL21(DE3) cells was a gift from Dr. Lee-Ho Wang (University of Texas Health Science Center, Houston, TX) and Dr. Shunsuke Ishii (Institute of Physical and Chemical Research, Ibaraki, Japan). LB-agar plates containing both ampicillin and chloramphenicol were used to isolate colonies.
Expression of Duox peroxidase domains
A 0.5-ml LB overnight culture of cells containing plasmid with the peroxidase domain from h-Duox or Ce-Duox was used to inoculate 50 ml of modified TB medium (Sandhu et al., 1993) containing 0.5 mM -aminolevulinic acid, 100 µg/ml ampicillin, and 25 µg/ml chloramphenicol in a 250-ml flask. Bacteria were grown at 37°C in a shaker at 200 RPM until the cell density measured 0.7 OD at 600 nm. Isopropyl-ß-D-thiogalactopyranoside (1 mM) was added, and the culture was continued at 25°C for 24 h at 150 RPM. Cells were pelleted at 4,500 g and resuspended in PBS containing 4-(2-aminoethyl)benzenesufnyl fluoride (2 µM), bestatin (130 nM), trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (1.4 nM), leupeptin (1 nM), and aprotinin (0.3 nM). The cell suspension was then sonicated on ice.
Activity assays
The TMB liquid substrate system (Sigma-Aldrich) was used to assay peroxidase activity (Holland et al., 1974). 100 µg of lysate protein from cells expressing either the human Duox1 peroxidase domain, Ce-Duox1 peroxidase domain, or a vector control was added to 1-ml aliquots of the TMB substrate system. The peroxidase reactions were performed in triplicate, and activity was monitored at 655 nm with a Beckman Coulter DU640B spectrophotometer. Some samples contained 30 µM aminobenzoic acid hydrazide, a peroxidase inhibitor (Kettle et al., 1995).
To assay tyrosine cross-linking, tyrosine ethyl ester (20 mM) was dissolved in 10 ml of PBS buffer supplemented with 80 µl of 3% H2O2. 100 µg of E. coli lysate protein was added to 1-ml aliquots, samples were incubated for 1 h, and the reaction was quenched using an equal volume of 12 M HCl. Samples were analyzed for di- and trityrosine as above.
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Footnotes |
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T. Lee's present address is National Creative Research Initiatives Center for Calcium and Learning, Pohang University of Science and Technology, 790-784 Pohang, Korea.
* Abbreviations used in this paper: Ce-Duox, Caenorhabditis elegans Duox; dsRNA, double-stranded RNA; FAD, flavin adenine dinucleotide; h-Duox, human Duox; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; RACE, rapid amplification of cDNA ends; RNAi, RNA interference; TMB, 3,3',5,5'-tetramethylbenzidine.
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Acknowledgments |
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This research was supported by National Institutes of Health grants (CA84138 and AR/GM 44419). D.B. Flaherty is supported by a fellowship from the American Heart Association, Southeast Affiliate, and W.A. Edens is supported by a National Institutes of Health fellowship (DK 07298).
Submitted: 28 March 2001
Revised: 2 July 2001
Accepted: 3 July 2001
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