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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Sheep airway mucus can potently scavenge hydrogen peroxide, an important mediator of airway inflammation. Here, the scavenging activity was identified as a peroxidase produced by goblet cells of the airway epithelium and secreted into the airway lumen. Ovine airway peroxidase activity was purified ~ 100-fold from airway lavage fluid in two steps, using cation exchange and lectin affinity chromatography, yielding an apparently homogeneous 82-kD glycoprotein. Ovine airway peroxidase represented about 1% of the total protein in airway mucus and thus was an abundant enzyme in airway secretions. The absorbance spectrum of the purified peroxidase showed a major peak at 412 nm indicative of a hemoprotein. The ratio of A412/A280 of the purified enzyme was 0.86. The absorption spectrum of ovine airway peroxidase, its ability to oxidize halides, its sensitivity to inhibitors and its apparent molecular mass on sodium dodecyl sulfate gels showed that airway peroxidase was similar to lactoperoxidase but distinguished from myeloperoxidase, eosinophil peroxidase as well as from glutathione peroxidases. Based on these observations, ovine airway peroxidase is a newly isolated and abundant enzyme of airway mucus which may function to control reactive oxygen species in the airway and to prevent infection by catalyzing the formation of biocidal compounds.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The surfaces of the airways are naturally exposed to a variety of potentially injurious airborne materials including inorganic and organic particulate and gaseous matter. Of
specific interest is the exposure of the airway to reactive
oxygen species (ROS) (1) which adversely affect tracheal
epithelial cells (2) and cause contraction of rat and
guinea pig intrapulmonary bronchi (5, 6) as well as hyper-responsiveness to carbachol in sheep (7). In light of their
adverse effects, it is surprising that superoxide radicals
(O2
) as well as hydrogen peroxide (H2O2) are produced
by the airway epithelium (1, 8, 9). In fact, these ROS seem
to be specifically released toward the luminal side of the
airways. The functional significance of epithelially-derived
ROS is not known but several groups have hypothesized
that they are involved in inflammatory processes in the
airways (for review, see 10).
In most tissues, H2O2 is spontaneously and constantly
formed from O2 (2 O2 + 2 H+ 
H2O2+ O2); in addition, it is formed enzymatically by the action of superoxide
dismutase. Although less reactive than O2, H2O2 can easily diffuse through membranes, gaining free access to all
intracellular compartments where it can be transformed into highly reactive hydroxyl radicals. Almost all cells possess enzymes to protect themselves against these reactive
oxygen molecules. In the airway, where epithelial cells are
known to produce ROS, the protective surface liquid of
the tracheobronchial tree is thought to act as an antioxidant (11).
Airway surface liquids are produced by specialized cells in the epithelium as a complex mixture of molecules including glycoproteins (mainly mucins), lipids, and a variety of enzymes. The H2O2-scavenging properties of airway secretions appear to be due in large part to the presence of an uncharacterized peroxidase (12) whose histochemical presence in secretory cells of the airway epithelium has been described (13) but not further investigated. The presence of a peroxidase in secreted mucus raises questions regarding its role in mucosal defense mechanisms. Based on properties and activities of known peroxidases, there are several possible functions for such an enzyme in the airway. They include: (1) removal of damaging ROS by H2O2 consumption; (2) formation of biocidal compounds (using H2O2 produced by the airway epithelium); and (3) oxidation of small molecules and/or catalysis of reactions which alter mediators of cellular activity. Before such functions can be examined, however, the nature of the peroxidase in the airways needs to be identified. Thus, the goal of this study was to isolate, purify, and characterize ovine airway peroxidase (APO).
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
Canine myeloperoxidase was a kind gift of Dr. Roger Fenna, University of Miami School of Medicine. Dithiothreitol (DT T) was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN); acrylamide, N,N'-methylene-bis-acrylamide, sodium dodecyl sulfate (SDS) and ammonium persulfate from BioRad (Richmond, CA); lectins (plain and coupled to agarose beads) from Vector Laboratories (Burlingame, CA); bovine lactoperoxidase (LPO) and all other chemicals from Sigma Chemicals (St. Louis, MO).
Sheep Tracheal Mucus Collection
Tracheal lavage. Sheep tracheal secretions were collected by lavaging the trachea as described previously (12, 16). Briefly, ewes were intubated under topical anesthesia with a double-cuff endotracheal tube providing a sealed tracheal chamber between the two inflated cuffs. This tracheal chamber was lavaged through two extramural catheters with 50 ml of pre-warmed phosphate-buffered saline (PBS), pH 7.4, every 2 h, five times in total, and the first wash was discarded. To pellet contaminating tracheal cells, the lavaged fluid was centrifuged at 300 × g for 10 min at room temperature and the supernatant again at 15,000 × g for 20 min at 4°C. The spun supernatant was stored at
80°C. After thawing, the samples were made
0.04% with cetyl-triethylammonium bromide (CeTAB),
adjusted to 10 mM NaPO4, pH 8.0, centrifuged (15,000 × g, 20 min, 4°C), and passaged through a 0.2-µm filter to remove any additional aggregates. Detergent (CeTAB) was
added to disrupt large mucin complexes and to prevent
nonspecific sticking to surfaces.
Bronchoalveolar lavage.
As an alternative to tracheal
lavages, bronchoalveolar lavages (BAL) were used to
measure APO activity and to purify the enzyme. The distal
tip of a specially designed 80-cm fiberoptic bronchoscope was wedged into a subsegmental bronchus and lavage was
performed by slow infusion and gentle aspiration of 3 × 30 ml aliquots of pre-warmed PBS via a syringe attached to
the working channel of the bronchoscope. Each lavage return was centrifuged at 420 × g for 15 min at room temperature. The supernatant was re-centrifuged at 15,000 × g for 20 min at 4°C and stored at 80°C.
Airway Peroxidase Activity Assays
Ovine APO activity was routinely assayed by oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). The assay conditions were: 50 mM sodium acetate, pH 5.2, 150 µM H2O2, 1.3 mM TMB at 37°C. Product was measured on a recording spectrophotometer by increases in absorption at 652 nm and activities were expressed as changes in absorbance per second during the initial 2 min of the reaction. The assay was linear with added mucus. Alternatively, assays were performed in microtiter plates at room temperature for 3 min. After the reaction was stopped by the addition of HCl to 400 mM, product was measured by changes in absorbance at 405 nm. Assays to determine pH optima were performed in a series of 50 mM buffers: sodium formate pH 3.5; sodium acetate pH 4.5, 5.2, and 6.0; and sodium phosphate pH 6.8, 7.5, and 8.0.
Activity of ovine APO was also measured by the triiodide formation assay developed for LPO (17). Assay conditions were: 4.5 mM KI, 0.4 mM H2O2, 0.1 M NaPO4, pH 7.0. Furthermore, oxidation assays were performed with the substrates 4-aminoantipyrene (1.25 mM 4-aminoantipyrene, 0.85 M phenol, 0.85 mM H2O2, 0.1 M NaPO4, pH 7.0) and o-phenylenediamine (4.5 mM o-phenylenediamine, 0.4 mM H2O2, 0.1 M NaPO4, pH 7.0).
Cytochemical Localization of Ovine APO
The presence of peroxidase activity in sheep tracheal mucosa was shown by histochemistry, modified from published methods (14). Rings of trachea were fixed in 1% paraformaldehyde, 1.25% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h. Following fixation, the tissue was washed in 50 mM Tris (pH 7.4) and incubated in 3,3'-diaminobenzidine tetrahydrochloride (DAB) in 50 mM Tris buffer (pH 7.4) for 30 min and then another 1 h in DAB, 50 mM Tris (pH 7.4) either in the presence or absence of 3 mM H2O2. The tissue was then washed in cacodylate buffer, postfixed in 2% cacodylate-buffered osmium tetroxide for 1 h, dehydrated in ethanol and finally embedded in Spurr's resin for cutting of both thick sections for light microscopy and thin sections for transmission electron microscopy. Oxidized DAB (a brown, highly insoluble indamine polymer) is visible in the light microscope and reacts with osmium tetroxide during postfixation to yield an electron-dense precipitate for ultrastructural studies. Thin sections were post-stained with uranyl acetate and lead citrate.
Purification and Characterization of Ovine APO
Cation exchange column. All procedures were carried out at 4°C. A 20-ml S-Sepharose® column was equilibrated with 0.15 M NaCl, 10 mM NaPO4, 0.04% CeTAB, pH 8.0. Lavage fluid (200-300 ml), prepared as described above, was fractionated on the column. The column was eluted with a salt gradient (0.15-0.5 M NaCl in 10 mM phosphate, pH 8.0), collecting fractions of 1 ml. Fractions with the highest peroxidase activity (as assessed by TMB oxidation) were pooled, evaluated by SDS gel electrophoresis (18, 19), adjusted to 0.5 M NaCl, 10 mM NaPO4 (pH 8.0), 0.04% CeTAB, and applied to a lectin column. Lectin affinity column. The pooled fractions containing peroxidase activity from the S-Sepharose® column were applied to a 2-ml Lens culinaris lectin-agarose column. The column was washed until the absorbance of the eluate at 280 nm returned to baseline. Then, the column was eluted with 0.5 M a-methyl mannoside, 0.5 M NaCl, 10 mM NaPO4 (pH 7.4) collecting fractions of 1 ml. The final purity of the fractions was evaluated by SDS gel electrophoresis (18, 19), peroxidase activity, and ratio of A412/A280 absorbance. Spectral characteristics. The peak fraction from the lentil-agarose chromatography was scanned in a 1-cm path-length cuvette thermostatted to 20°C, at 0.5 nm increments and the absorption signal was digitized and stored. To compare ovine APO to other known peroxidases and to characterize the reactive center, analysis of absorption spectra were performed between 260-800 nm. Staining of SDS gels for peroxidase activity. To stain for peroxidase activity of proteins resolved by electrophoresis (20, 21), SDS gels were submerged in 2 mM TMB, 75 mM sodium acetate, 30% methanol, pH 5.2 for 45 min. Then, H2O2 was added to a final concentration of 30 mM and the incubation continued for another 30 min. The reaction was stopped by exchanging the bathing solution with 30% isopropanol, 75 mM sodium acetate, pH 5.0 and gels were then photographed. Following longer incubation in 30% isopropanol, 75 mM sodium acetate, pH 5.0, to remove TMB oxidation product, the gels were stained with Coomassie blue. Amino acid sequencing. After SDS electrophoresis, homogeneous enzyme was transferred to PVDF membranes using a wet electrophoretic technique (22). The portion of the filter containing purified ovine APO was excised and sent to the Protein Core Facility at the University of Florida College of Medicine for amino-terminal sequence analysis on the Applied Biosystems Gas Phase Sequencer.| |
Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Peroxidase Activity
To directly demonstrate peroxidase activity in tracheal secretions, tracheal lavage fluid was assayed for its ability to transfer electrons from four classic peroxidase substrates (Figure 1). In the presence of H2O2, tracheal lavage fluid rapidly oxidized TMB, o-phenylenediamine, as well as 4-aminoantipyrene, and formed triiodide from KI in a concentration-dependent fashion (Figure 1). The oxidation of TMB was abolished by sodium azide at a concentration as low as 10 µM. The azide sensitivity suggested that the activity was unrelated to the glutathione peroxidase family (23, 24). Direct comparison of triiodide formation by tracheal lavage fluid to published values for bovine milk LPO (17) showed that tracheal lavage fluid contained approximately 0.7 U of LPO-like activity per mg of dry weight. BAL fluid also oxidized TMB, although with less activity per A280 than tracheal lavage (0.01- 0.001 times). The oxidation of all of the substrates required the presence of H2O2, conclusively demonstrating peroxidase activity in both tracheal lavage and BAL.
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Purification of Ovine APO
In an effort to isolate ovine APO from other glycoproteins in tracheal lavage, specific glycoproteins were removed from the processed tracheal lavage fluid by precipitation using Dolichos biflorus, Ulex europaeus, Arachis hypogaea, and Lens culinaris lectins. In the absence of detergent, all these lectins depleted ovine APO activity. After adding CeTAB to the lavages, however, only Lens culinaris lectin depleted ovine APO activity.
Ovine APO purification to apparent homogeneity was achieved by a two-step purification protocol using, first, cation exchange chromatography with S-Sepharose® and second, lectin affinity chromatography with Lens culinaris lectin coupled to agarose. During a typical purification procedure from tracheal lavage, the initial cation exchange chromatography yielded an ~ 20-fold increase in ovine APO specific activity. After Lens culinaris lectin affinity chromatography of the combined peak activity fractions from the cation exchange column, ovine APO was purified an additional ~ 5-fold for a total of ~ 100-fold with a yield of 50% of the activity in the starting lavage fluid (Table 1). Purified fractions of ovine APO could also be obtained from BAL, although total purification ranged from 200- 1,000-fold, consistent with the starting amount of peroxidase activity in BAL (see above).
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Characterization of Ovine APO
The purified ovine APO sample was analyzed on SDS gels after complete reduction with DTT and denaturation by heating at 100°C for 5 min in SDS. Coomassie blue staining revealed a single protein band at 82 kD (Figure 2a, lane 1) which could be distinguished from both highly purified bovine LPO and canine myeloperoxidase (MPO) (Figure 2a, [lanes 2 and 3, respectively]). By comparison, bovine LPO had an apparent molecular weight of 78 kD as reported by others (25). In the canine MPO lane, one of the two MPO subunits (59 kD) could be identified (26); the smaller subunit (13 kD) ran off the gel.
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Occasionally, the final fractions from the purification procedure displayed immunoglobulin contamination and these fractions were purified only about 50-60-fold from the starting material using the two chromatography steps. Such impure samples revealed two protein bands corresponding to the heavy and light chain of immunoglobulins in addition to the 82-kD protein. It was completely unpredictable whether immunoglobulins would be present in the samples or not. Thus, the purification variability appeared to depend mainly on the animal used for lavage. These preparations were not used for further studies.
To demonstrate that the 82-kD protein, found in the fully reduced and denatured ovine APO fractions, in fact had peroxidase activity, purified ovine APO was also run on SDS gels without prior reduction and heating in SDS, treatments which destroy catalytic activity. After incubation of such gels with TMB and H2O2 (21), peroxidase activity was detected at an apparent molecular weight of around 82 kD (Figure 2b, lane 1), but portions of the peroxidase activity did not enter the resolving gel or moved at intermediate sizes. Counterstaining of the gel shown in Figure 2b with Coomassie blue (Figure 2c, lane 1) revealed that the protein co-migrated with the peroxidase activity in Figure 2b, lane 1. In addition and similar to ovine APO, bovine LPO revealed peroxidase activity after electrophoresis without prior reduction and heating (Figure 2b, lane 2), and also ran as a heterogenous mixture of sizes despite being highly purified (see Figure 2a, lane 2). All of the Coomassie stainable material in the bovine LPO lane, like ovine APO, co-migrated with peroxidase activity (compare lanes 2, Figures 2b and 2c). Following identical treatment, canine MPO showed almost no activity (Figure 2b, lane 3) due to the fact that MPO is solely active when the subunits of this enzyme are associated. The purified canine MPO dissociated even under non-reducing conditions although a small portion that ran as a dimer revealed peroxidase activity (see minimal activity just below the 112-kD marker in Figure 2b, lane 3).
Amino-terminal amino acid sequence analysis of the purified ovine APO revealed that the 82-kD band was blocked at the N-terminus. Despite the block, some limited sequence information was obtained probably from a proteolytic fragment in the preparation. The sequence showed partial homology with bovine LPO, but not with MPO or eosinophil peroxidase (EPO).
The absorption spectrum of purified ovine APO exhibited the classic Soret type spectral absorption bands typical for heme-containing peroxidases. The major band for ovine APO was found at 412 nm and minor bands at 500, 544, and 635 nm (Figure 3). The purified ovine APO fraction had an A412/A280 absorbance ratio of 0.86.
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Because some peroxidases work best in a slightly acidic environment, the pH-dependence of ovine APO activity was evaluated in comparison to bovine LPO and canine MPO. Ovine APO showed a pH optimum between 4.0 and 5.0 similar to bovine LPO; a slight decrease in activity was found from pH 5.0 to pH 8.0 (Figure 4). Ovine APO and bovine LPO were clearly distinguished from canine MPO which showed maximal activity around pH 6.0.
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), bovine LPO (
), and canine
MPO (
) catalytic activity was
measured using TMB as a substrate in assays buffered at different pH values. Activity is expressed as the fraction of the
activity at the measured pH optimum.
Although secreted mammalian peroxidases share many
overall features in their reaction mechanisms, differences
in oxidation of halide or pseudohalide compounds allow
distinction among them (26). To evaluate such differences,
ovine APO, bovine LPO, and canine MPO were dialyzed
against 10 mM NaPO4 and assayed in the presence of
changing Cl or I concentrations. Ovine APO and bovine
LPO were stimulated 5-fold and 2-fold respectively at 1 mM I, while canine MPO was unaffected over this concentration range (Figure 5a). Chloride only slightly stimulated ovine APO and bovine LPO activity at concentrations between 1 mM and 200 mM, while canine MPO
activity was inhibited over the same concentration range
(Figure 5b), as reported by others (27).
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Ovine APO oxidation of TMB was inhibited by dapsone, azide, salicylhydroxamic acid (SHA) and aminotriazole. The inhibition of ovine APO could barely be distinguished from the inhibition of bovine LPO by the same compounds. In each case, however, canine MPO was differently inhibited than ovine APO or bovine LPO (Figure 6). The azide inhibition of ovine APO as well as its molecular size by SDS polyacrylamide gel electrophoresis (PAGE) and its absorption spectrum further rule out that ovine APO is related to glutathione peroxidases.
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Cytochemical Localization of Ovine APO
To identify cells in the trachea which may contribute to the secreted peroxidase activity, tracheal sections were fixed and then incubated in the presence of DAB and H2O2. Light microscopic examination showed that DAB reaction product was localized to a subset of epithelial cells (Figure 7a). Subsequent counterstaining of the very same sections with Mallory's stain revealed that the DAB-labeled cells were goblet cells (Figure 7b). No reaction product was seen when the tissues were incubated with DAB in the absence of H2O2 (Figure 7c), demonstrating that H2O2 was mandatory for formation of the reaction product. Electron microscopy of sheep tracheal epithelium showed peroxidase reaction product in granules and lamellar structures of the secretory pathway of cells after incubation in both DAB and H2O2 (arrows, Figures 8a and 8b) but not in the absence of H2O2 (Figure 8c).
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Peroxidase Activity
We have shown previously that ovine airway mucus can potently scavenge H2O2 (12). The H2O2 scavenging activity was proteinaceous, azide sensitive, and unlikely due to MPO contamination from luminal leukocytes or released catalase (12). Collectively, those data suggested that the scavenging activity was associated with a peroxidase, most likely produced locally by the airway epithelium. Here, we conclusively showed that peroxidase activity is present in tracheal lavage fluid since four classic peroxidase substrates (TMB, KI, o-phenylenediamine, 4-aminoantipyrine) served as electron donors in oxidation assays. Christensen and associates (13, 14) formerly described the presence of peroxidase activity in airway epithelial cells using cytochemical assays in guinea pigs and hamsters and suggested that this peroxidase was secreted into the airway lumen. We therefore designated the peroxidase activity found in tracheal secretions airway peroxidase (APO), following the naming suggestion of those investigators.
Avissar and colleagues (28) recently described the presence of a selenium-dependent glutathione peroxidase in the epithelial lining fluid of the airways. The peroxidase activity presented here could not be due to this enzyme because selenium-dependent glutathione peroxidases are azide insensitive (at > 1 mM azide) (23), whereas the activity of the presented peroxidase was completely abolished at azide concentrations as low as 10 µM. Selenium-independent glutathione peroxidases (or glutathione-S-transferases) have not been described in the airway epithelial lining fluid and the azide sensitivity (23) as well as the necessity of H2O2-addition to the oxidation assays rule out an association of the described activity with glutathione-S-transferase (these enzymes do not use H2O2 [23]). Taken together, the characteristics of the found peroxidase activity indicates it is most likely similar to secretory peroxidases produced in mammary or salivary glands.
The results from the triiodide formation assay showed that tracheal lavages contained approximately 0.7 U of activity per mg of dry weight based on the reported specific activity of purified bovine LPO (17). Given the fact that ovine APO has a similar size compared to bovine LPO and assuming a similar catalytic activity, 0.7 U represent approximately 10 µg of enzyme. This estimate predicted that ovine APO constitutes as much as 1% of all secreted macromolecules in tracheal lavage (per mg dry weight), most likely representing the major enzymatic activity of sheep tracheal secretions. The validity of these estimates were confirmed by the 100-fold purification of the enzyme from tracheal lavage to homogeneity.
This amount of enzyme would easily account for all of the previously observed H2O2 scavenging activity of airway mucus. Given the dilution of airway secretions by lavage buffer during collection (at least 20 -50-fold), the molar ovine APO concentration in mucus could possibly exceed the molar concentration of bovine LPO in milk by as much as 10 -100-fold (29). The fact that the amount of peroxidase activity in BAL was less than that in tracheal lavage could be explained by contamination of BAL with alveolar proteins and the reduced number of secretory cells in more peripheral airways.
Purification of Ovine APO
The strategy used for purification of ovine APO was based on the general properties of other secreted mammalian peroxidases. These peroxidases have high isoelectric points (pH 8-10) and can be partially purified on strong cation exchangers (30). Furthermore, we have previously shown that the H2O2 scavenging activity appeared to be associated with a glycoprotein (12), similar to other peroxidases. In fact, purification to apparent homogeneity was possible using cation exchange chromatography followed by Lens culinaris lectin affinity chromatography. Apparent homogeneity was confirmed by SDS gel electrophoresis evaluation and the A412/A280 absorbance ratio of 0.86 which is in the range of values obtained for other highly purified heme-containing peroxidases (31). The two-step purification procedure was somewhat variable with respect to final purity (50-100%) and yield. If present, the major contamination of the final fractions was immunoglobulin. The nature of this immunoglobulin has not been determined; however, IgM can be excluded from the position of the heavy chain appearing on the gels. It was completely unpredictable whether immunoglobulins would be present in the samples or not. Thus, the purification variability appeared to depend mainly on the animal used for lavage.
Characterization of Ovine APO
The absorption spectrum of purified ovine APO shows that it is a heme-containing enzyme. Thus and as expected from the features of the activity in tracheal lavage, ovine APO is distinct from a selenium-dependent glutathione peroxidase secreted into the airway lining fluid (28). This spectrum also distinguishes ovine APO from MPO, which has an absorption maximum at 430 nm, and suggests that ovine APO more closely resembles either LPO or EPO which cannot be distinguished by their spectra alone. The size and subunit structure of ovine APO, however, rules out that ovine APO was identical to EPO (32) as was expected based on the virtual absence of eosinophils in freshly collected lavage fluid from healthy animals. In addition, the pH dependence of ovine APO closely resembled bovine LPO but was distinct from canine MPO which showed a broad optimum between pH 6 and 7. The pH of the periciliary fluid in the airway lumen is slightly acidic with a pH as low as 6.85 under physiological conditions (33, 34) but is expected to be much more acidic during active bacterial infection where maximal activity of a potential biocidal function of ovine APO would be expressed.
More evidence for the similarity between ovine APO and bovine LPO stems from the partial amino acid sequence of the 82-kD protein, most likely obtained from a proteolytic fragment of the 82-kD protein (the N-terminus was blocked). This sequence revealed partial homology between ovine APO and bovine LPO. Furthermore, the stimulation and inhibition profiles suggested that ovine APO closely resembles bovine LPO with differences in halide stimulation which could be species specific. Ovine APO, however, was clearly distinct from canine MPO.
Cytochemical Localization of Ovine APO
So how does this peroxidase end up in the airway lining fluid? Our cytochemical results indicate that APO is probably made within goblet cells of the airway epithelium. Although the staining procedure used for histochemistry is nonspecific, the chosen reaction conditions exclude the involvement of several enzymes in DAB product formation. First, the necessity of H2O2 rules out the involvement of glutathione-S-transferase (or non-selenium-dependent glutathione peroxidase) which does not use H2O2 (23). Second, since glutathione peroxidase does not use DAB as a substrate (23, 35, 36), the activity inside goblet cells is not due to glutathione peroxidase (37). Third, the reaction conditions also exclude the involvement of catalase, mitochondrial enzymes (38), or product formation due to the presence of superoxide radicals (8, 41, 42). Thus, it is logical to infer that the major peroxidase activity found in the secretory pathway of goblet cells is identical to the activity secreted into the tracheal lumen, although the DAB reaction product inside goblet cells cannot be assigned to the purified ovine APO with total certainty. In support of this hypothesis, Christensen and colleagues (14) have shown that goblet cell granules with peroxidase activity may be discharged into the airway lumen.
The known peroxidases derived from leukocytes and epithelia are all made in the endoplasmic reticulum and pass through the lumen of the intracellular organelles on the way to secretion or to the plasma membrane. Membrane proteins and secreted proteins share this "secretory pathway" of biosynthesis which is distinct from the biosynthetic pathway of the proteins found in the cytoplasm including most glutathione peroxidases (with the exception of the secreted glutathione peroxidase [37]) and catalase. The finding of peroxidase activity in both the biosynthetic portion of the secretory pathway and in secretory granules rules out that the observed activity is due to endocytosed activity (e.g., EPO or MPO). Thus, our electron microscopy results are consistent with the conclusion that this secreted peroxidase is in fact made in goblet cells and are in agreement with those of Christensen and colleagues (13, 14).
In conclusion, the airway epithelium synthesizes and secretes a peroxidase which is distinct from canine MPO and from selenium-dependent and -independent glutathione peroxidases, but similar to bovine LPO. In sheep, the enzyme is an 82-kD glycoprotein which comprises about 1% of the total protein found in tracheal lavage. This activity is present in sufficient amount to account for all of the previously reported H2O2 scavenging activity in ovine airway secretions. The production of ROS by the airway epithelium, especially H2O2 (9), coupled with production of a peroxidase in goblet cells provides the airway with all the necessary components for a peroxidase-driven anti-microbial system. The exact function of this peroxidase remains to be determined; however, it may play a role both as a biocidal agent against bacteria, fungi, and viruses and as a scavenger of H2O2 during airway inflammation.
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Footnotes |
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Address correspondence to: Gregory E. Conner, Ph.D., Department of Cell Biology and Anatomy (R-124), University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136.
(Received in original form July 24, 1996 and in revised form October 2, 1996).
Acknowledgments: The writers thank Dr. Roger Fenna for supplying purified canine myeloperoxidase and for helpful discussions and suggestions. M. Salathe is a Howard Hughes Medical Institute Physician Postdoctoral Fellow. This work was supported by ALA/Florida, the Foundation for Fellows in Asthma Research, and NIH grant HL-20989.
Abbreviations
APO, airway peroxidase;
BAL, bronchoalveolar lavage;
CeTAB, cetyl-triethylammonium bromide;
DAB, 3,3' diaminobenzidine;
EPO, eosinophil peroxidase;
H2O2, hydrogen peroxide;
LPO, lactoperoxidase;
MPO, myeloperoxidase;
PBS, phosphate-buffered saline;
ROS, reactive
oxygen species;
O2, superoxide radical;
TMB, 3,3',5,5' tetramethylbenzidine.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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