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A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H2O2, and this study discusses the possible involvement of the chorion peroxidase in H2O2 formation by mediating NADH/O2 oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn2+. Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O2 leads to the production of H2O2, demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD+ oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD+ oxidoreductase and chorion peroxidase on generating H2O2 with NAD+ and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD+ and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O2 oxidoreduction may contribute to the formation of the H2O2 required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H2O2 production.
Peroxidase plays multiple physiological roles in living organisms. These enzymes occupy a special position in biochemistry and enzymology and are among the most extensively studied enzymes. In addition to their primary peroxidative reaction cycle with H2O2 as oxidizing agent, some peroxidases show an oxidase activity mediating the reduction of O2 to superoxide ( ) and H2O2 by substrates such as epinephrine, dihydroxyfumarate, reduced nicotinamide adenine dinucleotide (NADH) and indole-3-acetic acid [1–7]. There are also reports that discuss the catalase activity of the peroxidases [8,9], especially from fungi [10–12]. Data from our recent study demonstrate the presence of a specific peroxidase in mosquito eggs and the involvement of this enzyme in chorion protein cross-linking through dityrosine formation, which contributes to the formation of a hardened chorion in mosquitoes [13,14]. Floodwater mosquitoes, such as the yellow-fever mosquitoes of Aedes aegypti, oviposit on substrates at the edge of water, and the eggs hatch only after being flooded into water following adequate rainfall. The time period for these eggs in the environment varies enormously depending upon weather conditions. Consequently, formation of a hardened and protective chorion that is resistant to desiccation and other environment adversities is critical for the survival of these eggs before hatching.
The involvement of peroxidase in chorion protein cross-linking during chorion hardening is based on the progressive increase in peroxidase activity in the developing eggs, the localization of the enzyme within the chorion in mature eggs, and the detection of dityrosine in the hydrolysate of the hardened chorion [13,14]. The catalytic cycle of peroxidases is initiated with the oxidation of the resting enzyme by H2O2 to an active form termed compound I that is two oxidizing equivalents above its resting state, and two subsequent one-electron oxidations of reducing agents, involving the formation of compound II intermediate, return the compound I to its resting state . During peroxidase-mediated chorion hardening, the enzyme, after being oxidized by H2O2, oxidizes tyrosine residues on protein to tyrosine radicals that interact to form dityrosine, thereby resulting in chorion protein cross-linking that contributes to the formation of a rigid and insoluble chorion in mosquitoes. To be able to oxidize tyrosine, peroxidase must first be oxidized by H2O2; consequently, there must be mechanism(s) leading to the formation of H2O2 during chorion peroxidase-catalyzed protein cross-linking. In this communication, we provide data that suggest a feasible biochemical pathway for the formation of H2O2 involving both chorion peroxidase and a malate/NAD+ oxidoreductase (MAD) during mosquito chorion hardening.
Catalase (from bovine liver, 48 000 units/mg protein), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), disodium DL-malate, H2O2 (30%), horseradish peroxidase (HRP), manganese chloride (MnCl2), β-NAD+, β-NADH, phenazine methosulfate (PMS), phenylmethylsulfonyl fluoride (PMSF), superoxide dismutase (from bovine kidney, 4,950 units/mg protein) and L-tyrosine were from Sigma (St. Louis, MO). Dityrosine was synthesized enzymatically by mixing HRP with tyrosine and H2O2 , and purified by reverse-phase HPLC using a reverse-phase (C18) column (1.0×25 cm).
Aedes aegypti black-eyed Liverpool strain mosquitoes used in this study were reared according to a described method . Mosquito ovaries with mature eggs were dissected from A. aegypti at 72 h following a bloodmeal and placed in 10 mM phosphate buffer (pH 6.5). The procedures for the purification of chorion peroxidase were the same as those described in our recent study . Protein was determined by a colorimetric method .
A reaction mixture (0.3 ml) consisting of 0.1 mM NADH and varying amounts of purified chorion peroxidase (0, 2 and 5 μg) was prepared in 0.1 M phosphate buffer (pH 7.5) and incubated at 25°C. Oxidation of NADH in the reaction mixture was monitored spectrophotometrically at 340 nm. The effect of pH on peroxidase-mediated NADH oxidation was based on the rate of NADH oxidation in the NADH/chorion peroxidase reaction mixture (0.3 ml) prepared in 0.1 M citrate buffer (pH, 4.5–6.5), phosphate buffer (pH, 7.0–7.5) or Tris buffer (pH 8.0–8.5), respectively. The effect of Mn2+ on chorion peroxidase-catalyzed NADH oxidation was also based on the rate of NADH oxidation in the NADH/chorion peroxidase reaction mixtures (0.3 ml) in 0.1 M phosphate buffer (pH 7.5) containing 0, 40, 80, 160 and 240 μM MnCl2, respectively.
Formation of and H2O2 during NADH oxidation by peroxidase was based on production of dityrosine in a NADH/peroxidase reaction mixture after addition of tyrosine. A reaction mixture (0.3 ml) consisting of 0.1 mM NADH, 80 μM Mn2+ and 5 μg chorion peroxidase was prepared in 0.1 M phosphate buffer (pH 7.5), and incubated at 25°C. At 10 min after incubation, 0.1 ml of 1.5 mM tyrosine was added into the reaction mixture, and the reaction mixture was incubated for an additional 10 min. The reaction was stopped by mixing 0.4 ml of 0.8 M formic acid into the reaction mixture. The sample was centrifuged at 20 000×g for 15 min, and the supernatant was analyzed by HPLC with electrochemical detection (HPLC-ED) to determine the formation of dityrosine . and H2O2 formed in the reaction mixture may oxidize NADH directly or through peroxidative pathway, and their role in enhancing NADH oxidation was assessed by changes of NADH oxidation rate in the presence of either 40 units of catalase or superoxide dismutase in the reaction mixture.
Chorion sediments from 3000 ovary pairs were treated with 1% Triton X-100 plus sonication, and the solubilized chorion proteins were extensively dialyzed against 10 mM phosphate buffer (pH 7.5) containing 1 mM PMSF. The sample was chromatographed on a Q-cellulose column (2.5×12 cm), and proteins were eluted with a linear potassium phosphate (0–250 mM, pH 7.5). The active MAD fractions were pooled, washed and concentrated using a stirred cell with a membrane at molecular mass cut-off of 30 000 (Millipore). The concentrated enzyme fractions were chromatographed on an UNO-Q column (7×35 mm, Bio-Rad), and the active fractions that were devoid of peroxidase activity were concentrated and used for MAD activity assays. The presence of MAD in the concentrated fractions was further verified by native polyacrylamide gel electrophoresis of the sample with subsequent substrate staining in a solution containing MTT, PMS, NAD+ and malate .
The MAD activity was assayed spectrophotometrically at 340 nm. A reaction mixture (0.3 ml) consisting of 0.3 mM NAD+, 2 mM malate and 6 μg of MAD fraction was prepared in 0.1 M phosphate buffer (pH 7.5) and incubated at 25°C. Increase in absorbance at 340 nm was continuously monitored for 10 min. A reaction mixture with heat-inactivated MAD and a reaction mixture without malate served as controls. NADH is easily oxidized at the working electrode during HPLC-ED analysis. Therefore, accumulation of NADH in the above reaction mixtures was also verified by HPLC-ED at an oxidative potential (850 mV) of the working electrode.
The sequential actions of MAD and chorion peroxidase in mediating H2O2 formation with NAD+ and malate as initial precursors were determined by incubation of malate and NAD+ in the presence of chorion MAD fraction with subsequent addition of purified chorion peroxidase and tyrosine. The accumulation of H2O2 in the reaction mixture was based on detection of dityrosine. The ability to generate H2O2 by crude chorion proteins was also evaluated by mixing urea-solubilized chorion proteins with NAD+, malate and tyrosine at the same time and formation of H2O2 was based on the detection of dityrosine in the reaction mixture after a 40 min incubation.
There was no apparent decrease in absorbance at 340 nm in a NADH solution at weak basic conditions during a short incubation period, but a much rapid absorbance decrease at 340 nm was observed when chorion peroxidase was incorporated into the NADH solution (Fig. 1). The rate of absorbance decrease was approximately proportional to the amounts of chorion peroxidase in the reaction mixture (Fig. 1), indicating that the absorbance decrease of the reaction mixture at 340 nm results from chorion peroxidase-mediated NADH oxidation.
Based on the rate of NADH oxidation, two optimum pHs for chorion peroxidase-mediated NADH oxidation, one at weak acidic (pH 5.0) and the other at slightly basic conditions (pH 7.5), were determined (Fig. 2). At pH above 8.5, decrease in NADH was due primarily to the nonenzymatic NADH oxidation (not shown). Because pH 7.5 is close to physiological pH conditions, the subsequent NADH oxidation reactions were conducted in 0.1 M phosphate buffer at pH 7.5.
Mn2+ had no noticeable effect on enhancing NADH oxidation in the absence of chorion peroxidase (Fig. 3, line 1), but it significantly increased chorion peroxidase-mediated NADH oxidation at its applied concentrations of 40, 80 and 160 μM, respectively, with a maximum enhancing effect at 80 μM (Fig. 3, lines 4–6). However, a higher concentration of Mn2+ inhibited peroxidase-mediated NADH oxidation (Fig. 3, line 2).
At the applied reaction conditions, oxidation of NADH (0.1 mM final concentration in 0.3 ml) was completed at 10 min after incubation in the presence of 5 μg chorion peroxidase and 80 μM Mn2+ (Fig. 4A). Disappearance of NADH in the reaction mixture was also verified by HPLC-ED (not shown). When tyrosine was added to the above reaction mixture at 10 min after NADH oxidation and then incubated for an additional 10 min, production of dityrosine was observed in the reaction mixture (Fig. 4B). Oxidation of tyrosine by peroxidase requires H2O2; consequently, production of dityrosine demonstrates the accumulation of H2O2 in the reaction mixture. In contrast, no dityrosine was produced during a 20 min incubation in a reaction mixture containing tyrosine, chorion peroxidase and Mn2+ in the absence of NADH (Fig. 4C), indicating that H2O2 is derived from the NADH/O2 oxidoreduction process.
When the NADH/chorion peroxidase reaction mixture in the presence of 80 μM of Mn2+ was incubated in the presence of superoxide dismutase, the rate of NADH oxidation was decreased (Fig. 5). A similar effect was observed when catalase was incorporated into the NADH/chorion peroxidase/Mn2+ reaction mixture (Fig. 5).
Progressive increase in absorbance at 340 nm was observed when the concentrated MAD fractions devoid of chorion peroxidase activity after UNO-Q column separation were mixed with NAD+ and malate (Fig. 6A, line 1). Increase in absorbance at 340 nm was not observed when heat-inactivated MAD fraction was incorporated into the reaction mixture (Fig. 6A, line 2) or when malate was omitted in the reaction mixture (Fig. 6A, line 3). Analysis of the above reaction mixtures using HPLC-ED revealed the accumulation of NADH in the reaction mixture with active MAD fraction (Fig. 6B) and the absence of NADH in the reaction mixture with heat-treated MAD fraction (Fig. 6C). Native electrophoresis of the concentrated protein sample with subsequent MAD activity staining in a solution containing MTT, PMS, NAD+ and malate resulted in the detection of a positive MAD band (Fig. 6D, lane 1). The MAD band was not observed when the native gel with separated protein sample were incubated in the staining solution but without either malate or NAD+ (Fig. 6C, lanes 2 and 3). These data demonstrate the presence of a MAD in the mosquito chorion.
The sequential actions of MAD and chorion peroxidase in H2O2 formation with malate and NAD+ as starting substrates were demonstrated by the accumulation of NADH in a NAD+/malate reaction mixture in the presence of MAD fraction (Fig. 7A), the disappearance of NADH in the reaction mixture at 20 min after addition of chorion peroxidase and Mn2+ (Fig. 7B), and the production of dityrosine following addition of tyrosine into the same reaction mixture (Fig. 7C). Triton-solubilized chorion proteins had fairly high MAD activity, but this nonionic detergent was not efficient on solubilizing chorion peroxidase because of a low chorion peroxidase activity in the Triton-solubilized chorion proteins. In contrast, saturated urea was effective in solubilizing both MAD and chorion peroxidase, and both enzymes were active in the presence of the high concentration of urea. When malate, NAD+ and tyrosine were mixed with the urea-solubilized chorion proteins at the same time and incubated at 25°C, formation of dityrosine was also observed in the reaction mixture after 40 min incubation (Fig. 7D).
Peroxidase-catalyzed chorion protein cross-linking through dityrosine formation contributes to the formation of a hardened chorion in A. aegypti eggs . Involvement of peroxidase in eggshell protein cross-linking has also been suggested in Drosophila [18–20]. Although there have been no critical analysis of chorion hardening in other insects, peroxidase-mediated eggshell or chorion protein cross-linking is likely to be a critical physiological event in oviparous insects whose embryos require protection from environmental factors during embryonic development in the environment. H2O2 is necessary for peroxidase-catalyzed chorion protein cross-linking through dityrosine production; therefore, there must be pathway(s) leading to the production of H2O2 during chorion hardening in insects. Our data suggest that during chorion hardening H2O2 may be derived from NADH/O2 oxidoreduction mediated by chorion peroxidase in A. aegypti eggs or that this pathway may be one of the H2O2 generating mechanisms, and a MAD that also is present in the chorion of A. aegypti eggs may be responsible for the supply of NADH.
In plants, several peroxidase isozymes are present in the cell wall. These enzymes catalyze cell wall protein cross-linking and polymerization of cinnamic alcohols, the precursors of lignin [21–26]. Plant cell wall peroxidases were able to oxidize NADH to produce H2O2; therefore, these enzymes were suggested to be responsible for the production of H2O2 by mediating NADH/O2 oxidoreduction during lignification . There were also reports suggesting the presence of a cell wall-bound MAD and the role of the enzyme in H2O2 generation through the supply of NADH during lignification [27,28]. The ability of chorion peroxidase to enhance NADH oxidation and H2O2 production and the presence of a MAD in the solubilized chorion proteins provide a reasonable basis for suggesting a feasible H2O2 generating pathway involving both chorion peroxidase and MAD during chorion protein cross-linking in A. aegypti eggs.
The mechanism of the peroxidase-mediated NADH oxidation and H2O2 formation is likely to be similar to those described for HRP [29–31]. It is considered that H2O2, in catalytic amounts that may be derived from autoxidation of NADH if no exogenous H2O2 is added, is required to initiate NADH oxidation by peroxidase [29,30]. Once a small amount of H2O2 is present, it oxidizes peroxidase to compound I that in turn oxidizes NADH to NAD•. NAD•, once formed, can react with O2, leading to the formation of that can either dismutate to form H2O2, or react with NADH to produce NAD• and H2O2 In addition, NAD• could be oxidized by to produce relatively stable NAD+ and H2O2, respectively. The following describes the proposed overall reaction mechanisms involved in the chorion peroxidase-mediated NADH/O2 oxidoreduction (Eqs. 1–7).
Although NADH can be oxidized by O2, it proceeds slowly at physiological pH. However, the small amount of H2O2 derived from NADH autoxidation seems adequate enough to initiate a chain reaction of peroxidase-mediated NADH oxidation. In this process, peroxidase promotes H2O2 formation indirectly through its one-electron oxidation product of NAD• [29,30]. To be able to oxidize tyrosine, chorion peroxidase must be activated by H2O2 to compound I. Therefore, detection of dityrosine in the NADH/chorion peroxidase reaction mixture in the presence of tyrosine indicates not only the accumulation of H2O2, but also the formation of chorion peroxidase compound I in the reaction mixture.
In A. aegypti eggs, the overall chorion hardening process takes about 2–3 h after oviposition; therefore, a rapid production of H2O2 is necessary during chorion hardening. The ability of chorion peroxidase to mediate H2O2 formation by accelerating the NADH/O2 oxidoreduction process, and the formation of dityrosine in the NADH/chorion peroxidase reaction mixture in the presence of tyrosine provides a reasonable basis to suggest the involvement of chorion peroxidase in promoting H2O2 formation through NADH oxidation and O2 reduction during chorion hardening. In the mosquito chorion, the proximity between chorion peroxidase and its substrates and the possibility of H2O2 to defuse (from its site of production) within the chorion layer might allow H2O2 to be used more efficiently for protein cross-linking during chorion hardening.Mn2+ enhances NADH oxidation in the presence of chorion peroxidase, but it seems apparent that Mn2+ alone has no effect on facilitating NADH oxidation because it does not accelerate NADH oxidation in the absence of chorion peroxidase. The effect of Mn2+ in stimulating NADH oxidation may be due to its interaction with O2, in which Mn2+ reduces to H2O2 with itself being oxidized to Mn3+ and Mn3+ may then oxidize NADH to NAD• (Eqs. 8 and 9); thereby accelerating NADH oxidation and H2O2 formation .
However, this mechanism cannot explain why a relatively high concentration of Mn2+ inhibits NADH oxidation. It is possible that Mn2+ at high concentration may react more easily with H2O2 leading to the formation of hydroxyl radical (Eq. 10), which may inactivate the chorion peroxidase.
In summary, results from this study suggest that (i) chorion peroxidase in A. aegypti is capable of mediating NADH oxidation leading to the formation of H2O2, (ii) the NADH/O2 oxidoreduction mediated by chorion peroxidase represents a feasible pathway for H2O2 production required during chorion hardening, and (iii) a chorion MAD is present in the egg chorion, and the enzyme might be responsible for the supply of NADH for H2O2 generation during chorion hardening.
This work is supported by NIH Grant AI 37789.