Biol. Bull. 215: 108-114. (August 2008)
© 2008 Marine Biological Laboratory
Endonuclease Activity of Phenol Oxidase From Musca domestica Larvae
Shaoguang Sun1,2,
Weiquan Liu1,*,
Jigui Wang1,
Shuyan Yang1,
Ling Gu1,
Yan Hong1,
Dan Shang1,
Benxu Wang1,
Xiaoming Su1 and
Shunzhang Qi1
1 Department of Biochemistry and Molecular Biology, China Agricultural University, Beijing 100094, China
2 Department of Biochemistry, Hebei Medical University, Shijiazhuang 050017, China
* To whom correspondence should be addressed. E-mail: weiquan1{at}yahoo.com
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Abstract
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Phenol oxidase (PO), a copper-containing enzyme with oxygenase activity, can convert mono- or diphenol into quinone and plays an important role in the arthropod melanization reaction. Here, we report a new property of PO from Musca domestica larvae: a thermotolerant endonuclease activity, by which PO can degrade plasmid DNA even after being heated to 80° C for 20 min. We cloned PO cDNA, constructed the expression vector pVAX1-PO, and expressed it in HeLa cells. The expression product showed the same properties as purified PO. Our data indicate that PO is a bifunctional enzyme, exhibiting both oxygenase and endonuclease activity, suggesting new roles for this important molecule in the innate responses of M. domestica.
Abbreviations: ATA, aurintricarboxylic acid • L-DOPA, L-3,4-dihydroxyphenylalanine • PO, phenol oxidase • proPO, prophenol oxidase • PTU, 1-phenyl-2-thiourea
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Introduction
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Phenol oxidase (PO), also known as tyrosinase, is a copper-containing enzyme with monophenol monooxygenase activity and o-diphenol oxidase activity. PO catalyzes the oxidation of phenols to quinones; the quinone products then undergo non-enzymatic polymerization to form melanin. This reaction is called the melanization reaction and is observed at all infection sites in arthropods, where it contributes to wound healing and the control of pathogen growth.
PO, the active form of prophenol oxidase (proPO), is the terminal component of the proPO cascade, which functions in non-self recognition (Ratcliffe et al., 1984) and host defense in arthropods. The proPO cascade can respond to lipopolysaccharides, bacterial peptidoglycans, and fungal β-1,3-glucans (Ashida, 1990), which trigger the defensive melanization reaction. However, some studies have indicated that the proPO cascade may be a mechanism for pigmentation rather than defense (Vasse et al., 1999; Leclerc et al., 2006). Those results raise questions about the precise function of PO activation in defense against microbial invasion.
Here, we report an interesting, novel property of PO from the housefly Musca domestica, in which PO can degrade plasmid DNA even after being heated to 80 °C for 20 min. We purified PO from M. domestica larvae and characterized part of the novel physical-chemical natures of this protein. To better understand the novel characteristics of PO, we also constructed an expression vector (pVAX1-PO) and transfected HeLa cells to study the properties of the recombinant protein. The expression product and purified PO showed similar properties. Our data provide insight into the physiological role of PO in M. domestica.
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Materials and Methods
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Insect
Larvae of the housefly Musca domestica Linnaeus were reared on a moist mixture of wheat bran, white sugar, and milk powder. The humidity and temperature were maintained at 60% and 25 °C, respectively. Five-day-old larvae were used in this study.
Purification of phenol oxidase
Fly larvae were homogenized in phosphate-buffered saline (PBS), pH 7.4, containing 1% 2-mercaptoethanol. The slurry was heated to 80 °C for 20 min and then centrifuged at 11,000 x g for 10 min at 4 °C. The supernatant was brought to 35% saturation with solid ammonium sulfate. The resulting pellet was collected by centrifugation and dialyzed against water, after which the resulting solution was applied to a DEAE-52 (Pharmacia) column (30 x 5 cm) equilibrated with 50 mmol/l Tris-HCl (pH 8.6) buffer, and then eluted with 0.4 mol/l NaCl in the same buffer at a flow rate of 0.5 ml/min. The active eluant was collected and dialyzed against polyethylene glycol. Further purification was carried out on a Sephadex G-75 (Pharmacia) column (100 x 2.6 cm) using PBS as the column buffer. The active fractions were collected.
Endonuclease activity assay of purified phenol oxidase
Endonuclease activity was determined by incubating 1 µg plasmid (pVAX1, Invitrogen) with 19 µl of eluant fractions from the protein purification columns (DEAE-52 or Sephadex G-75) at 37 °C for 30 min. All reactions were analyzed by 1% agarose-gel electrophoresis and then photographed.
SDS-PAGE and peptide fingerprinting analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sambrook et al., 1989) was performed using Tris-glycine buffer, pH 8.3, with 5% and 12% acrylamide (w/v) in the stacking and separating gels, respectively. The purified proteins were then transferred to a polyvinylidene difluoride (PVDF; Millipore) membrane and run in 39 mmol/l glycine, 48 mmol/l Tris, 0.037% SDS, and 20% methanol at 100 V for 1 h. The PVDF membrane was stained with Coomassie brilliant blue R250 (Sangon), and proteins of interest were excised and subjected to peptide mass fingerprinting analysis.
Oxygenase activity assay of purified phenol oxidase
Oxygenase activity of PO was monitored in a U-721 spectrophotometer at 490 nm by dopachrome formation accompanying the oxidation of the substrate (L-tyrosine or L-3,4-dihydroxyphenylalanine [L-DOPA]). The reaction medium (3 ml) contained 20 µg purified PO and 2.0 mmol/l substrate in 50 mmol/l sodium phosphate buffer, pH 6.8. Each assay was carried out in triplicate at a constant temperature of 30 °C, with a 5-min incubation period.
SDS-PAGE was performed according to the method described above. The purified proteins were transferred to a nitrocellulose membrane (Gelman) under the same conditions, and the membrane was blocked by incubation with 50 mmol/l Tris containing 150 mmol/l NaCl, 5 mmol/l EDTA, 0.05% Triton X-100, and 0.25 % gelatin (pH 8.0) for 2 h at room temperature. The membrane was then incubated at room temperature in a buffer consisting of 100 ml 5 mmol/l CaCl2 containing 10 mmol/l L-DOPA (Sangon) mixed with 25 ml 0.3% 3-methyl-2-benzothiazolinone hydrazone (MBTH, dissolved in ethanol; Sigma) until color developed (Durran et al., 1993).
Effect of inhibitors on phenol oxidase oxygenase activity
The effect of inhibitors on PO oxygenase activity was determined spectrophotometrically at 490 nm in a U-721 spectrophotometer, with L-DOPA as substrate. The reaction medium (3 ml) contained 20 µg purified PO, 2.0 mmol/l inhibitor (aurintricarboxylic acid [ATA] or 1-phenyl-2-thiourea [PTU]), and 2.0 mmol/l L-DOPA in 50 mmol/l sodium phosphate buffer (pH 6.8). The assay was performed in triplicate at a constant temperature of 30 °C, with a 5-min incubation period.
Effect of inhibitors on phenol oxidase endonuclease activity
The effect of inhibitors on PO endonuclease activity was determined by incubating 1 µg plasmid (pVAX1, Invitrogen) with 20 µg purified PO and 2.0 mmol/l inhibitor (ATA or PTU) at 37 °C for 30 min. All reactions were analyzed by 1% agarose gel electrophoresis and then photographed.
Effect of pH and temperature
The thermotolerance of PO endonuclease activity was determined by measuring residual activity after incubating PO at different temperatures (30–100 °C, increasing by increments of 10 °C) for 10 min. The effect of pH on PO endonuclease activity was determined at different pH values (pH 2.0–13.0, increasing by increments of 1.0 pH unit). The endonuclease activity of samples was determined by incubating 2 µg plasmid (pVAX1) with 100 µl purified, heat-stressed PO at 37 °C for 30 min. After digestion with PO, the relative rates of DNA hydrolysis were quantified by fluorometric measurement of DNA concentration in a DU640 nucleic acid and protein analyzer (Beckman). The assay was performed in triplicate for each temperature and pH point. Activity of PO in Kunitz units was determined by incubating 5 µg plasmid DNA in PBS at 37 °C according to the method described by Ashida (Ashida, 1971).
Gene cloning and expression vector construction
Total RNA was extracted from M. domestica larvae using Trizol (Invitrogen) reagent, according to the manufacturer's recommendations. Total RNA was digested using RNase-free DNase I (TaKaRa) at 37 °C for 30 min; the sample was then incubated at 80 °C for 10 min to inactivate the DNase. The first cDNA strand was synthesized using 2 µg total RNA with oligo-(dT)18 primer (TaKaRa) in a 25-µl reaction system containing M-MLV reverse transcriptase (Promega). A cDNA fragment encoding the open reading frame of M. domestica larvae PO was obtained by reverse transcription polymerase chain reaction (RT-PCR). Primers were designed using Primer Premier 5.0, based on the cDNA sequence for M. domestica proPO available from the GenBank database (accession no. AY494738). The sense primer was 5'-TCTCGAGAATCCAAAGCATGTTCGG-3' (XhoI underlined), and the antisense primer was 5'-AGGGCCCGAAGGGCGCTGAATAAGG-3' (ApaI underlined). PCR was performed in a thermal cycler (Biometra) using the following cycling conditions: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 2.5 min, followed by a 10-min extension at 72 °C. The amplified cDNA fragment was cloned into pMD18-T vector (TaKaRa). After the pMD18-PO vector was digested with ApaI/XhoI (TaKaRa), the fragment of interest was recovered using a Gel DNA Extraction kit (TIANGEN) and then subcloned into a mammalian expression vector (pVAX1) digested with ApaI/XhoI.
Cell culture and transfection
HeLa cells were cultured at 37 °C in high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone). Cells were transfected with the expression vector pVAX1-PO or pVAX1 alone using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Western blot analysis
Western blots were performed according to a previously described method (Sambrook et al., 1989). Briefly, the cells and culture medium were collected after transfection for 24 h, and equal samples were examined by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane under the same conditions as described above, and the membrane was blocked using PBS, 5% milk, and 0.3% Tween 20 at room temperature. After three washes with PBS, the membrane was incubated for 2 h at 4 °C with affinity-purified guinea pig antibodies to M. domestica larval PO (1:100; produced in our laboratory). After three washes with PBS and one wash with washing buffer (150 mmol/l NaCl, 50 mmol/l Tris-HCl, pH 7.6), the membrane was subjected to a second incubation for 1 h at 4 °C with horseradish peroxidase (HRP)-conjugated goat anti-guinea pig IgG (1:200; Sangon). After three washes with washing buffer, the membrane was incubated in 0.01 mmol/l Tris-HCl (pH 7.6) with 0.6 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sangon), 0.3% CoCl2, and 0.1% H2O2 at room temperature and checked continuously for development. The result was photographed.
Endonuclease and oxygenase activity assay of recombinant-expressed phenol oxidase
Endonuclease and oxygenase activities of recombinant-expressed PO were assayed as described above.
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Results and Discussion
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Purification and identification of phenol oxidase
Two interesting phenomena have been noted in transgenic Musca domestica research. First, plasmid DNA becomes degraded after several injections with microinjection needles. Second, even after heating the slurry made from M. domestica to 80 °C for 20 min, which was intended to eliminate the DNase activity, plasmid DNA degradation still occurred. Based on these phenomena, we speculated that there might be a thermotolerant endonuclease in M. domestica larvae. Here, we purified such a thermotolerant endonuclease, which could degrade plasmid DNA after being heated to 80 °C for 20 min (Fig. 1A). Under denaturing conditions in SDS-PAGE, the purified active enzyme appeared as a single band with a relative molecular mass of about 60 kDa (Fig. 2B). Surprisingly, our results from peptide mass fingerprinting and bioinformatic analysis (data not shown) demonstrated that this protein was PO, showing monooxygenase activity with L-tyrosine as substrate (Fig. 3A) and o-diphenol oxidase activity with L-DOPA (Fig. 3A), and appearing as a differentially stained band with PO activity on nitrocellulose membranes after SDS-PAGE and the electrotransfer of proteins (Fig. 2A). This suggests that PO may act as a bifunctional enzyme, involved in both the biosynthesis of melanin in the melanization reaction and the degradation of DNA in vivo, although experimental support for this hypothesis is lacking. PO may be similar to DNase II, which functions in eliminating DNA from dead cells or non-self DNA in vivo.
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Figure 1. (A) Thermotolerance of purified phenol oxidase (PO) to plasmid DNA (pVAX1) degradation. Endonuclease activity was determined by incubating 1 µg plasmid DNA with 19 µl purified PO at 37 °C for 30 min. Lane M: plasmid DNA incubated with PBS. Lanes 1–6: plasmid DNA incubated with PO previously heated to 80 °C for 0, 5, 10, 15, 20, and 25 min respectively. (B) Effect of inhibitors on PO endonuclease activity. One microgram of plasmid DNA (pVAX1) was incubated with 20 µg purified PO and 2.0 mmol/l inhibitor (ATA or PTU) at 37 °C for 30 min. Lanes 1–3: plasmid DNA incubated, respectively, with purified PO and ATA, purified PO and PTU, and purified PO. Lane M: plasmid DNA incubated with phosphate-buffered saline. (C) Thermotolerance of expressed PO. Culture medium was heated to 80 °C for 20 min, and endonuclease activity was determined by incubating 1 µg plasmid DNA (pVAX1) in 50 µl culture medium at 37 °C for 30 min. Lanes 1–2: plasmid DNA incubated with culture medium from cells transfected with pVAX1-PO and pVAX1, respectively. Lane 3: pVAX1.
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Figure 2. Identification of phenol oxidase (PO). (A) PO bonded to nitrocellulose membrane was stained with L-DOPA and 3-methyl-2-benzothiazolinone hydrazone to detect PO oxygenase activity. (B) PO purification using SDS-PAGE under denaturing conditions. Sephadex G–75 eluant (lane 1) and DEAE–52 eluant (lane 2) were subjected to SDS-PAGE (12% gel) alongside molecular mass (lane M) markers. The gel was stained with silver nitrate. (C) Western blot analysis. Lanes 4–6: culture medium from, respectively, HeLa cells transfected with pVAX1-PO, with pVAX1 alone, or without transfection.
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Figure 3. (A) Oxygenase activity assay of purified phenol oxidase (PO). The effect of inhibitors on PO oxygenase activity was determined spectrophotometrically at 490 nm, using L-tyrosine or L-DOPA as substrate. The reaction medium (3 ml) contained 20 µg purified PO and 2.0 mmol/l substrate, or 2.0 mmol/l inhibitor (ATA or PTU) in 50 mmol/l sodium phosphate buffer, pH 6.8. The assay was carried out at a constant temperature of 30 °C. Line 1: PO and L-DOPA. Line 2: PO, PTU, and L-DOPA. Line 3: ATA and L-DOPA. Line 4: PO and L-tyrosine. (B) Oxygenase activity of expressed PO. The oxygenase activity of culture medium was determined spectrophotometrically in a U-721 spectrophotometer at 490 nm using L-DOPA (2.0 mmol/l) as a substrate in 50 mmol/l sodium phosphate buffer, pH 6.8. The assay was carried out at a constant temperature of 30 °C. Line 1: medium from cells transfected with pVAX1-PO. Line 2: medium from cells transfected with pVAX1 alone.
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POs have been isolated and characterized from many arthropods. Hemolymph PO is present as an inactive precursor known as proPO. The monomer of proPO has a relative molecular mass of 70–80 kDa, with an active PO of 60–70 kDa (Aspan and Söderhäll, 1991; Kopacek et al., 1995; Kwon et al., 1997). Active PO has been purified from the hemolymph of M. domestica larvae, with a relative monomeric molecular mass of about 61 kDa (Yamaura et al., 1980a, b). This finding is similar to ours. Interestingly, the relative molecular mass of PO from M. domestica is about 74 kDa, according to the deduced amino acid sequence (634 amino acid residues) available from the GenBank database. The slight reduction in size noted in our results may be because of a second cleavage that occurs before enzymatic activation. This is the case in Holotrichia diomphalia, where PO is cleaved to a 76-kDa product and then undergoes a second cleavage, resulting in an active end product of 60 kDa (Kim et al., 2002).
Effect of inhibitors
ATA, a general inhibitor of nucleases, inhibited the endonuclease activity of PO (Fig. 1B), but did not inhibit its oxygenase activity (Fig. 3A). PTU, an effective inhibitor of phenol oxidase, inhibited the oxygenase activity of PO (Fig. 1B) but did not inhibit its endonuclease activity (Fig. 3A). These results together suggest that the endonuclease active site may be separate from the oxygenase active site of PO, and PO may play different roles in different physiological conditions. It is well known that the primary function of PO is providing an oxygenase activity in the melanization reaction. Here, we presume PO might also provide an endonuclease activity in some other physiological condition, such as eliminating foreign DNA as part of the defensive mechanisms.
Effect of pH and temperature
We tested the effect of pH (2.0–13.0) on the endonuclease activity of PO. At each pH point, enzyme activity was measured in triplicate by incubating the reaction mixture at 37 °C for 30 min. Maximum activity was observed at about pH 8.0 (Fig. 4A). The effect of temperature (30–100 °C) on the endonuclease activity of PO was also tested in triplicate (Fig. 4B), and the results showed that PO was still active at high temperatures between 80 and 100 °C. However, the optimum pH and resistant maximum temperature for oxygenase activity have been reported to be 7.4 and 50 °C, respectively (Hara et al., 1993). This demonstrates that PO might display two different functions under certain conditions in vivo.
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Figure 4. Effect of pH and temperature on phenol oxidase (PO) endonuclease activity. (A) pH profile. (B) Thermotolerance profile. The relative rates of DNA hydrolysis were assessed by incubating 2 µg plasmid DNA with 100 µl heat- or pH-treated PO (0.5 Kunitz units) at 37 °C for 30 min.
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Gene cloning and expression vector construction
Most arthropod proPOs are synthesized without a hydrophobic signal sequence for endoplasmic reticulum localization; therefore, it is commonly assumed that they are released from cells upon rupture of the cell membrane (Cerenius and Söderhäll., 2004). In most cases, proPO is activated through proteolytic cleavage by a native proteinase, the cleavage site of the zymogen being between Arg and Phe (Fujimoto et al., 1995; Kawabata et al., 1995; Satoh et al., 1999; Kwon et al., 2000; Asano and Ashida, 2001; Asada et al., 2003). According to our results and the cDNA sequence information for proPO from M. domestica, we designed a sense primer (5'-TCTCGAGAATCCAAAGCATGTTCGG-3'), in which CGT (the codon of Arg50) was changed to ATG (the initiation codon). This ensures that the cDNA fragment obtained from RT-PCR encodes the open reading frame for larval PO. Sequence comparison analysis showed that 54 nucleotides were different between the cDNA we obtained and the Genbank cDNA sequence (Supplement 1, http://www.biolbull.org/supplemental/). Seven deduced residues differed between the two amino acid sequences (Supplement 2, http://www.biolbull.org/supplemental/).
Expression and Western blot analysis
To confirm that the cloned PO cDNA encodes a protein with thermotolerant endonuclease activity, HeLa cells were transfected with the mammalian expression vector (pVAX1-PO) or the vacant expression vector (pVAX1). After incubation at 37 °C for 24 h, Western blot analysis detected a band of about 74 kDa from the cell culture medium of cells transfected with pVAX1-PO (Fig. 2C). This result is consistent with the relative molecular mass of the deduced amino acid sequence of M. domestica PO in the GenBank database. However, we did not detect expressed PO in the remaining living cells in cultures transfected with pVAX1-PO. This could be because overexpression of PO in transfected cells caused cell death and the resultant release of PO into the culture medium, with the remaining living cells not having been transfected by pVAX1-PO because of the efficiency of transfection. This result suggests that overexpression PO might be poisonous to HeLa cells; however, the mechanism by which PO causes cell death is still unclear, but is being studied.
Endonuclease and oxygenase activity assay of recombinant-expressed phenol oxidase
Culture medium from HeLa cells transfected with pVAX1-PO oxidated L-DOPA to form dopachrome, resulting in an increase in absorbance at 490 nm (Fig. 3B), which demonstrated the presence of active PO in the medium. After the culture medium was heated to 80 °C for 20 min, plasmid DNA degradation was still observed (Fig. 1C). This property is consistent with that of PO purified from M. domestica larvae.
There are many precedents for a bifunctional enzyme with distinct catalytic capacities, such as phosphofructokinase II and fructose-2,6-bisphosphatase. However, to our knowledge, this is the first time that thermotolerant endonuclease activity of PO from M. domestica has been reported. Our results suggest that PO may be a bifunctional enzyme with both oxygenase and endonuclease activity, but the sites of the activities are different. Although it is well known that PO provides an oxygenase activity in the melanization reaction, the question of whether PO is involved in the degradation of foreign DNA by supplying an endonuclease activity in vivo remains unresolved. The roles of PO in the defensive mechanisms are still in dispute, but our report affords illumination for further elucidation of the precise function of PO.
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Acknowledgments
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We thank Dr. Ruqiang Yao and Guihua Wang for their kind assistance with this work. We also thank Dr. Haipeng Liu for revision of the manuscript.
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Footnotes
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Received 11 January 2008; accepted 27 February 2008.
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Abstract
Introduction
