Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Comparative Biochemistry and Physiology, Part B 151 (2008) 139–146 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology...

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Comparative Biochemistry and Physiology, Part B 151 (2008) 139–146

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p b

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae Congjing Feng a,⁎, Qisheng Song b, Wenjing Lü a, Jianfeng Lu c a b c

Department of Plant Protection, College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, Jiangsu Province, China Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA College of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, Anhui Province, China

A R T I C L E

I N F O

Article history: Received 30 March 2008 Received in revised form 20 May 2008 Accepted 20 May 2008 Available online 4 June 2008 Keywords: Kinetics Metal ion Ostrinia furnacalis Prophenoloxidase Secondary structure

A B S T R A C T Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for L-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA). Both Mg2+ and Cu2+ stimulated PO activity when compared with controls. The β-sheet content of PPO treated with Mg2+ and Cu2+ increased significantly (P b 0.05). The purified PPO has magnesium level of 5.674 ± 2.294 µg/mg and copper level of 1.257 ± 0.921 µg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein. © 2008 Published by Elsevier Inc.

1. Introduction Insects lack the elements that compose the adaptive immune response of vertebrates, but they possess an effective innate immune response system. This innate immune system includes both humoral and cellular reactions, which operate in a coordinated way to sequestrate and clear the foreign bodies from the hemocoel (Franssens et al., 2008). Phenoloxidase (PO) is one of the most important enzymes that are involved in the innate immune system of invertebrates (Cerenius and Söderhäll, 2004). PO is synthesized as an inactive zymogen, prophenoloxidase (PPO) and PPO is activated by a serine proteinase cascade triggered by microbial carbohydrates such as β-1,3-glucan, peptidoglycan and lipopolysaccharides (Söderhäll and Cerenius, 1998). In insects, PO catalyzes the hydroxylation of tyrosine to 3,4-dihydroxy-phenylalanine (DOPA) and the oxidation of phenolic substances to quinones needed for melanin formation. Melanotic

⁎ Corresponding author. Tel.: +86 514 87979344; fax: +86 514 87347537. E-mail address: [email protected] (C. Feng). 1096-4959/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.cbpb.2008.05.012

encapsulation plays an important role in innate immune response against large pathogens or parasites (Ling and Yu, 2005). To further clarify the molecular mechanism of PO in insect physiology and biochemistry, a detailed study on the enzyme is necessary. However, due to the instability and rapid loss of the activity of this enzyme during the purification process, more attention has been paid to the investigation of PPO. So far, PPO has been purified and characterized from only a small number of insect species including Lepidopteran, Dipteran, cockroaches and locusts (Durrant et al., 1993; Gillespie et al., 1997; Chase et al., 2000). The Asian corn borer, Ostrinia furnacalis Guenée, is an important component of the Lepidoptera pest complex of corn and cotton in China, and has been used as a model insect for studying the physiological interaction between this species and a polyembryonic parasitoid Macrocentrus cingulum Brischke (Hu et al., 2003). Although there have been a number of studies involving various functional aspects of insect PPO, our knowledge of this enzyme at the protein level is limited. For example, the exact site of synthesis, regulation of PPO, its activating enzymes and inhibitors are still controversial. Therefore, it is desirable to obtain more information dealing with the physicochemical properties of

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O. furnaclis PPO. In this paper, we report the purification and characterization of PPO from the hemolymph of O. furnaclis larvae. 2. Materials and methods 2.1. Insects O. furnacalis Guenée (Lepidoptera: Pyralidae) larvae were reared at 25 ± 1 °C, RH N 80% and a photoperiod of 14 L: 10 D. The larvae were fed on an artificial diet as described by Zhou et al. (1980) and the larval instars were determined by measuring the head-width. 2.2. Collection of hemolymph The first abdominal proleg of the 5th-instar O. furnacalis larvae was cut with a scissor. The hemolymph was allowed to drip onto a parafilm that was stretched over a smooth, ice-cold surface, and then transferred into 1.5 mL microcentrifuge tubes each containing 0.5 mL sodium cacodylate buffer (100 mmol/L CaCl2, 10 mmol/L Na2CAC, pH 6.5) in an ice-bath. After centrifugation (800 g, 10 min, 4 °C), the supernatant was transferred into new 1.5 mL microcentrifuge tubes and kept at 4 °C. 2.3. PPO assay Since PPO is devoid of any activity, it needs to be activated before detecting PO activity using the method described by Jiang et al. (1997b). For this purpose, a reaction mixture (1 mL) containing 2 mmol/L L-DOPA, 50 mmol/L sodium phosphate buffer, pH 6.0 and enzyme protein (5–10 µg) was incubated at 30 °C, and the increase in absorbance at 490 nm associated with the production of dopaminechrome was continuously monitored after activating the PPO by the addition of 10 μL 10% cetylpyridinium chloride (CPC). One unit was defined as 0.001 absorbance increase at 490 nm/min. 2.4. Protein determination Protein concentration in fractions was quantified by a dye-binding assay (Bradford, 1976). Standard curves were constructed using bovine serum albumin. 2.5. Purification of PPO All purification steps were performed at 4 °C in a cacodylate (CAC) buffer (20 mmol/L CaCl2, 10 mmol/L Na2CAC, pH 6.5) unless otherwise noted. Hemolymph (8 mL) was first diluted into 2:1 with the CAC buffer. The saturated ammonium sulphate solution was carefully added to hemolymph until reaching a saturation of 40%. The precipitate was spun down by centrifugation for 10 min at 12,000 g, and redissolved in 500 μL of 20 mmol/L sodium cacodylate solution (pH 6.5). The protein was dialyzed in 2000 mL CAC buffer overnight at 4 °C, and then applied to a Blue Sepharose CL-6B column (1.0 cm× 10 cm) pre-equilibrated with the CAC buffer. The column was eluted with an elution buffer (100 mmol/L CaCl2, 10 mmol/L Na2CAC, pH 6.5) at a flow rate of 1.5 mL/min. The fractions containing PPO from three simultaneous Blue Sepharose CL-6B chromatography were pooled (about 20× 1.5 mL) and immediately applied to a Phenyl Sepharose CL-4B column (0.8 cm× 12 cm) that was equilibrated with the CAC buffer. The column was washed with distilled water at a flow rate of 1.5 mL/min until the absorbance of fractions at 280 nm returned to zero. The fractions with PO activity were dried in a Heto FD3 Model Vacuum Cold Dryer. The purified proenzyme was stored in the presence of 0.02% NaN3 at −80 °C. 2.6. Native-PAGE, western blotting and two-dimensional gel electrophoresis Native PAGE was performed according to Tyagi et al. (1993) with 6% polyacrylamide gel. Proteins were fixed and stained with Coomassie

blue R-250. For western blotting analysis of PPO after separation by native PAGE, proteins were transferred to a PVDF membrane. The membrane was blocked with 5% dry skim milk, and then incubated with rabbit antiserum to Manduca sexta PPO (1:2000 dilution) donated by Dr. Haobo Jiang of Oklahoma State University. Antibody was bind to the goat anti-rabbit IgG conjugated by horseradish peroxidase (BioRad), and the ECL+Plus western blotting detection system (Amersham Pharmcia) was used to visualize the binding band. Two-dimensional (2-D) gel electrophoresis was performed according to O'Farrell (1975). For the first dimension, 7 μL of the purified PPO (500 µg) was diluted to a final volume of 350 μL in a rehydration solution (7 mol/L urea, 2% CHAPS, 65 mmol/L DTT, 0.2% Bio-Lyte, 0.001%(w/v) bromophenol blue). The sample was applied to the IPG-strip (7 cm, pH 3–10 linear; Bio-Rad) by rehydration. Isoelectric focusing was performed on a Protean® IEF cell (Bio-Rad) with a temperature of 17 °C and a maximum current of 50 μA/strip. Focusing parameters were as follows: active rehydration (50 V) for 12 h; step 1-gradient from 1 to 250 V over 30 min; step 2-gradient from 250 to 1000 V over 1 h; step 3-gradient from 1000 to 4000 V over 45 min; step 4—4000 V constant for 3 h; step 5—4000 V until complete 24,000 V h. Before the second dimension, proteins in the strips were reduced (10 mg/mL DTT) and alkylated (25 mg/mL iodoacetamide) in an equilibration buffer (6 mol/L urea, 2% SDS, 375 mmol/L Tris–Cl pH 8.8, 20% glycerol). The equilibrated IPG strips were applied directly onto the second dimension gel and the proteins were separated across 12% SDS-PAGE gels using a vertical system (Bio-Rad) and a standard Tris/glycine/SDS buffer. Gels were run at 20 mA/gel at 15 °C until the tracking dye left the gels. The gels were silver-stained (Oakley et al., 1980) and photographed. 2.7. N-terminal amino acid sequencing and amino acid composition analysis For determination of the N-terminal ends, samples were separated by native PAGE and transferred to a PVDF membrane. The PPO band was stained with amido black, then was excised and submitted to Edman degradation. The purified PPO yields no phenylthiohydantoin derivative of any amino acid, suggesting a blocked amino terminus. Then active PO was prepared from PPO after activation with trypsin and separated by SDS-PAGE (reduced condition). The protein bands of generated PO were subjected to automated protein sequencer (Procise, ABI). The N-terminal sequences alignment was constructed online (http://www.ebi.ac.uk./ Tools/clustalw2/index.html). After hydrolyzing with 6 mol/L HCl at 110 °C for 24 h (Simpson et al., 1976), amino acid composition of the purified PPO was analyzed with a Hitachi Model 835 amino acid analyzer by the method of Spackman et al. (1958). 2.8. Estimation of kinetic parameters Using different concentrations of L-DOPA or N-acetyldopamine or dopamine as a substrate, the PO activity was assayed. A typical reaction mixture containing 40 μL 0.1 mol/L CPC and 30 μL purified PPO (20 µg) was incubated at 30 °C for 10 min. After which, L-DOPA or N-acetyldopamine or dopamine prepared in a 10 mmol/L CAC buffer was added to the reaction mixture to provide an increasing concentration gradients in a final volume of 1 mL. Kinetic parameters (maximum velocity and apparent Km) were calculated. 2.9. Inhibition assay Different compounds including thiourea, phenylthiourea, dithiothreitol (DTT), ethylene diamine tetraacetic acid (EDTA), o-phenanthroline, ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid (EGTA), diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (TTHA), were tested for their inhibitory effect on the activity of the purified PPO. A solution containing 30 μL purified PPO (20 µg) was incubated for 10 min at 30 °C with 40 μL 0.1 mol/L CPC to obtain the active

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enzyme (PO). Then, a 830 μL solution with the indicated concentrations of inhibitors was added and incubated for 30 min at 25±1 °C. After adding 100 μL 2 mmol/L L-DOPA as a substrate, the PO activity was measured. 2.10. Effect of metal ions on the PO activity The fraction containing the purified PPO (10 µg) was added to solutions diluted respectively, with 100 mmol/L MgSO4, 100 mmol/L ZnSO4, 100 mmol/L MnCl2, 100 mmol/L FeCl2, 100 mmol/L CuSO4, and 100 mmol/L CaCl2 to various concentrations. Forty μL of 0.1 mol/L CPC and 100 μL of 2 mmol/L L-DOPA were added. The mixtures in a final volume of 1 mL were incubated at 30 °C for 30 min, and the increase in absorbance at 490 nm after 10 min was continuously monitored for calculation the PO activity. Fig. 1. Native PAGE (6%) of different fractions in the process of PPO purification. M: Marker of protein; lane 1: the precipitation of ammonium sulphate (3.5 µg); lane 2: the fractions of Blue Sepharose CL-6B (2 µg); lane 3: the fractions of Phenyl Sepharose CL-4B (2 µg); lane 4: hemolymph of O. furnacalis larvae (10 µg).

2.11. Effect of metal ions on the secondary structure of PPO The purified PPO was diluted to 0.01 mg/ml with 100 mmol/L MgSO4, 100 mmol/L ZnSO4, 100 mmol/L MnCl2, 100 mmol/L FeCl2, 100 mmol/L CuSO4, and 100 mmol/L CaCl2, respectively, and then incubated overnight at room temperature. The purified PPO with metal ion-free solution was used as control. The secondary structure of the purified PPO was determined using a Jasco J-810 spectropolarimeter equipped with a RTE bath/circulator (NESLABRTE-111) and purged with N2 gas at a flow rate of 3–5 mL/min. The spectra from 180 to 380 nm were recorded with a resolution of 0.2 nm and accumulation of six scans. The scan speed was at 100 nm/min and the response time was 1 s. Sample solutions were stored in 0.1-cm path length cells. The data were stored and analyzed with Standard Analysis Software. 2.12. The content of several metal elements in PPO Fifty mL of 50% HNO3 was added to a solution containing 3.5 mg purified PPO and the resulting mixture was then heated to boiling point. The 30% H2O2 solution was dropped continuously until the solution became clear. The control group was prepared under the same condition but without PPO. Inductively coupled plasma mass spectrometry (ICP-MS) instrument used in this work was the ELANDRC-e (USA). The ICP-MS measurement conditions (nebulizer gas flow, RF power and lens voltage) were optimized daily using a standard build-in software procedure. The dwell time for each isotope was 200 ms and six replicates were applied. The mass of several isotopes are as follows: copper 62.929, magnesium 23.985, manganese 54.938, iron 56.935, zinc 65.926, and barium 137.905. Standard curves of several metal elements were constructed using 2 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL solutions of various isotopes.

2.13. Statistics All data were analyzed by using the SPSS11.5.0 software (SPSS Inc., 2002). The differences between means were analyzed by independent samples t-test and one-way ANOVA. 3. Results 3.1. PPO purification The PPO was purified from the hemolymph of O. furnacalis larvae using a combination of ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The PPO purification results were shown in Table 1. The purification procedure yielded a total of 0.27 mg PPO from a starting sample of 8 mL hemolymph containing about 282.56 mg total protein. The PPO was purified 369.85-fold with a 35.34% total recovery of activity. Native PAGE (6% gel) analysis of the purified PPO eluted from the Phenyl Sepharose CL-4B chromatography revealed a single protein band with a relative molecular mass of 158 kDa (Fig. 1). The purified protein was also verified in western blotting analysis using anti-M. sexta PPO serum as primary antibody. (Fig. 2), suggesting that this zymogen was purified. To confirm the homogeneity of the PPO, we analyzed the purified PPO by 2-D gel electrophoresis and silver staining. The silver-stained 2-D gel map revealed two protein spots, one with a molecular mass of 80 kDa and pI of 5.70, and the other with a molecular mass of 78 kDa and pI of 6.50, respectively (Fig. 3), indicating that the PPO forms a dimer of two different polypeptides (named PPO1 and PPO2), which most likely represent different subunits of

Table 1 Purification of PPO from the hemolymph of O. furnacalis larvae Step

Total amount of protein (mg)

Hemolymph 282.56 (NH4)2SO4 77.83 Blue Sepharose CL-;6B 5.37 chromatography Phenyl Sepharose CL-4B 0.27 chromatography

Specific activity (U/mg)a

PO activity (U)b

Recovery Purification (%) fold

29.83 8429.30 100.00 87.09 6778.90 80.42 1209.6 6495.4 77.06 11032.6

2978.8

35.34

1.00 2.92 40.55 369.85

PO activity was determined by the activation of PPO after each purification step with CPC (cetylpyridinium chloride). a Expressed in ΔA490/min/mg of protein. b Expressed in ΔA490/min.

Fig. 2. Western blotting analysis of PPO purified from the hemolymph of O. furnacalis larvae. Using anti-M. sexta PPO serum as primary antibody. M: Marker of protein; lane 1: the fractions of Phenyl Sepharose CL-4B (2 µg).

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C. Feng et al. / Comparative Biochemistry and Physiology, Part B 151 (2008) 139–146 Table 2 Amino acid composition of PPO from O. furnacalis and its comparison with PPO from other invertebrates

Fig. 3. A silver-stained two-dimensional gel showing the purified PPO from the hemolymph of O. furnacalis larvae. Two spots named 1 and 2 on this gel indicated this enzyme was composed of two subunits with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively.

the PPO from O. furnacalis. The N-terminal amino acid sequences of two subunits are as follows: PPO1: FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2: FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The PPO2 N-terminal sequence exactly matches the cds for O. furnacalis PPO in GenBank (DQ333883). Microsequencing of the amino acids of the N-termini demonstrated that the two putative PPO subunits were distinct polypeptides. The alignment showed that the N-terminal amino acids of the purified PPO were different significantly from those of hemocyanins (Fig. 4). As shown in Table 2, the amino acid composition of the PPO from O. furnacalis seems to be very similar to that from the PPO of Galleria mellonella (Kopácek et al., 1995), however, differs significantly from the PPO of M. sexta (Aso et al., 1985) or the crayfish Pacifastacus leniusculus (Aspán and Söderhäll, 1991). 3.2. Kinetic properties The enzyme exhibited a typical Michaelis–Menten kinetics. The kinetic parameters calculated for substrate oxidation were Vmax =6.61 U/(mg protein.S) and Km =0.92 mmol/L for L-DOPA, Vmax =4.67 U/(mg protein.S) and Km =0.41 mmol/L for N-acetyldopamine, and Vmax =4.07 U/(mg protein.S) and Km =0.28 mmol/L for dopamine. The catalytic efficiency, calculated by the relation Vmax/Km showed that the affinity of the enzyme for dopamine was 2.0-fold higher than that for L-DOPA and about 1.3-fold higher than that for N-acetyldopamine (Fig. 5). 3.3. Effect of inhibitors on PO activity As shown in Table 3, significant inhibition of O. furnacalis PO activity was observed with the typical PO inhibitors such as phenylthiourea,

Amino acids

O. furnacalis (mol%)

G. mellonella (mol%)

B. mori (mol%)

M. sexta (mol%)

L. migratoria (mol%)

P. leniusculus (mol%)

Asp Thr Ser Glx Pro Gly Ala Cys1/2 Val Met Ile Leu Tyr Phe His Lys Arg Trp

13.6 5.2 7.1 5.8 7.1 4.5 5.8 n.d. 5.5 3.6 5.4 7.9 3.4 6.5 2.8 2.7 7.5 1.4

13.8 5.4 7.0 9.9 6.8 7.5 6.3 n.d. 5.4 2.6 4.4 7.7 3.2 5.1 2.7 4.1 8.5 n.d

14.8 5.2 7.1 10.1 6.0 5.5 6.0 0.5 5.7 3.0 4.9 7.7 3.3 6.2 2.5 2.7 7.4 1.4

4.9 4.8 9.1 9.1 n.d. 13.7 11.1 n.d. 8.0 1.3 5.6 8.1 2.9 5.2 2.1 10.0 5.6 n.d.

14.9 5.6 4.8 9.9 5.9 5.8 5.0 n.d. 6.8 2.0 4.9 8.9 3.5 6.3 2.3 3.9 8.8 n.d.

7.2 6.7 6.4 11.1 4.9 5.6 5.1 1.9 7.1 1.2 7.9 12.0 2.6 2.6 1.4 8.6 7.8 n.d.

Other species include insects Galleria mellonella (Kopácek et al., 1995), Bombyx mori (Ashida, 1971), Manduca sexta (Aso et al., 1985) and Locusta migratoria (Cherqui et al., 1996) and the crayfish Pacifastacus leniusculus (Aspán et al., 1995). n.d., not detected.

thiourea and EDTA. For example, both phenylthiourea and thiourea caused a 85% and 76% inhibition of the PO activity respectively at 10 μmol/L and a 100% inhibition at 20 μmol/L. The reducing agent, DTT, caused 60% inhibition of the PO activity at the concentration of about 0.1 mmol/L and N95% inhibition at 1 mmol/L. Both copper chelator diethyldithiocarbamate and magnesium chelator TTHA showed a poor inhibition of the PO activity while iron chelator o-phenanthroline and calcium chelator EGTA failed to inhibit the enzyme. 3.4. Effect of metal ions on PO activity To verify if the activation of the purified PPO is influenced by metal ions, we measured the activity of PO in the presence of MgSO4, ZnSO4, MnCl2, FeCl2, CuSO4 and CaCl2, respectively. Both Mg 2+ and Cu2+ stimulated the PO activity when compared with the control group. An approximately 25% increase in the PO activity was observed in the reaction mixtures containing 15 mmol/L of Mg 2+ or Cu2+. The PO activity was increased by 50% or higher when the Mg 2+ or Cu2+ concentration was increased to 30 mmol/L. Like Mg 2+ and Cu2+, Ca2+ was also able to increase the PO activity by up to 30% at the concentration of 15 mmol/L.

Fig. 4. Comparison of the Nterminal sequences of two subunits of PPO from O. furnacalis with other arthropod hemocyanins and some insect PO. OfPPO1. O. furnacalis hemolymph PPO subunit I; OfPPO1, O. furnacalis hemolymph PPO subunit II; BmPO1, B. mori PO subunit I; BmPO2, B. mori PO subunit II (Kawabata et al., 1995); HdPO, H. diomphalia hemolymph PO (Kwon et al., 1997); DmPO, D. melanogaster PO (Fujimoto et al., 1995); MsPO, M. sexta PO (Hall et al., 1995); BgHe, hemocyanin from B. giganteus in the presence of sodium dodecyl sulfate (Pless et al., 2003).; PlHe2, Crafish P. leniusculus hemocyanin 2 (Lee et al., 2004).

C. Feng et al. / Comparative Biochemistry and Physiology, Part B 151 (2008) 139–146

Fig. 5. Kinetics analysis of the purified PPO from the hemolymph of O. furnacalis larvae. Reaction mixtures containing L-DOPA or N-acetyldopamine, or dopamine, respectively, as substrates and CPC as activator were prepared and assayed as described in Materials and methods.

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Fig. 6. Effect of various metal ions on the activity of the purified PPO. Each point of the graph represents the mean ± standard deviation of six independent samples. Reaction mixtures containing L-DOPA as a substrate and CPC as an activator were prepared and assayed as described in Materials and methods.

3.6. The content of several metal elements in PPO 2+

However, when Ca concentration was increased to 25 mmol/L or higher, it became inhibitory. In the presence of Mn2+ or Zn2+, the PO activity was increased only by a moderate level. No significant addictive effect of Fe2+ ion on the PO activity was observed (Fig. 6). 3.5. Effect of metal ions on the secondary structure of PPO The result of circular dichroism spectra showed that O. furnacalis PPO contains about 37.28% α-helix and 17.00% β-sheet at 25 °C(Table 4). The β-sheet content of the PPO was increased to 37.98% and 36.87%, respectively, in the presence of Mg2+ or Cu2+ (P b 0.05). In addition, a marked increase in β-sheet content of the PPO was also observed when the PPO was exposed to Ca2+, Zn2+ and Mn2+. However, a significant decrease in β-sheet content of the PPO was observed with Fe2+ treatment (P b 0.05). Secondary structure studies indicate that the enhancement of the PPO activity after metal ion treatment is presumably due to the irreversible increase of β-sheet content.

Table 3 Inhibition of detergent-activated PPO purified from O. furnacalis Inhibitor

Concentration

Inhibition rate (%)

Phenylthiourea

20 μmol/L 10 μmol/L 1 μmol/L 20 μmol/L 10 μmol/L 1 μmol/L 10 mmol/L 1 mmol/L 0.1 mmol/L 10 mmol/L 1 mmol/L 0.1 mmol/L 10 mmol/L 1 mmol/L 0.1 mmol/L 10 mmol/L 1 mmol/L 0.1 mmol/L 10 mmol/L 10 mmol/L

100 85 72 100 76 63 100 95 60 83 72 45 46 34 27 38 33 15 0 0

Thiourea

Dithiothreitol

Ethylene diamine tetraacetic acid

Diethyldithiocarbamate

Triethylenetetramine hexaacetic acid

o-phenanthroline Ethylene glycol-bis (β-aminoethylether)N, N, N′,N′-tetraacetic acid

In this study, the purified PPO from the hemolymph of O. furnacalis was analyzed for the level of six metals. Mg (5.674 ± 2.294 µg/mg) was the most abundant metal among the elements quantified, followed by Cu (1.257 ± 0.921 µg/mg) and Ba (0.281 ± 0.211). The content of Fe, Mn and Zn was relative low but similar (approximately 0.05 µg/mg) in all tested samples (Table 5). 4. Discussion Because of the finding of homology between arthropod hemocyanin and insect hemolymph PPO (Fujimoto et al., 1993; Hall et al., 1995; Kawabata et al., 1995), intensive studies have been carried out, resulting in a rapid accumulation of our molecular biological knowledge on PPO. Hemocyanins are large multisubunit copper proteins composed of different subunit types and found freely dissolved in the hemolymph of arthropods and mollusks. Their primary biological function is the transport of oxygen. Hemocyanin, which is closely related to crustaceans and chelicerates, is absent in insects (Decker et al., 2001). The first PPO gene was cloned from the freshwater crayfish P. leniusculus (Aspán et al., 1995). To date, more than 30 PPO genes have been obtained from about 20 species (Cerenius and Söderhäll, 2004). Several investigators have also purified and characterized some different insect PPOs (Durrant et al., 1993; Gillespie et al., 1997; Chase et al., 2000). The major problem to analyze the PPO has been a lack of effective tools to identify and quantitate individual PPO isoforms.

Table 4 The effect of various metal ions on the secondary structure of PPO from O. furnacalis larvae (mean ± SD, n = 6) Metal ion

α-helix (%)

β-sheet (%)

β-turn (%)

Other (%)

Mg2+ Cu2+ Ca2+ Zn2+ Mn2+ Fe2+ CK

22.10 ± 1.14 a 32.62 ± 1.37 c 35.25 ± 2.20 de 35.52 ± 1.63 de 30.50 ± 1.22 b 33.52 ± 2.70 cd 37.28 ± 1.81 e

37.98 ± 1.56 e 36.87 ± 1.26 e 28.27 ± 1.73 d 23.10 ± 2.60 c 22.48 ± 1.49 c 10.80 ± 2.63 a 17.00 ± 1.65 b

21.11 ± 2.72 bc 19.10 ± 1.69 b 11.92 ± 1.38 a 18.80 ± 1.55 b 15.23 ± 2.16 a 23.95 ± 5.83 c 14.33 ± 1.39 a

18.82 ± 1.46 b 11.42 ± 0.74 a 24.65 ± 3.31 c 22.58 ± 2.62 c 31.78 ± 2.36 d 31.73 ± 3.58 d 31.38 ± 3.32 d

Values significantly different (one-way ANOVA, P b 0.05) from the control group (without metal ion treatment) are indicated by letters and values with the same letter are not significantly different from each other.

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Table 5 Average values of metal elements in the purified PPO from the hemolymph of O. furnacalis larvae (mean ± SD, n = 6) Element

Mg Cu Ba Fe Mn Zn

Purified PPO (µg/mg) Minimum

Maximum

Mean value

2.930 ± 0.772 0.641 ± 0.321 0.236 ± 0.181 0.016 ± 0.009 0.015 ± 0.012 0.014 ± 0.002

8.421 ± 3.293 1.872 ± 0.892 0.324 ± 0.284 0.092 ± 0.035 0.089 ± 0.046 0.098 ± 0.025

5.674 ± 2.294 1.257 ± 0.921 0.281 ± 0.211 0.054 ± 0.012 0.053 ± 0.031 0.051 ± 0.009

Since immunoaffinity chromatography was proven to be ineffective for purifying PPO (Kopácek et al., 1995), the conventional chromatographic method was used by several investigators (Chase et al., 2000). The use of Blue Sepharose for separation of PPO from its activating components was first introduced in the purification protocol of PPO from the crayfish blood cells (Aspán and Söderhäll, 1991) and proven to be an essential and very effective step. The key for PPO purification is to minimize enzyme activation during the purification process. Once purification of PPO is started it is essential to process the sample as rapidly as possible by avoiding sample dialysis or minimizing dialysis time (Li et al., 2005). In the present study, a combination of ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography was employed to purify the PPO from the hemolymph of O. furnacalis larvae. The PPO eluted from Phenyl Sepharose CL-4B column was 369.85-fold purified (Table 1). This indicated that our protocol using Blue Sepharose CL-6B followed by Phenyl Sepharose CL-4B was effective in the processing of purifying PPO. Native PAGE of the PPO purified from O. furnacalis larvae showed a single band in the gel. An anti-M. sexta PPO serum which has been proved to be useful as a reagent for detection of PPO from mosquitoes (Jiang et al., 1997a) cross-reacted significantly with purified protein from O. furnacalis (Fig. 2), indicating that the purified 158 kDa protein contained PPO at least. To further confirm the O. furnacalis PPO protein has been purified, N-terminal amino acid sequencing was used. We found that the purified PPO has a blocked terminus. Then the purified protein was digested with trypsin to generate PO and submitted to Edman degradation. Two different N-terminal amino acid sequences were determined and compared with those of arthropod hemocyanins and some insect PO with Clustal W2 online. The differences of Nterminal sequences between OfPPOs and hemocyanin indicated that the PPO from O. furnacalis has been purified to homogeneity (Fig. 4). Under two-dimensional gel electrophoresis the purified PPO was separated to two protein spots, one with a molecular mass of 80 kDa and pI of 5.70, and the other with molecular mass of 78 kDa and pI of 6.50. These data showed the purified PPO consisted of two subunits. This result is similar to that of the native PPO obtained from Holotrichia diomphalia using gel chromatography (Kwon et al., 1997). In insect species, the presence of different isoforms of PPO has been detected. For example, there are two isoforms in the G. mellonella (Kopácek et al., 1995) and Bombyx mori (Yasuhara et al., 1995), six in the mosquito, Anopheles gambiae (Müller et al., 1999), and three in the fruit fly, Drosophila melanogaster (Fujimoto et al., 1993). However, only a single isoform has been characterized from Hyalophora cecropia (Andersson et al., 1989), Blaberus discoidalis (Durrant et al., 1993), Locusta migratoria (Cherqui et al., 1996) and Sarcophaga bullata (Chase et al., 2000). The PPO1 and PPO2 polypeptides of M. sexta and B. mori have been shown to be the constituent subunits of the native PPO heterodimer (Aso et al., 1985; Yasuhara et al., 1995; Jiang et al., 1997b). In these species, analysis of PPO dimers revealed the presence of two isoforms (Doucet et al., 2008). Whether or not the native PPO of O. furnacalis forms a homo or heterodimer of PPO1 and PPO2 remains to be determined. It seems likely that more detailed studies on the

structure of PPO subunits I and II will reveal minor heterogeneity in the subunit I and/or subunit II. The physiological significance of the presence of multiple PPO isoforms in these organisms still remains to be studied. A conclusive answer to these questions will come from the complete nucleotide sequence. The discrepancy of PPO characters in various insects may caused by the PPO gene duplication occurred after the divergence of holometabolous and hemimetabolous insects. When L-DOPA was used as a substrate, the Km value of O. furnacalis PPO is comparable to those reported for Heliothis virescens (Lockey and Qurth,1992), Spodoptera littoralis (Lee and Anstee,1995), D. melanogaster (Asada and Sezaki, 1999), and Apis mellifera (Zufelato et al., 2004), with 2.25, 1.35, 1.30, and 0.17 mmol/L, respectively. The affinity of the PPO from O. furnacalis for L-DOPA (apparent Km was 0.92 mmol/L) was higher than that reported for the H. virescens (Lockey and Qurth, 1992), S. littoralis (Lee and Anstee, 1995), D. melanogaster (Asada and Sezaki, 1999). Although L-DOPA has been traditionally used as a substrate for characterization of POs from arthropods in general (Durrant et al., 1993; Kopácek et al., 1995; Lee and Anstee, 1995; Brivio et al., 1996; Cherqui et al., 1996), our data showed that the affinity of O. furnacalis PO for dopamine is 2-fold higher than that for L-DOPA, a result similar to what have been reported in other insect species (Hall et al., 1995; Sugumaran, 1998; Asada and Sezaki, 1999; Chase et al., 2000). Thus, insect POs seem preferring dopamine as a substrate than L-DOPA. Traditionally, estimates of the binding energy between substrate and enzyme are based on Km values. The PPO binding affinity is significantly affected by the nature of the mature part of the PPO. Different substrates can adopt the appropriate conformation to interact with the PPO protein. There is no information at this time on the substrate binding pocket in PPO. The differences in the substrate binding pockets between the different insects are probably the result of differences in substrate-protein contact points or differences in the size of the substrate binding pocket. Since EDTA is a chelating agent for divalent cations while DTC is a specific chelator for copper, copper presence in PPO may explain the inhibition of O. furnacalis PO by EDTA and DTC. Inhibition of O. furnacalis PO by DTC was similar to that found in POs of H. virescens (Lockey and Qurth, 1992), Aedes aegypti, Anopheles quadrimaculatus (Nayar and Bradley, 1994), and other arthropods such as Limulus polyphemus (Nellaiappan and Sugumaran, 1996) and brown shrimp Penaeus californiensis (Gollas-Galván et al., 1999). Aspán and Söderhäll (1991) reported that crayfish PPO contains two copper atoms per molecule, and the copper binding sites are similar to those in crustacean hemocyanin. While in contrast, although phenylthiourea is a strong inhibitor of PPO activity in insects and in the horseshoe crab L. polyphenmus (Nellaiappan and Sugumaran, 1996), it did not have inhibitory effect on shrimp PO activity. It is not known whether these differences are reflected in the molecular structure of the enzyme. While TTHA, a kind of chelator for magnesium, caused the obvious inhibition, suggesting that the binding of some magnesium atoms is necessary in the activating center of O. furnacalis PPO. As EGTA is a specific calcium chelator and o-phenanthrocine is a chelator for iron cations, failure of inhibition of O. furnacalis PO activity by EGTA and o-phenanthrocine indicates that few calcium and iron atoms exist in the PPO. PO activity from O. furnacalis was increased in the presence of the divalent ion Mg2+, Cu2+ (Fig. 6). The stimulation of PO activity by equimolar concentrations of Mg2+ and Cu2+ ions suggested that each one could substitute for the other during activation. An increase in PO activity in presence of both mono- and divalent metal ions has been observed for PPO from Hyalophora cerepia (Andersson et al., 1989). Calcium-mediated PO activity enhancement has been reported for a variety of insect species, e.g. B. mori (Ashida et al., 1983), Schistocerca gregaria (Dularay and Lackie, 1985), Blaberus craniifer (Leonard et al., 1985), L. migratoria (Brehé1in et al., 1989) and Lymantria dispar and G. mellonella (Dunphy, 1991). Interrelationships among secondary structure, metal ion binding and PO activity are investigated. The binding of metal ions has only a slight effect on the secondary structure of some peptides, but for certain other peptides some metal ions can significantly change secondary structure (Li et al., 2000). The secondary structures of the

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purified PPO were found to be low in β-turn (14.33%) and high in αhelix content (37.28%) (Table 4) based on calculation using CD spectra. When Mg2+ and Cu2+ ions were added to a solution of the purified O. furnacalis PPO, the CD was dramatically changed, and the β-sheet content augmented (Table 4). Meanwhile, the PO activities increased too. This raises a key question whether the content of β-sheet in this protein could affect the activation of PPO. The zymogen could, therefore, be a least partially sheety. This finding supports the hypothesis that the binding sites of metal ions might have a strikingly different structure as part of the enzyme. This ability to change conformation in solution might explain how the enzyme enhances its activity. Trace metal elements play numerous roles in biology notably as integral parts of enzymes or protein structures (Smith et al., 1997). Thus the knowledge of the binding of trace metal elements to the PPO is required. We are not aware of any published studies on the trace metal speciation in PPO. In the present study, we found that the contents of magnesium and copper were higher than other trace metal elements. Therefore, PPO should be considered as a kind of metalloprotein (a copper, magnesium-protein), and small changes at the metal center and its ligands can be amplified by the protein to perform complex biological processes. To understand the similarities as well as differences in molecular characterization and physiological function among these arthropod PPOs, it is necessary to conduct more accurate, qualitative and quantitative analyses by cloning and transcriptional or translational detection of PPO. Acknowledgement This research was supported by National Natural Science Foundation of China (grant No. 30571248). References Andersson, K., Sun, S.C., Boman, H.G., Steiner, H., 1989. Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation. Insect Biochem. 19, 629–637. Asada, N., Sezaki, H., 1999. Properties of phenoloxidase generated from prophenoloxidase with 2-propanol and the natural activator in Drosophila melanogaster. Biochem. Genet. 37, 149–158. Ashida, M., 1971. Purification and characterization of prephenoloxidase from hemolymph of the silkworm, Bombyx mori. Arch. Biochem. Biophys. 144, 749–762. Ashida, M., Ishizaki, Y., Iwahana, H., 1983. Activation of pro-phenoloxidase by bacterial cell walls or beta-1, 3-glucans in plasma of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 113, 562–568. Aso, Y., Kramer, K.J., Hopkins, T.L., Lookhart, G.L., 1985. Characterization of haemolymph protyrosinase and a cuticular activator from Manduca sexta (L.). Insect Biochem. 15, 9–17. Aspán, A., Huang, T., Cerenius, L., Söderhäll, K., 1995. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. USA. 92, 939–943. Aspán, A., Söderhäll, K., 1991. Purification of prophenoloxidase from crayfish blood cells, and its activation by an endogenous serine proteinase. Insect Biochem. 21, 363–373. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brehé1in, M., Drif, L., Baud, L., Boemare, N., 1989. Insect haemolymph: co-operation between humoral and cellular factors in Locusta migratoria. Insect Biochem. 19, 301–307. Brivio, M.F., Mazzei, C., Scari, G., 1996. proPO system of Allogamus auricollis (Insecta): effects of various compounds on phenoloxidase activity. Comp. Biochem. Physiol. B 113, 281–287. Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116–126. Chase, M.R., Raina, K., Bruno, J., Sugumaran, M., 2000. Purification, characterization and molecular cloning of prophenoloxidase from Sarcophaga bullata. Insect Biochem. Mol. Biol. 30, 953–967. Cherqui, A., Duvic, B., Brehelin, M., 1996. Purification and characterization of prophenoloxidase from the hemolymph of Locusta migratoria. Arch. Insect Biochem. Physiol. 32, 225–235. Decker, H., Ryan, M., Jaenicke, E., Terwilliger, N., 2001. SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and Cancer magister. J. Biol. Chem. 276, 17796–17799. Doucet, D., Béliveau, C., Dowling, A., Simard, J., Feng, Q., Krell, P.J., Cusson1, M., 2008. Prophenoloxidases 1 and 2 from the spruce budworm, Choristoneura fumiferana: molecular cloning and assessment of transcriptional regulation by a polydnavirus. Arch. Insect Biochem. Physiol. 67, 188–201. Dularay, B., Lackie, A.M., 1985. Haemocytic encapsulation and the prophenoloxidaseactivation pathway in the locust Schistocerca gregaria Forsk. Insect Biochem. 15, 827–834.

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