Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

Comparative Biochemistry and Physiology, Part B 158 (2011) 235–241

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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 ev i e r. c o m / l o c a t e / c b p b

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus Inês A. Costa a, Richard I. Samuels b, Thaís D. Bifano c,1, Walter R. Terra c, Carlos P. Silva d,⁎ a

Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28030-600, Campos dos Goytacazes, RJ, Brazil Laboratório de Entomologia e Fitopatologia, Centro de Ciências e Tecnologia Agropecuária, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28015-620, Campos dos Goytacazes, RJ, Brazil c Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, 05513-970, São Paulo, SP, Brazil d Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, CP 476, 88040-900, Florianópolis, SC, Brazil b

a r t i c l e

i n f o

Article history: Received 26 October 2010 Received in revised form 8 December 2010 Accepted 8 December 2010 Available online 15 December 2010 Keywords: Hemiptera Perimicrovillar membranes Insect digestion Enzyme purification

a b s t r a c t The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AβNA and AsnMCA (Kms 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. © 2010 Elsevier Inc. All rights reserved.

1. Introduction The midgut cells of Hemiptera and Thysanoptera are ensheathed by a lipoprotein membrane, the perimicrovillar membrane (Terra, 1988), which extends toward the luminal compartment of the midgut. The perimicrovillar membrane maintains a constant distance from the inner microvillar membrane and delimits a closed compartment, the so-called perimicrovillar space. According to Silva et al. (1995), the perimicrovillar membranes are formed in the Golgi areas of epithelial cells as internal membranes originating from double membrane vesicles. The double membrane vesicles migrate to the cell apex, where the outer vesicle membrane fuses with the microvillar membrane and the inner vesicle membrane fuses with the perimicrovillar membrane. This model is supported by immunolocalization of α-glucosidase (a marker enzyme of perimicrovillar membranes) in midgut cells of the cotton seed sucker ⁎ Corresponding author. Fax: +55 48 3234 4069. E-mail address: [email protected] (C.P. Silva). 1 Present address: Laboratório de Bioquímica e Imunologia de Artrópodes, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900, São Paulo, Brazil. 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.12.002

bug Dysdercus peruvianus (Silva and Terra, 1995; Silva et al., 1995). However, further studies of other biochemical markers are still necessary to explain the origin of this system. Aminopeptidases (EC 3.4.11) catalyze sequential hydrolysis of amino acid residues from the N-terminal region of peptides or polypeptides, and are classified according to their dependence on metallic ions (generally Zn2+ or Mn2+) and their substrate specificity. Aminopeptidase N (EC 3.4.11.2) has a wide specificity, although it removes preferentially alanine and leucine residues, while aminopeptidase A (EC 3.4.11.7) prefers aspartic acid and glutamic acid as substrates (Norén et al., 1986; Ferreira and Terra, 1984, 1986). In insects, these enzymes are found in the midgut and play an important role in the intermediate and final digestion of proteins, since in these animals they are in general more active than carboxypeptidases (Terra and Ferreira, 1994, 2005). Insect aminopeptidases possess pH optima between 7.2 and 9.0, molecular masses between 90 and 130 kDa and Km values between 0.78 and 0.13 mM when using LpNA as a substrate (Terra and Ferreira, 2005). Aminopeptidases known from insect sources possess wide specificity, being capable of hydrolyzing a variety of β-naphthylamide substrates,

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except acyl-amino-β-naphthylamides, which indicates that they are in general N type aminopeptidases (Terra and Ferreira, 1994). An exception is the soluble aminopeptidase associated to the glycocalix of Rhynchosciara americana (Diptera) that is similar to aminopeptidases of vertebrates. This enzyme liberates N-terminal residues of aspartic or glutamic acid from peptides that are not efficiently attacked by other aminopeptidases (Klinkowstrom et al., 1994). In less evolved insects (Orthoptera, Hemiptera, Coleoptera Adephaga), aminopeptidases are mainly found in the soluble form, while in more evolved insects (Coleoptera Polyphaga, Diptera and Lepidoptera), the aminopeptidases are generally associated with the microvillar membranes of the midgut cells (Terra and Ferreira, 1994, 2005). In the majority of these insects, microvillar aminopeptidases are the main proteins present in the membranes. They compose 55% of microvillar proteins in larval Tenebrio molitor according to Cristofoletti and Terra (1999), being the target of δ-endotoxins produced by the bacterium Bacillus thuringiensis. These toxins, after binding to aminopeptidases and to cadherin receptors, form channels that cause cell breakdown and eventually insect death (Knight et al., 1995; Gill et al., 1995; Denolf et al., 1997). In Hemipterans, aminopeptidases are found in soluble form and are imprisoned in the perimicrovillar space, as demonstrated in R. prolixus (Ferreira et al., 1988), D. peruvianus (Silva et al., 1996; Damasceno de Sá et al., 2007) and in Eurygaster integriceps (Allahyari et al., 2010). In accordance with these results, aminopeptidases could be used as biochemical markers of the perimicrovillar space, whilst α-glucosidase is a perimicrovillar membrane marker and β-glucosidase, a microvillar membrane marker (Ferreira et al., 1988; Silva and Terra, 1995; Silva et al., 1996; Damasceno de Sá et al., 2007; Allahyari et al., 2010). The present work was performed to extend our knowledge on the aminopeptidase activity present in the midgut epithelium of D. peruvianus. We now describe the purification of the major soluble midgut aminopeptidase and detail its enzymatic characteristics (catalytic properties, substrate specificities and its susceptibility to inhibition) and molecular properties (molecular mass and partial sequence). 2. Material and methods

in double distilled water (unless otherwise stated) by using a pestle. The homogenate was centrifuged at 15,000 g for 30 min at 4 °C. The supernatant was adjusted to 1 mL and the correspondent pellet was dispersed in double distilled water and the final volume adjusted to 1 mL. 2.4. Protein determination and hydrolase assays Protein concentration was determined according to Bradford (1976), using ovalbumin as a standard. Most of the enzymatic assays for detecting aminopeptidase were carried out using the synthetic substrate leucine p-nitroanilide (LpNa) at a concentration of 1 mM according to Erlanger et al. (1961). The reaction volume for the aminopeptidase assay was of 100 μL of enzyme source, 300 μL of substrate in 100 mM Tris/HCl, pH 7.0 and the reaction was interrupted with 200 μL 30% acetic acid and absorbance of free p-nitroaniline was read at 410 nm. The assays against RpNA (arginine p-nitroanilide) were accomplished using the same conditions as described for LpNA. Assays against AβNA (alanine β-naphthylamine) were accomplished in a reaction medium containing 100 μL of the enzyme source plus 200 μL of the substrate. After different intervals of time, the reactions were interrupted with 500 μL of 2% HCl in ethanol and revealed with 500 μL of 0.06% p-dimethylaminocinamaldehyde in ethanol. Absorbance was read at 540 nm. The fluorimetric assays with AsnMCA (asparagine methyl-coumarin) was accomplished in a reaction medium containing 50 μL of enzyme source added to 1.95 mL of the substrate/buffer solution (final volume of reaction of 2 mL). Fluorescence was measured for the free release of methyl-coumarin using a Hitachi F2000 fluorimeter at 440 nm after an excitation at 380 nm. For each determination, the mixture of the reagents was kept at 30 °C for at least four different time periods and initial velocities were calculated. Controls without enzyme or substrate were carried out and treated in a similar way as the experimental groups. One unit of enzymatic activity was defined as the amount of enzyme that hydrolyses one μmol of substrate per minute. 2.5. Ion-exchange chromatography and hydrophobic interaction chromatography

2.1. Animals Stock cultures of the cotton stainer bug D. peruvianus (Hemiptera: Pyrrhocoridae) were maintained in plastic bottles, covered with a piece of cloth, under natural photoperiod conditions at a relative humidity of 60–70% and a temperature of 25 ± 2 °C. The insects in the colony had ample access to water and cotton seeds (Gossypium hirsutum). 2.2. Dissection of insects Adult females were individually immobilized by placing them on crushed ice (8–10 min) and then dissected in cold 215 mM NaCl. D. peruvianus midgut is divided into three main sections or first ventriculus (V1), second ventriculus (V2) and third ventriculus (V3) and is linked through V4 (the fourth ventriculus) to the hindgut (Silva and Terra, 1994). Entire guts were removed and rinsed in distilled water. The midgut was separated from the foregut and hindgut, and the midgut contents were removed after cutting open the midgut first ventriculus (V1) lengthwise. Groups of V1 tissues were pooled as a replicate group and stored at − 20 °C for use as enzyme sources in the experiments. 2.3. Preparation of samples Due to the fact that V1 tissue is known to have the highest enzymatic activities (Silva and Terra, 1994), it was used in the preparation of samples. V1 tissues of fed animals were homogenized

The crude extract of V1 tissue was applied onto a column (5 mL) of Econo-Q (BioRad) equilibrated with 20 mM Tris/HCl buffer, pH 7.0. The column was eluted with 25 mL of a linear gradient of 0–0.5 M of NaCl, followed by 10 mL of a 0.5–1.0 M gradient and 10 mL of 1 M NaCl. The flow was of 1 mL/min and 1 mL fractions were collected. The fractions were assayed for aminopeptidase activity and for protein determination. The fractions obtained from the Econo-Q column with aminopeptidase activity were pooled and diluted twice with a solution of 2 M ammonium sulfate (final concentration of 1 M). The sample was applied onto a phenyl-agarose column (1.0 × 5.5 cm i.d.) equilibrated with Tris 20 mM HCl buffer, pH 8.0 containing 1 M ammonium sulfate. The column was eluted with 40 mL of a gradient of 1–0 M of ammonium sulfate, followed by 5 mL of an isocratic elution with the same buffer without ammonium sulfate. The flow rate was 1 mL/min and 50 fractions of 1 mL were collected and assayed for the aminopeptidase activity and for determination of proteins. 2.6. Polyacrylamide gel electrophoresis Electrophoresis was performed under denaturing conditions (SDSPAGE) as follows. Samples were combined with the sample buffer containing 500 mM Tris–HCl buffer, pH 6.8, 2.0% (w/v) SDS, 5% (w/v) β-mercaptoethanol, 10% (v/v) glycerol and 0.005% (w/v) bromophenol blue. Samples were heated for 5 min at 95 °C in a water bath before being loaded onto a 12% polyacrylamide gel slab containing

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0.1% SDS (Läemmli, 1970). Native electrophoresis was carried out under the same conditions, but without SDS and also in the absence of β-mercaptoethanol and the samples were not boiled. Gels were run at a constant voltage of 150 V and stained for protein using a silver staining method (Blum et al., 1987). 2.7. Kinetic studies Enzyme preparations were pooled from fractions obtained after native electrophoresis and enzyme assays. To obtain the fractions from native gel electrophoresis, the gels were cut into 2 mm wide sections which were subsequently homogenized in water using the Potter–Elvehjem homogenizer. The effect of substrate concentration on the activity of aminopetidase was determined in 100 mM Tris HCl, pH 7.0, at 30 °C, using 10 different substrate concentrations. Km and Vmax values were determined by a weighted linear regression (Wilkinson, 1961) using the EnzFitter® program. Lineweaver–Burk plots were always verified and the determinations were performed with concentrations of the substrates in the range of 0.2 to 2 Kms. 2.8. Gel filtration Gel filtration was performed in a Smart System (PharmaciaAmersham Biotechnology, Sweden) using a Sephadex G150 column (1 × 54 cm, i.d.) equilibrated and eluted in 10 mM sodium phosphate containing 100 mM NaCl, pH 7.0. Fractions of 0.2 mL were collected at a flow rate of 0.4 mL/min. Mr values were calculated by using the following proteins as standards: cytochrome c (Mr 12,400), lysozyme (Mr 14,000); trypsinogen (Mr 24,000); ovalbumin (Mr 45,000), and bovine serum albumin (Mr 66,000). 2.9. Molecular cloning of the midgut aminopeptidase from D. peruvianus peruvianus (DpAPN) and sequence analysis Total RNA was extracted from D. peruvianus V1 midgut epithelium following the manufacturers (Invitrogen) instructions, which are based on Chomczynski and Sacchi (1987), and the extracts were sent to Vertis Biotechnologie AG (Germany), in order to construct a normalized cDNA library. At Vertis Biotechnologie AG the mRNAs were isolated, divided into two equal samples and used in cDNA synthesis with a poly-T and a random primer. Finally, the two cDNA pools were mixed (1:1) and non-directionally inserted in the vector λ ZAPII. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of a cDNA library obtained from D. peruvianus midgut. All analyses including open reading frame, translation, Clustal W alignments were performed using ExPASy Proteomics tools (http:// expasy.org/tools/). The partial sequence was also analyzed for the presence of N- and O-glycosylation sites using NetNGly1.0 program (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGly3.1 program (http://www.cbs.dtu.dk/services/NetOGlyc/).

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Table 1 The primers used for cloning and expression experiments. Oligonucleotide Aminopeptidase Forward primer Reverse primer rRNA1 Forward primer Reverse primer

DNA sequence 5′-GGGGCAGTTTCAAGAGTAAT-3′ 5′-GCTTCCTTTAGGCAGCCTTT-3′ 5′-TGGTGCATGGAATAATGGAA-3′ 5′-GCTTTCGCTCTAGTGCGTCT-3′

ruled out by the lack of sequence amplification observed when the initially extracted RNA was used in PCR reactions. The primers used were shown in Table 1. PCR reactions was performed after initial denaturation at 94 °C (2 min) using TAQ DNA polymerase (Invitrogen) in a reaction buffer containing 1.5 mM MgCl2. PCR conditions were: 25 cycles of 45 s at 94 °C (denaturation), 30 s at 53 °C (annealing) and 2 min at 72 °C (synthesis). The number of cycles was chosen after several trials so that the amplification was in log phase, resulting in a clearly visible amplified band. The size of the RT–PCR products agreed with that expected from RNA. This ruled out the possibility of DNA contaminants in the preparations. 3. Results 3.1. Aminopeptidase purification A soluble aminopeptidase associated with the intestinal epithelium of D. peruvianus (DpAPN) was purified using ion-exchange chromatography (Fig. 1), hydrophobic interaction chromatography (Fig. 2) and preparative native electrophoresis. The specific activity of DpAPN following each purification stage and the evaluation of the SDS-PAGE fractions is shown in Table 2 and Fig. 3 respectively. The recovery of DpAPN activity and the purification factor for hydrophobic interaction chromatography in relation to the crude extract were relatively low (3.4% and 7.8 times respectively). This was partly due to the fact that the enzyme is relatively unstable and lost activity during hydrophobic interaction chromatography probably due to the high concentration of ammonium sulphate used during this step. Moreover, the aminopeptidase is a low abundant protein in the intestinal epithelium of D. peruvianus, with many other proteins of similar molecular mass coeluted with this enzyme. Only a single symmetrical peak for aminopeptidase activity was detected using the substrate LpNA following semi-preparative

2.10. Semi-quantitative reverse transcription (RT)-PCR Female insects immobilized on ice were dissected in cold 215 mM NaCl using gloves, sterile forceps and glassware previously treated with diethylpyrocarbonate. The dissected tissues were maintained in an ethanol-ice bath and eventually were stored at −80 °C. RNAs were extracted from the tissues using Trizol (Invitrogen) according to the manufacturer's protocol, which is based on Chomczynski and Sacchi (1987), and used to synthesize the corresponding cDNAs with the aid of a reverse transcriptase present in the Superscript™ First Strand Synthesis System kit for RT-PCR (Invitrogen) with samples free of contaminating genomic DNA. The resulting cDNAs were used as templates for amplifying sequences by PCR with specific primers for the aminopeptidase. The presence of contaminating genomic DNA was

Fig. 1. Ion-exchange chromatography of the soluble fraction from midgut homogenate from Dysdercus peruvianus using an Econo-Q column. The column was equilibrated with 20 mM Tris/HCl buffer, pH 7.0 and eluted with a gradient of 0–0.5 M NaCl, followed by 10 mL of 0.5 M NaCl and a gradient 0.5–1 M NaCl. Fractions of 1 mL were collected at a flow rate of 1.0 mL/min. Substrate used: LpNA. Aminopeptidase activity (●), protein (○). Insert: native electrophoresis in 7% polyacrylamide gel of the active fractions (20–29, from left to right) obtained from the Econo-Q column. Arrow indicates the position of the bands of aminopeptidase.

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Fig. 2. Hydrophobic interaction chromatography on phenyl-agarose column of the active fractions (21–25) from the Econo-Q column. The column was equilibrated with 20 mM Tris/HCl buffer, pH 7.0 plus 1 M ammonium sulfate and eluted with a gradient of 1–0 M ammonium sulfate in the same buffer. Fractions of 1 mL were collected at a flow rate of 1.0 mL/min. Aminopeptidase activity (●), protein (○). Insert: native electrophoresis in 7% polyacrylamide gel of the active fractions (30–39, from left to right) obtained from the phenyl-agarose column. Arrow indicates the position of the bands of aminopeptidase.

polyacrylamide gel electrophoresis under native conditions (Fig. 4) and gel filtration chromatography (Fig. 5). These results indicate that there is only one molecular form of aminopeptidase present in the epithelium from D. peruvianus V1 midgut compartment. 3.2. Determination of the molecular mass of the aminopeptidase The molecular mass of the native aminopeptidase estimated by gel filtration was 106 kDa (Fig. 5). The molecular mass of the aminopeptidase under denaturing and reducing conditions estimated by SDSPAGE was 55 kDa (Fig. 3). These results suggest that the native aminopeptidase is a dimer. 3.3. Determination of the kinetic parameters of DpAPN The Vmax and Km values of DpAPN toward the substrates LpNA, RpNA, AβNA and AsnMCA are shown in Table 3. The ratio of Vmax/Km showed that the enzyme hydrolyzes LpNA more efficiently than RpNA. The aminopeptidase was inhibited by EDTA (not shown) and 1,10-phenantroline. The Ki value for the inhibitor 1,10-phenantroline using LpNA as substrate was 0.51 ± 0.03 mM. In this case, inhibition of the simple hyperbolic competitive type was observed with the value of the parameter α equal to 39.6 ± 4.0 (Fig. 6).

Fig. 3. 12% SDS-PAGE of aminopeptidase from Dysdercus peruvianus midgut tissue at different stages of purification. Lane 1, homogenate of V1 midgut tissue; lane 2, Econo-Q fraction; lane 3, phenyl-agarose fraction; lane 4, preparative electrophoresis fraction; lane M, molecular weight markers.

The partial sequence contained 795 bp encoding a 265 amino acid protein (Fig. 7) and its GenBank accession number is HQ378156. Some computational analyses of the deduced amino acid sequence were carried out to detect the presence of signature sequences. Five potential N-glycosylation sites and one potential O-glycosylation site were found. Besides that, the four cysteine residues, which are highly conserved in eukaryotic aminopeptidases N were found in the DpAPN sequence. As the DpAN sequence lacks the final portion of the C-terminal, it was not possible to verify the occurrence of zinc-binding/gluzincin and glycosyl phosphatidyl inositol (GPI) motives, probably because of the soluble nature of the D. peruvianus aminopeptidase. RT-PCR analyses of the transcripts of the aminopeptidase gene revealed that the corresponding mRNA was expressed in whole midgut epithelium extracts, epithelia of V1, V2 and V3, Malphigian tubules, and fat body, but it was not expressed in the salivary glands (Fig. 8).

3.4. Sequence coding and expression of DpAPN A cDNA library prepared from D. peruvianus midgut RNA was randomly sequenced and the identities of the assembled sequences were analyzed. One partial sequence of BLAST-identified aminopeptidase was found and showed a maximum identity of 26% to Bombyx mori aminopeptidase N (GenBank accession number BAA33715), 25% to a noctuid moth Achaea janata AjMgAPN (ABH07377) and 24% to Helicoverpa armigera aminopeptidase N4 (AAO23562).

Table 2 Purification of aminopeptidase from V1 epithelium of Dysdercus peruvianus. Fraction

Total activity Total protein Specific activity Purification Yield (mU) (mg) (mU/mg) factor (%)

Crude extract 1023 Econo-Q 175 Phenyl-agarose 35 Electrophoresis 25

16 1.4 0.07 0.04

64 125 500 625

1.0 1.9 7.8 9.7

100 17 3.4 2.4

Fig. 4. Electrophoretic migration of the aminopeptidase under native conditions. An enzymatic fraction of phenyl-agarose chromatography was concentrated and applied onto two lanes of a 7% polyacrylamide gel and submitted to the electrophoresis under native conditions. One of the lanes was stained for protein and the remaining lane was fractioned in portions of 1 mm that were incubated with LpNA. (●), activity of the aminopeptidase against LpNA.

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B 10 8 6 4 2 0

Slope

90 80 70 60 50 40 30 20 10 0

0

2

4

6

[1,10-Phenantroline], mM

C 0

4

8

1/[LpNA], mM-1

12

1/Δ Slope

1/V, (mU/mL)-1

A

6 4 2

0 -3

2

7

12

17 22

1/[1,10-Phenantroline], mM-1

Fig. 5. Elution profile of Dysdercus peruvianus aminopeptidase after gel filtration on a Sephadex G-150 column. The column was equilibrated and eluted in 20 mM Tris/HCl buffer, pH 7.0, at a flow rate of 0.5 mL/min and fractions of 1 mL were collected. Activity of the aminopeptidase against LpNA (●), RpNA (□) and AβNA (w).

4. Discussion Lane and Harrison (1979) suggested that the perimicrovillar membranes were formed by the incorporation of lipoproteins synthesized by cisterns of the endoplasmic reticulum located close to the microvilli of the intestinal cells. However, Andries and Torpier (1982) suggested that these membranes might originate from multimembrane vesicles and that these vesicles could incorporate their internal membranes to the perimicrovillar membranes after the fusion of the external membrane of the vesicle with the microvillar membrane. These observations were corroborated by the immunolocalization of α-glucosidase within the perimicrovillar membranes and in internal membranes of double membrane vesicles from the Golgi complex in D. peruvianus (Silva et al., 1995). Based on these observations, a model has been suggested in which the perimicrovillar membranes may be organized initially from internal membranes of double membrane vesicles after the fusion of the external membrane of these vesicles with the microvillar membrane, followed by the fusion of the internal membrane of the vesicles with the perimicrovillar membrane (Silva et al., 1995). The use of enzyme markers is an important tool in the elucidation of the intracellular origin of the perimicrovillar membranes. Cellular fractionation and the separation of microvillar and perimicrovillar membranes fractions by isopicnic centrifugation of midgut cells of R. prolixus (Ferreira et al., 1988) and D. peruvianus (Silva et al., 1996), demonstrated that an aminopeptidase was located in the perimicrovillar space. The hypothesis that the aminopeptidase is soluble within this space is supported by the fact that this enzyme is easily liberated during

Table 3 Kinetic parameters of Dysdercus peruvianus aminopeptidase towards synthetic substrates. Substrate

Km (mM)

Vmax (mU/mL)

Vmax/Km (mU/mL × mM)

LpNA RpNA AβNA AsnMCA

0.65 ± 0.03 0.14 ± 0.06 0.68 ± 0.01 0.74 ± 0.13

32 ± 1 4 ± 0.7 10 ± 1 748 ± 58

49.2 ± 4 30 ± 1 14.5 ± 1 1010 ± 85

The enzyme was incubated with ten different concentrations (7–1000 μM) of each substrate in 20 mM Tris/HCl, pH 7.0. Kinetic parameters were determined by a weighted linear regression by the Enzfitter® computer program. Figures are means and standard deviations obtained from four independent replicates. LpNA, L-leucine-pnitroanilide; RpNA, arginine-p-nitroanilide; AβNA, alanine β-naphthylamide; AsnMCA, asparagine methylcoumarin.

Fig. 6. Inhibition of Dysdercus peruvianus aminopeptidase by 1,10-phenantroline. (A) Curves of Lineweaver–Burk of the activity against LpNA using different concentrations (mM) of 1,10-phenantroline (●, 0; w, 0,05; ○, 0,1; □, 0,2; △, 0,5; ♦, 1; ▲, 2 and ■, 5). (B) Replot of the slopes from the curves of Lineweaver–Burk against different concentrations of 1,10-phenantroline. (C) Secondary curves of the inverse of the inclination (inclination of the curve with inhibitor less inclination of the curve without inhibitor) against the inverse of the concentration of 1,10-phenantroline.

the homogenization of the tissue and recovered in the final supernatant after differential centrifugation (Ferreira et al., 1988; Silva et al., 1996; Damasceno de Sá et al., 2007; Allahyari et al., 2010). The purification of D. peruvianus midgut aminopeptidase was a laborious task due to the amount of “contaminating proteins” that coeluted with the aminopeptidase during the chromatographic separations and due to the fact that it is a low abundant protein in the midgut tissue. Data obtained originally by Silva and Terra (1994) demonstrated that the midgut aminopeptidase of D. peruvianus possesses a pH optimum of 7.0 and a molecular mass under nondenaturing conditions of 72 kDa as determined by gel filtration and 87 kDa as determined by ultracentrifugation using a glycerol gradient. The aminopeptidase purified in the present study had a molecular mass of approximately 106 kDa as determined by gel filtration and 55 kDa as determined by SDS-PAGE. Our results showed that there was only one molecular form of aminopeptidase in the intestinal epithelium of D. peruvianus and that it probably exists as a dimer (Figs. 1–5). The absence of more than one intestinal aminopeptidase isoform is common in Coleoptera, in contrast to the situation in Diptera and Lepidoptera, which possess multiple aminopeptidase isoforms, with a range of specificities toward different substrates (Terra and Ferreira, 2005; Cristofoletti et al., 2006). No soluble insect aminopeptidases have been sequenced to date, although sequences are available for membrane bound aminopeptidases. In Lepidoptera, where aminopeptidases are mainly bound to the microvillar membrane, sequences were obtained from Epiphyas postvittana, B. mori, Heliothis virescens, Heliothis punctigera, Heliothis armigera, Manduca sexta and Plutella xylostella, among others (Knight et al., 1995; Gill et al., 1995; Denolf et al., 1997; Hua et al., 1998; Chang et al., 1999; Oltean et al., 1999; Yaoi et al., 1999; Garner et al., 1999; Emmerling et al., 2001; Rajagopal et al., 2003; Cristofoletti et al., 2006). These papers showed that aminopeptidases have a conserved signal peptide RLP close to the N-terminal, a zinc binding area HEXXHX18E, a conserved motif GAMEN and a long hydrophobic C-terminal tail containing a glycosil-phosphatidyl-inositol anchor. In T. molitor, a RLP motif was also found close to the N-terminal of the microvillar aminopeptidase, but there were no other residues in common with other well-known insect aminopeptidases (Cristofoletti and Terra, 1999; Budatha et al., 2007). The aminopetidase from D. peruvianus hydrolyses the synthetic substrates LpNA, AlaβNA, ApNA and AsnMCA (Table 2). The values for the Vmax/Km ratios showed that the D. peruvianus intestinal aminopeptidase hydrolyzed LpNA more efficiently than ApNA, but the preferred substrate was AsnMCA. Similar results were obtained

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Fig. 7. Clustal W alignment of aminopeptidase putative protein sequences from Helicoverpa armigera (H. armigera, AAO23562), Bombyx mori (B. mori, BAA33715), Achaea janata (A. janata, ABH07377) and Dysdercus peruvianus (DpAPN, ADP55362). DpAPN potential N-glycosylation sites are indicated by arrows and the O-glycosylation site is indicated by a ball. The four cysteine residues are framed at the positions 58, 65, 89 and 125. Black shades indicate total identity among amino acid sequences and grey ones indicate similarity. Only relevant part of the sequences is displayed (C-terminal portion).

for T. molitor intestinal aminopeptidase (Cristofoletti and Terra, 1999), where a higher kcat/Km ratio for LpNA was obtained when compared to ArgpNA. It is probable that the DpAPN is an “N” type aminopeptidase due to the fact that it efficiently hydrolyses peptide bonds with alanine and leucine residues at the N-terminus. Metal binding agents or chelators are known to inhibit metalloenzymes by the removal of metal ions from the active site or by the formation of an inactive ternary complex (Terra and Ferreira, 1994). In the present study, we showed that the D. peruvianus aminopeptidase was inhibited by EDTA and 1,10-phenantroline. The inhibition by 1,10-phenantroline is of the partial competitive type (or hyperbolic), in which EI and ESI complexes are formed. This type of inhibition occurs when: substrate and inhibitor bind to the enzyme at different sites; and the substrate binds to the free enzyme with a greater probability than the EI complex and when the ESI complex is capable of generating products with the same facility (Segel, 1993). In other words, substrate and inhibitor bind at the enzyme active site, so that when one of them binds the enzyme, it decreases the binding of the other by the interaction factor alpha. This suggests that phenanthroline bound to the enzyme through hydrophobic interactions of its ring system with the enzyme pocket that houses the lateral chain of the leucine moiety. If phenanthroline bound to the active zinc, the inhibition would be a simple competitive one or of the

irreversible type like EDTA. The midgut aminopeptidase from Melolontha melolontha (Wagner et al., 2002) and the aminopeptidase from M. sexta pharate adult moulting fluid (Samuels et al., 1993) were also inhibited by 1,10-phenantroline, suggesting the presence of metallic co-factors. However, certain insect aminopeptidase are not inhibited by these chelators, as was the case for B. mori midgut aminopeptidase (Parenti et al., 1997). In the majority of insects so far studied, aminopeptidases are associated with the microvillar membrane of the midgut epithelium via a glycosylphosphatidylinositol-anchor (Terra and Ferreira, 2005; Cristofoletti et al., 2006), but in less evolved insects (Hemiptera, Coleoptera Adephaga and Orthoptera) soluble aminopeptidases are predominant, as demonstrated here for D. peruvianus, where the aminopeptidase gene was expressed along the whole length of the midgut, as well as in the Malpighian tubules and fat body (Fig. 8). Future studies concerning intracellular trafficking and compartmentalization of the aminopeptidase in the perimicrovillar space will increase our understanding of the formation of the perimicrovillar membrane in hemipterans. Acknowledgements We thank Cristovão Barros Pinheiro for his technical assistance. This work was funded by grants from FAPESP, FAPERJ, FAPESC and CNPq. Richard I. Samuels, Walter R. Terra and Carlos P. Silva are CNPq research fellows. Thaís D. Bifano is a graduate fellow from FAPESP. Walter R. Terra is also member of the Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. References

Fig. 8. Relative amount of amplified transcripts of Dysdercus peruvianus aminopeptidase relative to amplified rRNA transcripts in different tissues by semiquantitative RT-PCR. Equal amounts (5 μg) of total RNA from different tissues were extracted and treated with DNAse and used as template for RT reaction. The product of this reaction was used for amplification in a thermocycler, using primers specific for each sequence of cDNA and the ribossomal RNA (see Table 1). The sizes of the amplified bands are in the left and appear in the tissue when the corresponding mRNA is transcribed. MT, Malpighian tubules; SG, salivary gland, FB, fat body, MG, midgut, V1, ventriculus 1 of the midgut; V2, second ventriculus, V3, third ventriculus; rRNA and ribosomal RNA.

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