The enzymatic component of Drosophila melanogaster chorion is the Pxd peroxidase

ARTICLE IN PRESS Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 35 (2005) 1043–1057 www.elsevier.com/locate/ibmb

The enzymatic component of Drosophila melanogaster chorion is the Pxd peroxidase Ourania A. Konstandia, Issidora S. Papassideria,, Dimitrios J. Stravopodisa, Christos A. Kenoutisb, Zulfiqar Hasanc, Theodoros Katsorchisa, Ron Weverc, Lukas H. Margaritisa a

Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, 15784 Athens, Greece b Institute of Biology, National Center for Scientific Research (NCSR) ‘‘Demokritos’’, Athens, Greece c Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, 1018WS Amsterdam, The Netherlands Received 31 January 2005; accepted 27 April 2005

Abstract In the present study, we demonstrate the isolation and characterization of the Pxd cDNA clone, which codes for the Drosophila melanogaster chorion peroxidase. This specific peroxidase is involved in the chorion hardening process, through protein crosslinking mediated by the formation of di- and tri-tyrosine bonds. The Pxd gene product has been identified in crude protein extracts from adult flies as three immunoreacting, with the anti-rAePO polyclonal antibody, bands of 77, 67 and 55 kDa, while in larvae and purified chorions as a unique 55 kDa band. Moreover, the mature form of the Pxd recombinant protein was specifically recognized by the anti-rAePO antibody as a 77 kDa band, while in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties. Northern blotting analysis of total RNA preparations revealed distinct molecular weight patterns of the Pxd RNA transcripts among adult flies, ovaries and larvae. The in situ hybridization clearly shows that the Pxd mRNA is specifically expressed in follicle cells during the late stages of oogenesis 11–14, while the reverse transcription reactions dictate the stage-specific developmental regulation of the Pxd gene. The immunolocalization approach, using the anti-rAePO polyclonal antibody, has revealed that the Pxd peroxidase is selectively localized in the chorion structures and particularly in the endochorion and innermost chorionic layer (ICL). r 2005 Elsevier Ltd. All rights reserved. Keywords: Chorion; Di-tyrosine; Drosophila melanogaster; Follicle cells; Oogenesis; Peroxidase; Pichia pastoris; Pxd

1. Introduction The Drosophila eggshell is a specialized extracellular matrix that is synthesized between the oocyte and overlaying somatic follicle cells during the late stages of oogenesis. Largely proteinaceous, the eggshell is a highly organized multilayered structure with regional specialization designed to perform a variety of functions. The production of a physiologically developed eggshell is characterized by the: (a) differentiation of the Corresponding author. Tel.: +30 210 7274546; Fax: +30 210 7274742. E-mail address: [email protected] (I.S. Papassideri).

0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2005.04.005

follicle cell subpopulations in response to ovarian signals, (b) directed migrations of the follicle cells within the developing egg chamber, (c) expression of eggshell structural genes by the follicle cells in a defined temporal and spatial order, (d) post-depositional modifications of the eggshell proteins, including several temporally regulated proteolytic cleavage events and (e) directional trafficking of several eggshell proteins in the assembling structure (Margaritis and Mazzini, 1998). By exploiting the genetic advantages of Drosophila and using evolution as a guide, the eggshell provides an excellent experimental model system to study, in vivo, the molecular mechanisms governing protein–protein specific interactions and the apoptotic cell death pathways,

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all together controlling the function of such a complex extracellular architecture in a developing organism (Trougakos and Margaritis, 1998a). The eggshell consists of several successive layers that in the main body of the mature egg are divided into two major overlays. Outward from the oocyte, the vitelline membrane and the chorion are easily observed. Furthermore, in the chorion four successive layers are recognized, namely the wax layer, the crystalline innermost chorionic layer (ICL), the amorphous tripartite endochorion and the fibrous exochorion (Margaritis, 1985a). Eggshell formation is a gradual process involving intense synthetic and secretory activities of the follicular epithelium and has been divided into nine (11–14) successive stages of oogenesis (Margaritis, 1986). The first layer to be formed is the vitelline membrane, while consequently the follicle cells are engaged in the secretion and morphogenesis of the chorionic layers during stages 11–14. Consecutive chorionic layers are largely proteinaceous structures (Regier and Kafatos, 1985), the only exception being the fibrous exochorion, which is mainly composed of polysaccharides. Distinct groups of chorion proteins are deposited in overlapping succession according to an accurate developmental program, which is presumably responsible for the intricate morphogenesis of the chorion. As many as 20 different polypeptides can be detected following two-dimensional electrophoresis of purified Drosophila melanogaster chorions (Waring and Mahowald, 1979). Among them, six proteins represent the major chorionic components, being encoded by differentially amplified genes and classified as developmentally ‘‘early’’ (s36, s38), ‘‘middle’’ (s16, s19) and ‘‘late’’ (s15, s18) structural proteins. The developmentally regulated activity of the six chorion genes is asynchronous in the distinct follicle cell subpopulations and this is observed for all genes studied so far (Parks and Spradling, 1987; Trougakos and Margaritis, 1998b). Following the complicated protein intercalation events occurring during eggshell formation, the final step in producing a completely functional assembled eggshell is the insolubilization process, or hardening, which in Drosophila is mainly catalyzed through eggshell peroxidase mediated di- and tri-tyrosine bonds formation among the chorion protein components. This enzyme has been histochemically detected in the chorion of Drosophila (Mindrinos et al., 1980; Trougakos and Margaritis, 2002), as well as in the chorion of Ceratitis capitata, Bactrocera oleae (Margaritis, 1985b) and Eurytoma amygdali (Mouzaki and Margaritis, 1994). The activation of the enzyme is triggered by the production of endogenous H2O2 (hydrogen peroxide) during the last stage of oogenesis (stage 14). The catalytic reaction cycle of the chorion peroxidase crude extract shows significant similarities with the ones of the

heme-containing peroxidases. In vivo experiments, where insects were treated with the peroxidase inhibitor phloroglucinol, demonstrated complete inhibition of the chorion peroxidase activity and production of nonmatured and finally non-survived eggs (Keramaris et al., 1996). In the present study, we report for the first time the isolation and characterization of the chorion peroxidase cDNA from D. melanogaster follicle cells, using a set of primers designed according to conserved regions among different heme peroxidase genes. We also demonstrate the identity of our protein to the known peroxidase Pxd, with specific emphasis on the protein function as a chorionic component with enzymatic activity and its topology in distinct areas of the chorion. Additional evidence, concerning its expression profile, obtained by RT-PCR and in situ hybridization approaches, revealed a stage-specific expression pattern during late oogenesis. Our observations strongly suggest for the essential role of the chorion peroxidase Pxd into the chorion structure and function and its potential significance as a crucial protein target for future agricultural applications.

2. Materials and methods All tissues used in this study (adults, embryos, follicles and eggs) originated from a wild-type strain Oregon-R D. melanogaster flies culture being collected in Ringer’s medium at 25 1C, in a walk-in environmental chamber. Strains and growth media: DH5a and XL1-Blue Escherichia coli strains were used for the cloning steps and final propagation of all the recombinant constructs. Pichia pastoris KM71 was selected as the most appropriate host strain of the expression plasmid pPICZaA (Invitrogen) carrying the isolated (from the folliclespecific library) Pxd cDNA. Growth medium components and culture conditions of P. pastoris cells are described in the supplier’s manual (Invitrogen). Isolation of the cDNA clone: poly[A+] RNA was extracted from mixed staged 11–14 follicles, using the Dynabeads kit (Dynal) according to the manufacturer’s instructions. The library was constructed using the Smart cDNA library construction kit (Clontech). A fragment of 0.980 kb was first amplified via a PCR approach, using a forward primer pQUE 50 -TTCCAG GAGGCTCGAAAG-30 , corresponding to a conserved region among insect and mammalian peroxidase genes and a reverse primer pCDSrev 50 -CGAGGCGGCGGC GGCCGACATG-30 , corresponding to the 30 -end library primer. Consequently, the 50 -end 1.5 kb fragment of the cDNA was isolated by a PCR approach using a reverse primer pQUErev 50 -GCAATGTTGATCTTT CGCCTCCTG-30 , corresponding to a conserved region among insect and mammalian peroxidase genes and a forward primer PC5 50 -AAGCGTGGTATCAACGCA

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GAT-30 , corresponding to the 50 -end library primer. PCR reactions were performed using the Finzymes DyNAZyme DNA polymerase, in a Mini Cycler thermocycler (MJ research), at 55 1C as the annealing temperature (94 1C for 1 min, 55 1C for 40 s and 72 1C for 1.5 min, 30 cycles). PCR products were analyzed in a 1.2% agarose gel, extracted using a gel extraction kit (Genome) and subcloned via the pGEM-T-easy ligation kit (Promega). The 2.2 kb fragment corresponding to the full-length Pxd cDNA was isolated via a PCR approach using a forward primer pPxdfor 50 -ATGATAAGGG CACGAGATCTTCTG-30 and a reverse primer pPxdrev 50 -CTTCTTTCCGTAGTGAGACGGATC-30 corresponding to the 50 -end and the 30 -end of the Pxd cDNA (1 min at 95 1C, 1 min annealing at 58 1C, 2.5 min at 72 1C for 30 cycles). The PCR product was subcloned into the pBSKS+ plasmid using the T4 ligase (Promega). DNA sequencing: Sequencing reactions were performed by the MWG-Biotech Company. The obtained results were analyzed using the BLAST software from NCBI. Southern blotting analysis: Genomic DNA was prepared from D. melanogaster adult flies (females without ovaries and males), larvae, embryos, laid eggs and ovaries according to Holmes and Bonner protocol. Purified DNA was digested with the appropriate restriction endonucleases, size fractionated in a 1% agarose gel and finally blotted overnight onto a nylon membrane according to Sambrook et al. (1989). The radioactive 32P-dATP cDNA probe was generated using a random priming labeling kit (Promega), following the manufacturer’s instructions and finally added into a Church hybridization buffer. All the DNA–DNA hybridizations were performed under high stringency conditions (65 1C). Northern blotting analysis: Total RNA was extracted from D. melanogaster adult flies (females without ovaries), larvae, embryos and ovaries, using the method described by Bouhin. The RNA extracts were separated in a 1% denaturing formaldehyde agarose gel and consequently blotted overnight onto a nylon membrane, according to Sambrook et al. (1989). The radioactive 32 P-dATP cDNA probe was labeled by a random priming labeling kit (Promega) and finally added into a Church hybridization buffer. All the DNA–RNA hybridization reactions were performed under high stringency conditions (65 1C). Semi-Quantitative RT-PCR: Staged (11–14) follicles (20/stage) were hand-dissected in Ringer’s solution and consequently subjected to RT-PCR (Reverse Transcription-PCR), using the Cell to cDNA kit (Ambion), according to the manufacturer’s recommendations. 100 ng of total RNA were used for each RT-PCR reaction. Specific primers for the Pxd cDNA were used; pQUE as mentioned above and pQUErev 50 -GAGTCCGCAGAATTCCCG-30 (94 1C for 1 min,

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56 1C for 40 s and 72 1C for 1 min, 30 cycles). The pRibf and pRibr, specific for the Rpb1 gene, were selected as the control primers for RNA extracts quantitation. In situ hybridization: Riboprobes were generated against the Pxd cDNA and in situ hybridization reactions were performed as following: ovaries were dissected into D. melanogaster Ringer’s solution and fixed in solution I (2 mM MgSO4, 0.1 M Hepes pH 6.9, 1 mM EDTA and 4% formaldehyde), for 30 min, at room temperature. After rinsing in PBS-T for 20 min, the follicles were incubated for 20 min in PBS-T containing 4 mg/ml non-predigested Proteinase K (Sigma). Prehybridization and initial washes in hybridization buffer (50% formamide, 5X SSC, 0.1% Tween, 100 mg/ml Salmon sperm and 100 mg/ml heparin) were all performed at 56 1C. Alkaline phosphatase (AP)conjugated anti-digoxigenin antibody (Boehringer, Manheim) was used in a dilution of 1:2000. Following rinsing (PBS-T following APB) and staining reactions [APB (100 mM NaCl, 50 mM MgCl2, 100 mM Tris–HCl pH 9.5, levamisole and 0.1% Tween 20, BCIP (5-bromo4-chloro-3-indonyl-phosphate) 0.38 mM and NBT (4-nitrobluetetrazolium-chloride) 0.41 mM)], follicles were spread on a poly-L-lysine coated slide overlaid with mounting media. Samples were examined with an OLYMPUS BH-2 light microscope (LM). Overexpression, purification and enzymatic activity assay of the recombinant Pxd (rPxd) protein: The ovarian 2.2 kb Pxd cDNA was initially subcloned into the pBLKS+ vector and consequently subjected to PCR using the sense primer SfiFor 50 -TTCCCAGCCGGC CAAAAGTTTCCTCTGGC-30 and the antisense one KpnRev 50 -GCGGTACCTTACTTCTTTCCGTAGT GAG-30 . The approximately 2.1 kb amplified product was digested with the SfiI and KpnI restriction endonucleases and consequently ligated into the pPICZaA expression vector, resulting into the final recombinant construct pPICZaA-Pxd. In order to efficiently transform the P. pastoris KM71 strain, the pPICZaAPxd expression vector was linearized with the restriction endonuclease PmeI, purified and finally dissolved in a concentration of 2 mgr/ml. The P. pastoris KM71 transformants were analyzed for fragment integration by conventional PCR screening. Large-scale fermentation of the Pxd-containing clones was performed as suggested by the suppliers. To induce the Pxd cDNA overexpression, 0.5% methanol was daily added in the culture medium, for a period of 5 days. After the centrifugation of the induced transformed cells, the obtained supernatant, containing the secreted Pxd peroxidase, was concentrated by a CentriconTM filter PM50 and consequently separated through a DEAEsephacyl resin column (Amersham). The high salt eluted protein fraction was loaded onto a Mono-Q exchange column and the eluted (through a linear NaCl gradient) proteins were tested for peroxidase activity, in the

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presence of 1 mM o-dianisidine and 0.8 mM H2O2. Only the fractions containing high levels of this enzymatic function were collected together, concentrated and finally stored in 20 1C. The recombinant and purified Pxd protein was resolved in a 14% SDS-PAGE denaturing gel electrophoresis and consequently transferred onto a PVDF membrane. The amino-terminal sequence analysis of the protein band (visualized by Ponceau staining), after it had been excised from the membrane, was performed by the phenylthiohydantoinderivatization procedure, in a Beckman LH 2600 gasphase sequencer. A small volume of the reaction mixture, containing 1 mg of the purified rPxd protein, or 4 mg of the HRP enzyme (negative control), 1 mM tyrosine and 0.65 mM H2O2, in 50 mM Tris–HCl pH 6.0, was incubated at 30 1C, for short periods of time (0–12 min). The di-tyrosine moieties formation was detected by measuring the increase of their absorbance at 315 nm (Li et al., 1996), in a Cary 50 spectrophotometer. An enzymatic reaction mix lacking H2O2 (or tyrosine) was used as a negative control. Western blotting analysis: Fruit flies, larvae, ovaries and isolated chorions were homogenized in a solubilization buffer, containing 6 M urea, 10 mM Tris–HCl pH 8.4, 1% SDS and 4% b-mercaptoethanol. The homogenates were centrifuged at 12.500 rpm for 10 min, in order to obtain soluble proteins. A 12% SDS-PAGE denaturing gel electrophoresis was performed, as described by Laemmli (1970), on a BIORAD miniprotean apparatus. The separated proteins were electrotransferred onto a nitrocellulose membrane with 25 mM Trisbase, 200 mM glycine, 20% methanol and 0.04% SDS. The Pxd peroxidase was detected using a rabbit antirAePO polyclonal antibody, at a final dilution of 1:2000 in blocking buffer (TBS-T and 5% non-fat milk) and visualized using the ECL system (Amersham-Pharmacia). The recombinant and purified Pxd (rPxd) peroxidase was resolved through SDS-PAGE denaturing gel electrophoresis and consequently processed for Western blotting analysis exactly as described above. Immunolocalization (LM and TEM): Ovaries were dissected in Ringer’s solution and processed as previously described by Trougakos and Margaritis (1998b). After an initial fixation in PBS containing 4% formaldehyde, ovaries were dehydrated through a graded series of ethanol solutions. Follicles were infiltrated with Unicryl resin (BBInternational) in a graded resin 100% series (1:2, 2:1, absolute resin), at room temperature, for 60 min and finally embedded in pure resin for 72 h, at 4 1C, under UV (Ultraviolet) lamps. For LM immunodetections, unicryl resin sections were placed onto polyL-lysine covered slides and, after equilibration and blocking, they were incubated for 60 min, at room temperature, with the rabbit anti-rAePO polyclonal antibody, diluted 1:10 in blocking buffer. After rinsing the sections in TBS-T, the secondary antibody [goat

anti-rabbit IgG/HRP-conjugated (Amersham NA-934)] was applied, diluted 1:30 in blocking buffer. The immunoreacting areas were developed with DAB solution, containing 0.03% H2O2 and 0.06% diaminobenzidine (DAB), in 50 mM Tris–HCl pH 7.6, for 5 min, without hematoxylin counterstaining. Alternatively, an anti-rabbit IgG conjugated to a fluorescent molecule was also used as the secondary antibody. Mounted slides for LM immunodetections were examined with an OLYMPUS BH-2 LM. For transmission electron microscopy (TEM) immunolocalizations (Polak and Pristley, 1990), thin ovarian sections were mounted on nickel grids coated with formvar and, after equilibration and blocking, they were incubated with the rabbit anti-rAePO polyclonal antibody, diluted 1:200 in blocking buffer. After rinsing the sections in TBS-T, the secondary antibody [goat antirabbit IgG conjugated with 10 nm gold particles (Sigma, G 7402)] was applied, diluted 1:300 in blocking buffer. Following washes in TBS-T and TBS, the grids were extensively rinsed with ddH2O and counterstained for 10 min, in aqueous solution of 7% uranyl acetate. Mounted slides for TEM immunolocalizations were examined using a Philips EM 300 electron microscope operating at 80 kV. Routine procedures used as negative controls for LM immunodetections and TEM immunolocalizations, to demonstrate the specificity of the rabbit anti-rAePO polyclonal antibody, included omission of either the primary antibody or the secondary one. In both cases, no immunoreactivity was ever detected.

3. Results 3.1. Cloning and structural characterization of the Pxd cDNA Two specific primers were designed, the forward pQUE and the reverse pQUErev, according to sequences corresponding to conserved regions among insect and mammalian peroxidase genes (Figs. 1A and B). The PCR product amplified from the pQUErev and the Pc5 primers is a 1.5 kb fragment, while the PCR product amplified from the pQUE and the pCDSrev primers is a 0.980 kb fragment (data not shown). The final construct is a 2.2 kb fragment corresponding to the full-length Pxd cDNA and it was amplified using the pPxdfor and pPxdrev primers. The strategy for the stage-specific (11–14) follicle cDNA library construction and the cloning scheme for the chorion peroxidase cDNA isolation are illustrated in Fig. 1A. The cDNA sequence analysis revealed 100% identity with the Pxd cDNA clone, previously isolated by Ng et al. (1992), bearing only one nucleotide difference but without any effect on the amino acid sequence of the protein (GenBank accession number: AY 541497). Extensive

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residues forming the surrounding heme microenvironment have been also clearly identified in distinct protein areas and sites (Fig. 1C) (Daiyasu and Toh, 2000). The tertiary structure of the protein has been predicted via the SWISS PROT Prediction Model Software. The molecular structure is similar to the MPO one, mainly characterized by two chains, a light and a heavy one, forming a homodimer (data not shown).

comparative analysis demonstrated that our putative ovarian peroxidase was also highly homologous with the mosquito Aedes aegypti peroxidases (CPO 50%, AePO 78%), as well as the mammalian myeloperoxidase (MPO 44%), thyroid peroxidase (TPO 43%) and eosinophil peroxidase (EPO 42%) (data not shown). The similarity increases to 70% with the mammalian peroxidases in regions which are supposed to harbor the functional domains. The Pxd cDNA clone earlier reported by Ng et al. was isolated from a D. melanogaster embryonic cDNA library, while our clone was extracted from a D. melanogaster stage-specific (11–14) follicle cDNA library. Computational studies of the deduced amino acid sequence disclosed the presence of a putative leader peptide for secretion, mainly defined in the aminoterminal 21 amino acids. The putative calcium binding sites, the cystein residues forming the intra-chain disulfide bonds, the multiple glycosylation sites and the

3.2. Pxd gene amplification and copy number Extracts of purified genomic DNA, isolated from adult female flies without ovaries, adult male flies, larvae, embryos, ovaries and laid eggs, were digested with the restriction endonuclease EcoRI, separated in 1% agarose gel and transferred onto a nylon membrane (Sambrook et al., 1989). As it is illustrated in Fig. 2A, there are no detectable differences of the obtained hybridization patterns among the various DNA

Staged (11-14) Follicies poly[A+] mRNA

3′-poly[A+]

5′ 5′

GGG pCDSrev

5′

3′-poly[A+]

GGG CCC

5′

3′-poly[A+]

GGG CCC

pQUE

Amplification

Pxd cDNA

pQUErev

ds DNA

PCR pQUE

pCDSrev

pPxdfor

pQUErev

PC5

pPxdrev

(A) Fig. 1. Isolation of the chorion peroxidase cDNA and primary structure analysis of its putative product. (A) Cloning strategy for the Pxd cDNA isolation. pQUE and pQUErev primers, designed according to a conserved region among insect and mammalian peroxidase genes (B), were used in PCR reactions, for the isolation of peroxidase-like clones specifically expressed in D. melanogaster staged (11–14) follicles. pCDSrev and PC5 primers were synthesized according to the pTriplex (library vector) cloning sites. An ovarian 2.2 kb cDNA clone, with 100% identity (except of one nucleotide difference) to the Pxd cDNA sequence, was finally isolated. (C) Schematic representation of the Pxd protein primary structure. Each ‘‘domain’’ of the putative Pxd peroxidase is indicated by a box filled with a distinctive pattern. The correspondence between ‘‘domains’’ and patterns is explained in the bottom of the figure. The CD box pictures the catalytic ‘‘domain’’ of the enzyme. The putative positions of the intra-chain disulfide bridges are illustrated by ‘‘S’’, the glycosylation sites by ‘‘G’’, the calcium binding sites by ‘‘Ca’’ and the histidine residue binding heme by ‘‘H’’.

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Fig. 1. (Continued)

extracts, suggesting that the Pxd genomic locus is not specifically amplified during oogenesis, as it has been clearly demonstrated for the six major chorion genes (gS38, gS36, gS19, gS18, gS16 and gS15). A single copy Pxd gene is also presented to occur in the genome of D. melanogaster, according to the Southern blotting analysis of genomic DNA purified from adult flies, consequently digested with a variety of different restriction endonucleases, separated in 1% agarose gel electrophoresis, blotted overnight onto a nylon membrane and finally hybridized against a 0.5 kb PCR fragment directly amplified from the Pxd cDNA clone isolated from choriogenic-specific follicles (Fig. 2B). 3.3. Tissue expression pattern of the Pxd gene The levels of transcriptional expression of the Pxd gene were assessed in RNA extracts isolated from adult (F/), larvae and ovarian tissues. Preparations of total RNA (15 mg each) were purified as described in Materials and Methods, consequently electrophorized in 1% denaturing agarose gel and finally hybridized (Sambrook et al., 1989) against a 0.5 kb PCR fragment (pQUE and pQUErev primers) directly amplified from the follicle-specific Pxd cDNA, under high stringency conditions (65 1C). Strong transcriptional activity of the Pxd gene was clearly detected in adult flies samples (females without ovaries), as one major thick band (2.9 kb) and at least a second minor one (1.7 kb). Due to the strong signal, the molecular sizes of the two

RNA transcripts can be only approximately estimated as 2.9 and 1.7 kb each, without excluding the possibility of, in between existing, hidden transcripts (such as a 2.2 kb transcript). The positive signals observed in the ovary and larva preparations represent an approximately 2.2 kb Pxd RNA transcript (Fig. 3). Positive hybridization reactions have been also obtained by using total RNA extracts isolated from male flies on dot-blot RNA assays (data not shown). The detection of distinct Pxd RNA transcripts, of various molecular sites (2.9, 2.2 and 1.7 kb), in the somatic and germ-line tissue total RNA extracts, indicates the presence of a differentiation-dependent regulatory pathway controlling Pxd gene activity. In other words, the function of the Pxd peroxidase could play an essential role in the physiology of numerous cell types during distinct stages of development. The 2.2 kb mRNA transcript expressed in the ovaries codes for the chorion peroxidase, while no definitive suggestions could be done for the larvae, or the adult flies (F/), yet. Since the Pxd peroxidase gene is a single copy gene (Fig. 2B), it is very likely that the 2.2 kb mRNA transcript is produced through an alternative splicing tissue-specific ovarian mechanism. 3.4. Stage-specific transcriptional regulation of the Pxd gene during choriogenesis Semi-quantitative RT-PCR reactions, performed on staged (11–14) follicles, using specific primers (pQUE

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Fig. 2. Pxd gene amplification and copy number determination. (A) Genomic DNA preparations isolated from adult female flies without ovaries (F/), adult male flies (M), ovaries (Ov), larvae (L), embryos (E) and laid eggs (Eg) were digested with the restriction endonuclease EcoRI and consequently resolved in 1% agarose gel electrophoresis. After the transfer, the nylon membrane was hybridized, under high stringency conditions (65 1C), using as probe a 0.5 kb fragment, directly amplified from the Pxd cDNA clone (pQUE and pQUErev). The intensity similarities among the positive signals (approximately 4.5 kb bands), in all the analyzed tissues, strongly suggest for the absence of any amplification event in the Pxd genomic locus. (B) Genomic DNA (gDNA) extracted from adult flies was digested with various restriction endonucleases, consequently followed by Southern blotting and hybridization, under high stringency conditions (65 1C), against a 0.5 kb fragment (pQUE and pQUErev), directly amplified from the Pxd cDNA clone. The obtained hybridization pattern clearly indicates the presence of a single-copy Pxd gene in D. melanogaster genome.

and pQUErev) designed from the ovarian Pxd cDNA, revealed a developmentally regulated expression pattern, which is clearly illustrated in Fig. 4. The amplified, 0.5 kb, PCR products (pQR: pQR11–pQR14) of all the four developmental stages were analyzed in 1% agarose gel and the resulting image was processed and quantitated through the Gel analyzer image analysis system (Biosure). Pxd RNA expression levels significantly increase from the developmental stage 11–13, with a detectable reduction in stage 14 (Fig. 4A). The approximately 5-fold (5  ) augmentation of the Pxd

RNA transcript levels detected at stage 13 (compared to stage 11) (Fig. 4B) could be due to either an enhanced transcriptional gene activity or to distinct post-transcriptional events, such as differential splicing and/or RNA stability. The presence of the cis-regulatory element TCACGT, also known as ‘‘chorion box’’, in the proximal promoter region of the Pxd gene (data not shown), strongly suggests for the occurrence of a regulatory master mechanism controlling the coordinative transcriptional activity of the Pxd gene and chorion structural genes, as well (see Discussion). The chorion

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Fig. 3. Tissue expression pattern of the Pxd gene. Fifteen micrograms of total RNA preparations, purified from adult female flies without ovaries (F/), ovaries (Ov) and larvae (L), were fractionated in 1% agarose formaldehyde denaturing gel. The used probe was a 0.5 kb fragment directly generated from the Pxd cDNA clone (pQUE and pQUErev primers). All the analyzed RNA extracts were carefully quantitated based on the 28S and 18S band intensities, after the ethidium bromide staining. The detection of a single size Pxd RNA transcript, with an estimated molecular length of 2.2 kb, in both the ovarian and larva extracts is evident.

hardening process is launched at late stage 13, when elevated Pxd peroxidase activity is likely required and is finally completed at late stage 14. 3.5. Pxd in situ hybridization during follicle development Follicles of stages 10–14 were hand-dissected in Ringer’s solution and subjected to whole-mount in situ hybridization. The sense and antisense dig-labeled RNA probes were generated using as a template the Pxd cDNA clone isolated from D. melanogaster ovaries. Hybridization reactions were performed under standard conditions, following manufacturer’s instructions, at 55 1C, in a humidity chamber. Positive signals were detected with immunostaining using the NBT and BCIP reagents. Low expression levels of the Pxd RNA transcripts were observed in follicle cells of stage 11 egg chambers, but they were substantially increased during stages 12 and 13 (Fig. 5). At stage 12, the already elevated transcriptional activity of the Pxd gene is specifically enriched into the posterior pole

Fig. 4. Stage-specific regulation of the Pxd gene during choriogenesis. (A) Total RNA preparations from different stages (11–14) of choriogenic follicles were subjected to semi-quantitative RT-PCR, using specific for the Pxd gene internal set of primers, finally resulting into the production of a 0.5 kb fragment (pQR: pQR11-pQR14). Amplification of a D. melanogaster riboprotein gene (Rpb1) fragment (pRib) was also performed, as a quantitative control of the RT-PCR reactions. (B) Image analysis (Gel analyzer software) of the obtained RT-PCR products revealed a 5-fold (5  ) increase in the generation of the Pxd mRNA transcripts during developmental stage 13 (pQR13), when compared to stage 11 (pQR11).

and the respiratory filaments of the developing follicle. Furthermore, no positive reaction was ever detected in the oocyte, indicating the presence of a follicle cell-specific regulatory mechanism controlling the Pxd gene activity. Every time we were using the sense riboprobe for hybridization, as a negative control, no detectable signals were developed, documenting the specificity of our technical protocol. The stageand cell-specific Pxd gene expression is tightly and directly associated with the enzymatic activation of the chorion peroxidase, which functions at the last stage of oogenesis, when the chorion is completely assembled.

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Fig. 5. The Pxd gene transcriptional activity is restricted to follicle cells. Stage-specific (11–14) follicles were subjected to in situ hybridization analysis, using sense and antisense RNA probes, specifically generated from the ovarian Pxd cDNA clone. The Pxd RNA transcripts are selectively localized in the follicle cells of all choriogenic stages and they are being continuously accumulated from stages 11 to 14. (A) Pxd RNA sense probe, used as a negative control. (B) Pxd RNA antisense probe. (a) Stage 11 follicles, (b) stage 12 follicles, (c) stage 13 follicles, (d) stage 14 follicles. (i) Magnification of the anterior pole (arrows) of a stage 12 follicle (Bb), (ii) Magnification of the posterior pole (arrow) of a stage 12 follicle (Bb). Fc: follicle cells, Nc: nurse cells, Oc: oocyte. Bar: 100 mm (insert 70 mm).

3.6. Biochemical characterization and functional expression of the Pxd chorion peroxidase The rabbit anti-rAePO polyclonal antibody was used to immunodetect the chorion peroxidase in vivo, through Western blotting analysis. Whole protein extracts, obtained from adult male flies, adult female flies without ovaries, larvae, oocytes and purified chorions, were subjected to SDS-PAGE denaturing gel electrophoresis and consequently transferred onto a nitrocellulose membrane. The positive signals of 5-days, adult male and female without ovaries, flies correspond to three different proteins with molecular weights of approximately 77, 67, and 55 kDa each. On the other hand, a single 55 kDa protein was clearly observed in all other tissues, except from the oocyte preparation, where no signal was ever detected even after significantly increasing the exposure time (data not shown) (Fig. 6). The 55 kDa peroxidase protein identified in the chorion and larva extracts is 22 kDa smaller compared to the putative one from the cloned Pxd cDNA, suggesting for the presence of a tissue-specific ovarian post-translational modification mechanism. More specifically, a distinct proteolytic process could be directly involved in the production of the 55 kDa chorionic component, likely generated by a tissue-specific cleavage of the 77 kDa precursor protein. Interestingly, as it has been shown previously by Zhao et al. (2001), the A. aegypti

Fig. 6. The Pxd 55 kDa protein belongs to the family of chorionic structural components. Whole protein extracts, prepared from adult male flies (M), female flies without ovaries (F/), isolated pure chorions (C), larvae (L) and oocytes (Oc), were fractionated and separated in a 12% SDS-PAGE denaturing gel electrophoresis. After protein electrotransfer, the incubation of the nitrocellulose membrane with the rabbit anti-rAePO polyclonal antibody resulted in the appearance of three immunoreacting zones in (M) and (F/) extracts (arrows) and only one zone, with a molecular weight of 55 kDa, in chorion (C) and larva (L) preparations (asterisks and arrow). The absence of positive signal in the oocyte (Oc) protein extracts demonstrates the specificity of our immunodetection process. The comparative quantitation analysis among all the loaded samples was accomplished by the conventional Ponceau staining (data not shown).

AePOx peroxidase can be also proteolytically processed finally resulting into the generation of a 55 kDa protein product. As expected, the anti-rAePO polyclonal antibody could specifically recognize the recombinant Pxd protein, produced by the P. pastoris KM71 transformed clones overexpressing a slightly modified version of the

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initially isolated ovarian Pxd cDNA. More specifically, the pPICZaA-Pxd recombinant construct was designed in such a way that the first 66 nucleotides corresponding to the authentic signal peptide were completely removed, in order to be selectively expressed only the mature form of the Pxd peroxidase. This manipulation allowed the synthesis of a hybrid molecule, containing in the amino-terminal part of the Pxd mature peroxidase the signal peptide of the Saccharomyces cerevisiae a mating factor. The fusion protein was specifically processed by the KEX2 endoprotease and the STE13 gene product (Julius et al., 1983) and finally, after the removal of the yeast signal peptide, the mature form of the Pxd peroxidase was secreted in large amounts into the P. pastoris growth medium. However, eight additional amino acid residues have been introduced in between the prepro-a-factor and the putative mature form of the Pxd peroxidase, due to significant restrictions of the cloning strategy. The amino-terminal sequencing of the recombinant and purified Pxd protein (rPxd) has unambiguously identified a single and unique amino acid sequence (N-Lys–Val–Ser–Ser–Gly-C),

which displays 100% identity with the putative protein deduced from the cloned ovarian Pxd cDNA (data not shown). This observation clearly demonstrates that we have indeed overexpressed and purified the mature form of the Pxd peroxidase. As it is obviously illustrated in Fig. 7A, the mature form of the recombinant and purified Pxd (rPxd) peroxidase could be specifically immunorecognized by the rabbit anti-rAePO polyclonal antibody as a single band of 77 kDa. This is, as expected, in absolute agreement with either the estimated molecular weight of the putative protein deduced from the cloned Pxd cDNA, or the 77 kDa protein form (the highest molecular weight band) detected in adult flies (M and F/) crude extracts, as it is shown in Fig. 6. In order to imitate the functional ability of the chorion peroxidase to catalyze the di- and tri-tyrosine bonds formation in vivo, we tested the peroxidase activity of the recombinant and purified Pxd protein in vitro, by using tyrosine residues as the specific enzymatic substrates. So, in the presence of 1 mM tyrosine and 0.65 mM H2O2 (enzymatic activator), the mature form

Fig. 7. Generation of di-tyrosine bonds by the H2O2-activated 77 kDa recombinant and purified Pxd protein. (A) The extract containing the mature form of the recombinant Pxd (rPxd) protein, directly purified from the pPICZaA-Pxd transformed P. pastoris KM71 strain (see Materials and Methods), was resolved in a 12% SDS-PAGE denaturing gel electrophoresis and consequently processed to Western blotting analysis, using the rabbit anti-rAePO as the primary polyclonal antibody in a final concentration of 1:2000. The recombinant and purified Pxd peroxidase was clearly visualized as a 77 kDa immunoreacting zone (arrow) (a), while the specificity of our immunodetection procedure was demonstrated by the complete absence of any detectable signal in the case of protein extracts produced from either non-transformed (data not shown) or transformed with the empty parental vector (pPICZaA) P. pastoris KM71 strain (b). (B) The 77 kDa recombinant and purified Pxd (rPxd) protein, in the presence of single tyrosine residues as the specific substrates and H2O2 as the authentic activator, at a temperature of 30 1C, resulted into a significant progressive increase in the absorbance at 315 nm, directly indicating the production of di-tyrosine moieties in vitro (a). On the other hand, when the purified HRP enzyme was added in the reaction mixture, instead of the rPxd protein, under the same incubation conditions, no detectable increase in the absorbance at 315 nm was ever observed, demonstrating the inability of the HRP peroxidase to generate di-tyrosine bonds in vitro (b). Additionally, there was no di-tyrosine bonds formation detected, when the H2O2 (or the tyrosine) was missing from the reaction mixture already containing the rPxd peroxidase (data not shown).

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of the Pxd recombinant protein was able to efficiently accomplish the formation of di-tyrosine bonds, as it is clearly demonstrated in Fig. 7B. More specifically, the significant increase observed in the absorbance at 315 nm strongly indicated the production of di-tyrosine moieties, as it was previously shown (Han et al., 2000b; Heinecket et al., 1993). On the other hand, no generation of di-tyrosine molecules was ever detected when either H2O2 (or tyrosine) was absent from the reaction mixture (data not shown), or HRP enzyme was used as the source of peroxidase activity (Fig. 7B). 3.7. Pxd peroxidase constitutes a minor structural component of the chorion The rabbit anti-rAePO polyclonal antibody was used to immunodetect the chorion peroxidase of staged (choriogenic) follicles, through the use of light and electron microscopy sections. The anti-rabbit IgG conjugated to HRP, or to a fluorescent molecule, was used as a secondary antibody on LM sections. Positive

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signals are observed in the chorion and respiratory filaments, as well (Fig. 8A). The anti-rabbit IgG conjugated to 10 nm gold particles was used as a secondary antibody for the Pxd immunolocalization on electron microscopy sections. Positive signals are easily observed in the follicle cells, the ICL and the endochorion. More specifically, highly enriched and enhanced signals are selectively identified in the ICL and the floor of the endochorion, with lower representation in the roof of the endochorion and the pillars (Fig. 8C). The same experiment was also performed using as primary antibody the anti-S38, an antibody raised against one of the major chorionic structural proteins. A number of particles were clearly detected in the roof and the pillars of the endochorion, but not in the floor and the ICL (data not shown). The same experimental protocols for both light (Fig. 8B) and electron microscopy procedures (Fig. 8D) were also performed without adding the primary antibody (negative control). As expected, no signals were ever obtained in both cases.

Fig. 8. Immunolocalization of the Pxd peroxidase in distinct areas of the chorion structure. (A) The Pxd peroxidase was mainly detected in the respiratory filaments (Rf), in the micropyle area (Mp) and in the chorion structure (Ch) of a stage 14 follicle, as it was clearly observed by light microscopy (LM) sections specifically incubated with the anti-rAePO polyclonal antibody. The secondary antibodies used were either the anti-rabbit IgG conjugated to the HRP enzyme (main section of A), or the anti-rabbit IgG conjugated to a fluorescent molecule (insert of A). (B) No positive signal was observed when the primary polyclonal antibody anti-rAePO was omitted from the immunodetection procedure (negative control). (C) Immunoelectron microscopy localization of the Pxd peroxidase in a stage 13 follicle. One can easily observe the distribution and topology of the 10 nm gold particles. The enzyme is clearly detected into the follicle cells (double arrows) and is also localized in the floor of the endochorion (f) and in the ICL as well (arrows). (D) No positive signal was ever observed when the primary antibody was omitted from the immunolocalization procedure (negative control). For (C) immunoreaction the primary polyclonal antibody used was the anti-rAePO, while the secondary one in (C) and (D) was the goat anti-rabbit IgG conjugated to 10 nm gold particles. (A) and (B) LM sections. (Insert) fluorescent microscopy sections. (C) and (D) transmission electron microscopy (TEM) sections. Bars: (A) 200 mm (insert: 100 mm), (B) 200 mm, (C) 0.5 mm, (D) 0.5 mm.

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4. Discussion Peroxidases manifest multiple physiological roles in living organisms. In insects, they have been identified histochemically, by enzymatic activity assays and also by molecular cloning techniques. Many reports have provided evidence for peroxidase activities in fruit flies (Margaritis, 1985b; Ng et al., 1992; Nelson et al., 1994), mosquitoes (Li et al., 1996; Ribeiro and Nussenzveig, 1993; Ribeiro et al., 1994; Ribeiro, 1996; Ribeiro and Valenzuela, 1999), Lepidoptera (Mathews et al., 1997), Orthoptera and Segestidae (Douroupi et al., 2001). Additionally, a peroxidase function plays a fundamental role in the chorion hardening process during oogenesis of D. melanogaster (Mindrinos et al., 1985), B. oleae (Keramaris et al., 1991, 1996) and mosquito A. aegypti (Li et al., 1996). The eggshell of D. melanogaster is completely assembled during the last stage of oogenesis and is essentially involved in several physiological functions, including sperm entry, waterproofness of the oocyte, respiration, mechanical protection and hatching of the embryo (Margaritis, 1985a). The chorion peroxidase catalyzes the formation of di- and tri-tyrosine bonds, which are directly associated with the chorion hardening process. In the present study, we report for the first time the identification of the Pxd gene product as the essential enzymatic component of the D. melanogaster chorion structure. The nucleotide sequence analysis of a 2.2 kb peroxidase cDNA clone, isolated through a 50 -end and 30 -end PCR approach from a cDNA library constructed from staged (11–14) follicles, revealed 100% identity with the Pxd cDNA clone primarily isolated from an embryonic cDNA library (Ng et al., 1992). The Pxd gene product exhibits great homology, especially in distinct functional domains, with the mammalian peroxidases and more specifically the MPO (Bakkenist et al., 1978), the TPO (Kimura et al., 1987) and the EPO (Wever et al., 1981) (data not shown). The deduced amino acid sequence has been analyzed in detail and important structural characteristics, such as putative calcium binding sites and glycosylation sites, have been clearly identified. The Pxd protein also manifests significant modular sequence similarities with distinct invertebrate peroxidase family members, including the AePOx (Zhao et al., 2001), the Pxt (Vasquez et al., 2002) and the A. aegypti CPO protein (Li et al., 2004) (data not shown). Interestingly, as it has been recently shown, the CPO peroxidase is involved in the chorion crosslinking process during oogenesis (Li et al., 2004). Additionally, the Pxt protein is preferentially expressed during oogenesis and more specifically is exclusively detected in the oocyte and nurse cells, as well. The Southern blotting analysis clearly revealed that the Pxd genomic locus is represented by a single copy

gene, which does not follow the gene amplification rule that characterizes the other six major chorion genes. This, developmentally regulated, amplification mechanism allows the synthesis of large amount of chorion proteins (S38, S36, S19, S18, S16 and S15) that play essential roles in chorion assembly and organization during oogenesis. On the other hand, since the Pxd gene codes for an enzyme (a peroxidase), which is also considered as a minor structural component of the chorion, the demands for peroxidase production are really limited and thus a Pxd gene amplification process is not required for the physiological completion of oogenesis. The Pxd gene transcriptional activity is not restricted to certain tissues, since positive signals are clearly detected in total RNA extracts from adult flies (at least two RNA transcripts), larvae and ovaries (one RNA transcript). Having shown that Pxd is a single copy gene, it is strongly suggested that the different RNA species are generated through a tissue-specific alternative splicing mechanism. It is very likely that the 2.2 kb Pxd RNA transcript, detected in the ovarian RNA extract, is selectively produced into the follicle cells through differential RNA maturation–splicing events. This conclusion can be further supported by the observation that two (in contrast to the rest) of the Pxd exon–intron junctions do not follow the GT–AG rule (data not shown) (also see the Pxd genomic locus organization in the D. melanogaster genome) that ensures the accuracy and efficiency of the splicing process (Sharp, 1985). The comparative structural analysis of the six chorion genes proximal promoter regions has revealed the presence of a cis-regulatory sequence, TCACGT, which is 100% conserved not only in many Drosophila species but in other insects as well, such as C. capitata and Bombyx mori (Sourmeli et al., 2003). Interestingly, the TCACGT ‘‘chorion box’’ has been also pinpointed in the Pxd proximal promoter region (-61), along with additional common cis-regulatory elements (data not shown), likely controlling the transcriptional expression of the Pxd gene during choriogenesis. This is a really important observation that probably indicates the presence of common regulatory mechanisms directly governing the transcriptional activation pathways of all the above genes. The Pxd gene activity is developmentally regulated throughout the early (stage 11) and middle–late (stages 13–14) phases of choriogenesis. Minor RNA expression levels are detected at stage 11, with a 5-fold (5  ) increase at stage 13. During stage 14, the RNA levels are lower, since the choriogenesis is almost completed, but still when compared to the stage 11 ones they are 2-fold (2  ) up. The highest accumulation of the Pxd RNA transcripts at stage 13 is directly associated with the completion of follicle maturation, since the chorion

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hardening process occurs at the late phase of choriogenesis and the immunohistochemistry data demonstrate that the chorion peroxidase functions at the end of oogenesis. The in situ hybridization approach has essentially confirmed the developmentally regulated profile of the Pxd transcriptional activity, which has been clearly demonstrated by the RT-PCR reactions. Moreover, the in situ hybridization analysis has revealed the follicle cell-specific expression of the Pxd RNA transcripts, but has also shown their complete absence from the developing oocyte. The combination of the RT-PCR results, the in situ hybridization data and the putative molecular organization of the Pxd proximal promoter region strongly suggest that the differential Pxd gene expression during development could be mainly regulated by stage-specific transcriptional initiation events, even though post-transcriptional RNA stabilization phenomena could not be excluded. Both approaches have clearly identified that the developmental period of the highest Pxd RNA transcripts accumulation is defined at stage 13, while stage 11 is characterized by minor production levels. The respiratory filaments and the micropyle structure are highly enriched in Pxd RNA transcripts. This observation is directly related to the strong color staining we can obtain in these areas, when isolated follicles are treated in vitro with synthetic compounds detecting peroxidase activity, such as guaiacol and o-DAB (data not shown). Moreover, all the above data are in absolute agreement with the strong Pxd protein immunodetection observed in the respiratory filaments and the micropyle area, as well as in the ICL and the floor of the endochorion (Fig. 8). When the Pxd chorion peroxidase was characterized by Western blotting analysis, in the male and female without ovaries flies, one major band and two minor ones could be readily detected, with approximate molecular sizes of 77, 67 and 55 kDa, respectively. On the other hand, in the chorion and larva extracts only one protein band was clearly identified at 55 kDa. The 67 and 55 kDa proteins could be likely generated by distinct proteolytic cleavages of the 77 kDa precursor molecule, differentially occurring in various cell populations during development. Moreover, we strongly believe that the follicle cells could selectively produce the 55 kDa unique protein fragment through the specific activation of highly efficient proteolytic mechanisms during oogenesis. As it is clearly illustrated in Figs. 6 and 7, the 77 kDa native protein (Fig. 6) and the recombinant and purified mature form of the Pxd (rPxd) peroxidase (Fig. 7) can be both specifically recognized by the anti-rAePO polyclonal antibody with comparable affinities, revealing two strong immunoreacting bands with an identical molecular weight of 77 kDa each. These observations strongly suggest that the 77 kDa major band unambigu-

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ously corresponds to the mature form of the Pxd peroxidase. As expected from the in situ hybridization analysis, the oocyte did not express any of the Pxd protein forms, demonstrating their cell-specific biosynthetic profile. The 77 kDa chorionic protein could be probably synthesized by the 2.2 kb alternatively spliced RNA product, which could be generated into the follicle cells of the developing egg chambers, during the late stages of oogenesis. The proteolytically processed (from the 77 kDa precursor molecule) 55 kDa Pxd peroxidase form could be likely secreted from the follicle cells to the pre-assembled chorion during the late stage 14, catalyzing the hardening of the chorion structure, through the formation of di- and tri-tyrosine covalent bonds among the various chorionic proteins. The novel property of the Pxd peroxidase to perform crosslinking activities, using distinct chorionic components as the authentic in vivo substrates, can be directly disclosed by the functional ability of the recombinant and purified Pxd (rPxd) protein to efficiently catalyze the in vitro conversion of single tyrosine residues to di-tyrosine moieties. As it is shown in Fig. 7B, the progressive increase in absorbance at 315 nm is directly associated with the recombinant Pxd-mediated formation of dityrosine covalent bonds, which are also detected in the D. melanogaster chorion structure, during the hardening process, at the last stage of oogenesis. The presence of H2O2, which has been well documented as the authentic endogenous regulator of the chorion peroxidase, can specifically and efficiently trigger the in vitro activation of the recombinant Pxd peroxidase, allowing the generation of numerous di-tyrosine products. This enzymatic property, along with the specific immunodetection of the 55 kDa Pxd protein in isolated pure chorion extracts, indicate the dual regulatory fundamental role of the Pxd peroxidase in chorion anatomy, organization and physiology. The immunoelectron microscopy approach clearly reveals that the Pxd peroxidase can be specifically detected in relatively dispersed locations on the chorion structure during the late stages of oogenesis (Fig. 8C). The scattered (in ICL and floor of the endochorion) profile of the Pxd peroxidase topology could be likely attributed to the ability of each peroxidase molecule to efficiently convert more than one (probably numerous) substrates (single chorionic components) to complete products (crosslinked chorionic components probably forming a structural network). This specific distribution is in absolute agreement with the weak immunodetection profile of the Pxd peroxidase molecules on the chorion structure, as it has been visualized by LM approaches (Fig. 8A). On the other hand, the respiratory filaments and to a lower extent the micropyle area are able to produce enriched immunofluorescent signals (insert Fig. 8A), implying the significantly

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enhanced Pxd protein synthesis and peroxidase activity in these particular regions. Conclusively, we strongly believe that the 55 kDa Pxd protein constitutes not only the, so-far unidentified, essential chorionic enzymatic component with peroxidase activity, but also a necessary structural element for the chorion scaffold organization and assembly. Additionally, the ability of the rabbit anti-rAePO polyclonal antibody, primarily originated from the mosquito A. aegypti, to specifically crossreact with a distinct chorionic protein of D. melanogaster promulgates that the enzymology of the chorion hardening mechanism is evolutionarily highly conserved among various insect species. This conclusion is further substantiated by the strong sequence homologies observed in between the Pxd (D. melanogaster) peroxidase and AePOx, CPO (A. aegypti) peroxidases (data not shown).

Acknowledgements We would like to thank Dr. R. Renerie and N. Tanaka (Department of Molecular Chemistry, University of Amsterdam) for helpful information and support. O. Konstandi would like to thank all members of Prof. Koomen lab. for their invaluable help and to express her gratitude to T. Douroupi (member of Prof. Margaritis lab.) for her friendship and excellent collaboration during their stay in Amsterdam. The rabbit anti-rAePO polyclonal antibody, raised against the A. aegypti AePox peroxidase, was kindly provided by Professor B. Christensen. We are also grateful to the Associate Professor K. Komitopoulou for the generous gift of the pRibf and pRibr primers. O. Konstandi was supported by a fellowship from the Greek Foundation for Fellowships. This work was funded by a TMR Grant No. EBR4061PL970047, awarded to Professor L.H. Margaritis. References Bakkenist, A.R.J., Wever, R., Vulsma, T., Plat, H., Van Gelder, B.F., 1978. Isolation procedure and some properties of myeloperoxidase from human leucocytes. Biochim. Biophys. Acta 524, 45–54. Daiyasu, H., Toh, H., 2000. Molecular evolution of the myeloperoxidase family. J. Mol. Evol. 51, 433–445. Douroupi, T., Konstandi, O., Kathirithamby, J., Margaritis, L.H., 2001. Histochemical and molecular evidence of peroxidase activity in Segestidea novaeguineae (Brancsik) (Orthoptera) and Stichitrema dallatorreanum (Hofeneder) (Strepsiptera). Tijdschr. Entomol. 144, 197–202. Han, Q., Li, G., Li, J., 2000. Purification and characterization of chorion peroxidase from Aedes aegypti. Arch. Biochem. Biophys. 378, 107–115. Heinecket, J.W., Li, W., Daehnke 3rd., H.L., Goldstein, J.A., 1993. Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase–hydrogen peroxidase system of human neutrophils and macrophages. J. Biol. Chem. 268, 4069–4077.

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