Insect Biochemistry and Molecular Biology 39 (2009) 430–439
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A lepidopteran pacifastin member: Cloning, gene structure, recombinant production, transcript profiling and in vitro activity Bert Breugelmans a, *, Gert Simonet a, Vincent van Hoef a, Sofie Van Soest a, Guy Smagghe b, Jozef Vanden Broeck a a b
Department of Animal Physiology and Neurobiology, Zoological Institute K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2008 Received in revised form 20 March 2009 Accepted 23 March 2009
Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.e. the silkworm Bombyx mori. The bmpp-1 gene contains 17 exons and codes for two pacifastin-related precursors of different length. The longest splice variant encodes 13 inhibitor domains, more than any other pacifastinlike precursor in arthropods. The second transcript lacks two exons and codes for 11 inhibitor domains. By studying the expression profile of the Bombyx pacifastin-like gene a different expression pattern for the two variants was observed suggesting functional diversification. Next, several PI domains of BMPP-1 were produced and, contrary to locust pacifastin peptides, they were found to be potent inhibitors of both bovine trypsin and chymotrypsin. Surprisingly, the same Bombyx PI are only weak inhibitors of endogenous digestive peptidases, indicating that other peptidases are the in vivo targets. Interestingly, the Bombyx PI inhibit a fungal trypsin-like cuticle degrading enzyme, suggesting a protective function for BMPP-1 against entomopathogenic fungi. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Inhibitor Peptide Pacifastin Serine peptidase Bombyx mori Gene structure
1. Introduction According to the MEROPS database (http://merops.sanger.ac.uk) (Rawlings et al., 2008) members of the pacifastin family (I19) are inhibitors of the largest family of serine peptidases (S1 family). All pacifastin members that have been characterized at the molecular level are precursor peptides composed of an N-terminal signal sequence followed by a variable number of inhibitor domains. These domains are designated as PLDs (Pacifastin Light chain Domains) in reference to the light chain of pacifastin, the first member of the pacifastin family that was found in the crustacean species, Pacifastacus leniusculus (Hergenhahn et al., 1987; Liang et al., 1997). All other pacifastin-related precursors (PPs) that have been identified by cDNA cloning originate from only two insect orders: Orthoptera and Hymenoptera (Kromer et al., 1994; Parkinson et al., 2002; Simonet et al., 2002a, 2002b, 2004a, 2004b, 2005) and only in locusts, the structural and biochemical characteristics of pacifastin-related inhibitors have been intensively studied by 1H NMR, crystallography,
* Corresponding author. Tel.: þ3216324260; fax: þ3216323902. E-mail address:
[email protected] (B. Breugelmans). 0965-1748/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2009.03.005
site directed mutagenesis and activity studies (Boigegrain et al., 1992; Mer et al.,1994,1996; Kellenberger et al.,1995; Hamdaoui et al., 1998; Malik et al., 1999; Roussel et al., 2001; Gaspari et al., 2002; Simonet et al., 2005; Fodor et al., 2005). However, previous in silico data mining studies have predicted additional pacifastin members in various other insect orders (Simonet et al., 2003a; Gaspari et al., 2004; Breugelmans et al., 2008b). Conforming to the presence of putative dibasic cleavage sites between inhibitor domains, nearly all identified insect PPs are believed to be processed into smaller inhibitor peptides (Kellenberger et al., 1995; Hamdaoui et al., 1998; Simonet et al., 2002a, 2002b, 2004b, 2005). All PLD-related domains are characterized by a conserved pattern of six cysteine residues (Cys1 – Xaa9–12 – Cys2 – Asn – Xaa – Cys3 – Xaa – Cys4 – Xaa2–3 – Gly – Xaa3–6 – Cys5 – Thr – Xaa3 – Cys6). Detailed analysis of the 3-D structure shows that these six residues form three disulfide bridges (Cys1–4, Cys2–6, Cys3–5), giving members of the pacifastin family a typical fold and remarkable stability (Mer et al., 1994, 1996; Roussel et al., 2001; Gaspari et al., 2002; Kellenberger et al., 2003). Each pacifastin-like inhibitor domain contains a reactive site (P1–P10 ) that is involved in the binding of the target peptidase and inhibitor activity studies on locust pacifastin-like peptides have
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confirmed that the peptidase specificity is mainly determined by the residue at the P1-position (Kellenberger et al., 1995; Hamdaoui et al., 1998; Malik et al., 1999; Roussel et al., 2001; Gaspari et al., 2002; Simonet et al., 2005). Although all available data indicate that members of the pacifastin family share a common conformation, the family can be divided into at least two separate groups (I & II) because of different intramolecular interactions in the inner core region (Kellenberger and Roussel, 2005). The variability in the reactive site (P1–P10 ), the inner core structure and additional peptidase interaction sites contribute to a different peptidase specificity and species selectivity between members of the two groups. Contrary to the detailed knowledge on both the structure and the inhibitory activity of locust pacifastin-like members, very little is known about their in vivo function(s). In crayfish, pacifastin itself is involved in the regulation of an immune response related serine peptidase-dependent cascade (the prophenoloxidase cascade) (Liu et al., 2007). In insects, on the other hand, no direct evidence is available on the possible function(s) of the pacifastin-related. Detailed transcript profiling studies (Northern blot experiments and real-time PCR studies) on locust pacifastin-related precursors led to a number of hypotheses about possible functions in insects (Vanden Broeck et al., 1998; Simonet et al., 2002a, 2004a, 2004b, 2005; Franssens et al., 2008; Breugelmans et al., 2008a). These profiling studies showed that the locust PP transcripts are differentially regulated in (i) a variety of tissues (fat body, brain, gonads, ventral nerve cord, foregut and hindgut), (ii) during development (larval stages, in adults), (iii) between genders, (iv) depending on the phase (gregarious versus solitarious) and (v) upon immune challenge with fungal components. Therefore, pacifastin-related inhibitors are believed to have multiple functions in locusts as regulators of diverse serine peptidase-dependent processes (Hamdaoui et al., 1998; Malik et al., 1999; Simonet et al., 2003b). In this study, a pacifastin-like member of the order of the Lepidoptera was cloned from the silkworm, Bombyx mori. In addition, the structure as well as the expression profile of the Bombyx pacifastin-like gene was studied. Furthermore, Bombyx pacifastinrelated precursor domains were produced and the in vitro inhibitory activity of these multi-headed PI was analyzed. 2. Methods 2.1. Experimental organisms Larvae of the silkworm, B. mori (Daizo strain), were reared on fresh mulberry leaves at 25 C under 12 L:12 D photoperiod. In addition, an entomopathogenic fungus, Beauveria bassiana was grown and stimulated to secrete cuticle degrading enzymes (Donatti et al., 2008).
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Table 1 Overview of gene specific primers. Different sets of primers are designed to clone the ORF of the pacifastin-related precursor in B. mori. The sequences of the restriction sites are underlined and stop codons are in bold. Primers corresponding to a specific variant of BMPP-1 are named accordingly. Construct
Primer
Primer sequence
Restriction site
U1 D1
50 -AAACAGTGTTGTGATTGAGGATTG-30 50 -TGGCTTATGCGGCGTTT-30
U2 D2a D2b
50 -GATAACGGTCTGGGCCTATGCTC-30 50 -CAGGGTGTGTTAGGTATTTCGGT-30 50 -CGCGACGCAAACGATTGCTT-30
U3a
50 -ACCGAAATACCTAACCACACCCTG-30
BMPP-C
U3b D3
50 -GATATCGTTTGCGTCGCGAACC-30 50 -AAGCTTGGCTTATGCGGCGTTTATG-30
EcoRV HindIII
BMPP-N
U D
50 -GAAGAAATTCGGAAGTGGAATGCCTGGC-30 50 -AAGCTTAGATCAGCTCAGACTTCTCGGGAG-30
XmnI (PdmI) HindIII
primers allowing for a directional ligation of the amplification of fragments into an expression vector (cf. 2.4). PCRs (50 ml) contained 5 ml 10 PCR Buffer (provided by the manufacturer), 1 ml of a 10 mM dNTP mix, 1 ml of each primer (10 mM), 2 ml whole body cDNA and 0.2 ml platinum Taq DNA polymerase (Invitrogen Life Technologies). Hot-start PCR was run for 35 cycles in a ‘triothermoblock TB-1’ thermocycler (Biometra). Each cycle consisted of a denaturation step for 1 min at 94 C, an annealing step for 1 min at a specific temperature according to the selected primer set and an extension step for 1 min at 68 C, with a final extension step of 7 min at 68 C. PCRs were analyzed by horizontal agarose gel electrophoresis. The specific PCR fragments were subcloned and sequenced as outlined below. 2.2.3. Cloning, sequencing and sequence analysis The PCR products were separated on an agarose gel by electrophoresis and purified with the GenElute Gel Extraction Kit (Sigma). The purified PCR fragments were then subcloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen Life Technologies). After plasmid isolation (GenElute HP Plasmid Miniprep Kit: SIGMA), the inserts were sequenced on the 3130 Genetic Analyzer (Applied Biosystems) and all nucleotide and deduced amino acid sequences were compared with the AlignX software (InForMax, Inc., Invitrogen Life Technologies). To study the intron–exon organization of the Bombyx pacifastin gene in more detail, EST and cDNA sequences were aligned with the genome sequence using Splign (http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi). 2.3. Real-time RT-PCR analysis
2.2. Cloning of the Bombyx pacifastin-related precursor 2.2.1. mRNA extraction and cDNA synthesis Decapitated bodies of last larval silkworms were added to reaction tubes containing ‘Green Beads’ (Roche, Indianapolis, IN, USA). Then, semi-automated homogenization of the samples was performed in the ‘MagNA Lyser’ (Roche) instrument according to the manufacturer’s instructions. Subsequently, total RNA was extracted from the homogenates utilizing the ‘RNeasy Lipid tissue mini kit’ (Qiagen, Valencia, CA, USA). The resulting RNA was reverse transcribed (M-MLV RT, Invitrogen Life Technologies) utilizing random hexamers as described in the provided protocol. 2.2.2. Primer design and polymerase chain reaction (PCR) Different sets of gene specific primers were designed based on PP-encoding EST sequences. The sequences of the primers (SIGMA) are presented in Table 1. Restriction sites were added to particular
2.3.1. Experimental samples In order to study the transcript distribution of pacifastin-related precursor transcripts (BMPP-1a and BMPP-1b) in Lepidoptera, B. mori larvae were synchronized and at days 2 and 6 of the last larval stage different tissues were dissected (10 animals per pooled sample; three independent samples per condition): foregut (Fg), midgut (Mg), hindgut (Hg), Gonads (Gon), head (H), ventral nerve cord (VNC), silk glands (SG) and tracheae (Trach). In addition, fat body (Fb) was dissected at day 6 of the last larval stage. All insect tissues were microdissected under a binocular microscope and immediately collected in RNAlater (Ambion) solution to prevent degradation. Until further processing, these pooled tissue samples were stored at 20 C. 2.3.2. Total RNA extraction and cDNA synthesis The total RNA samples were prepared as described above (2.2.1). In combination with this extraction procedure, a DNase treatment
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(RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination. After spectrophotometric quantification and verification of the RNA quality via the Agilent 2100 Bioanalyser (Agilent Technologies), the resulting total RNA was reverse transcribed (Superscript II, Invitrogen Life Technologies) utilizing random hexamers as described in the provided protocol. To minimize variations during the cDNA synthesis step, all RNA samples were reverse transcribed simultaneously. Furthermore, negative control reactions, i.e. without the reverse transcriptase, were prepared and analyzed in parallel with the experimental cDNA-samples during the quantitative PCR-assay. 2.3.3. Primer design, PCR amplification and cDNA quantification PCRs were performed in a 25 ml reaction volume following the manufacturer’s instructions for the SYBR Green assay (Applied Biosystems). The final concentration of the primers was 300 nM. Primers for the two different BMPP target sequences, as well as for the endogenous control (Rp49), were designed by means of the Primer Express software (Applied Biosystems) (Table 2). To guarantee a specific amplification of the BMPP-1b transcript and to exclude the amplification of genomic DNA, a primer set was chosen at the exon/exon junction of exons 11 and 12 and in the BMPP-1b exclusive region (exon 12). For the amplification of the shorter BMPP-1a transcript, primers were chosen at the exon/exon junction of exons 14 and 15 and at the exclusive junction between exons 11 and 14 (Supplementary file 1). All reactions were run in triplicate on an ABI PRISM 7000 Sequence Detection System (ABI PRISM 7000 SDS, Applied Biosystems) using the following thermal cycling profile: 50 C (2 min), 95 C (10 min), followed by 40 steps of 95 C for 15 s and 60 C for 60 s. After 40 cycles, samples were run for the dissociation protocol (i.e. melting curve analysis). In order to compensate for differences in loading and RT-efficiency, Rp49 was used as an endogenous control of silkworm BMPP transcripts. Thus, for both BMPP transcripts, values were analyzed and normalized relative to Rp49 transcript levels by means of the 7000 System SDS (Sequence Detector Software) (version 2.3, Applied Biosystems). For all samples, three technical replicates were run on the ABIPRISM 7000 SDS, as described above, and the entire assay was repeated twice with biological replicates. In all negative control samples, no amplification of the fluorescent signal was detected, proving that the extraction procedure, including the DNase treatment, effectively removed genomic DNA from all RNA samples. A dissociation protocol was run at the end of the PCR to verify the specific amplification of one amplicon, excluding the formation of primer dimers. In addition, a validation experiment was performed to verify whether both BMPP amplification reactions and the endogenous control have similar PCR-efficiencies. Therefore standard curves were generated for the different transcripts with a serial (5) dilution of a cDNA mixture of the different tissues. Then relative transcript levels were determined with the DDCt
Table 2 Primers for Q-RT-PCR of BMPP transcripts and endogenous control in Bombyx mori. The bold font corresponds to the location of the primers on both BMPP-1 transcript variants (BMPP-1a and BMPP-1b) at exon–exon junctions or at a particular exon to guarantee their specific amplification. Primer
Primer sequence
Exon
BMPP-1aU BMPP-1aD
50 -AAAGGAACCGAAATACCTAACCAC-30 50 -GTTGTCCTTTGGCAGTTTTGG-30
XI–XIV XIV–XV
0
0
BMPP-1bU BMPP-1bD
5 -GGAACCGAAATACCTGAGTTAAGG-3 50 -TTAGCAAACGCAGCGCT-30
Rp49-U Rp49-D
50 -TGGTTACGGTTCCAACAAGAAGA-30 50 -CTAAGGCATTCCAGGATCAAGTGTT-30
XI–XII XI
method according to ABI user bulletin #20 (Bloch and Robinson, 2001). Statistical analysis of the three biological replicates was performed by means of non-parametric Mann–Whitney–Wilcoxon (MWW) tests. 2.4. Recombinant production of Bombyx pacifastin-like inhibitors 2.4.1. Cloning strategy and construction of fusion plasmids Based on the cDNA sequence of BMPP-1, two sets of primers were designed to amplify the sequences coding for the N- and C-terminal regions of the Bombyx BMPP-1 precursor, BMPP-1N and BMPP-1C. Distinct restriction sites were added at the 50 end of forward and reverse primers, allowing directional ligation in the pMAL-p2p vector as previously described by Simonet et al. (2003a,b) (Table 1). After the ligation, the two BMPP expression constructs (1–10 ng) were transformed to BL21 Escherichia coli cells (Novagen). Following overnight incubation (37 C) on an LB agar plate containing 100 mg/ml ampicillin, individual clones were checked for the presence of the correct insertion and the fragment by means of sequence analysis. 2.4.2. Growing and harvesting of bacterial cells 2 ml Overnight Express Instant TB Medium (Novagen) was inoculated with 100 ml of an overnight culture of bacterial cells containing the fusion plasmid and incubated at 37 C (300 rpm). When an OD600 of approximately 0.5 was reached, 10 ml pre-heated medium was inoculated with 500 ml of the grown bacterial cell volume. This stepwise inoculation was repeated until a suitable volume (typically 500 ml) was reached. After 16 h of incubation (37 C, 300 rpm), the cells were harvested by centrifugation (10 000 g,10 min), the supernatant was discarded and the pellet was weighed and placed on ice. 2.4.3. Cell lysis The pellet was re-suspended in 1 BugBuster Protein Reagent (Novagen) (5 mg/g pellet), resulting in a mild lysis of the bacterial cells. Simultaneously, per ml BugBuster reagent, 1 ml Benzonase (Novagen) was added. In addition, lysozyme was added (1 KU per ml BugBuster, Novagen) to improve the disruption of the bacterial cell walls. Complete tablets were added (1 tablet per 50 ml, Roche) to avoid proteolytic degradation. Subsequently, the mixture was incubated (15 min at room temperature, gently shaken). Finally, after centrifugation (16 000 g, 20 min, 4 C) the supernatant, containing the fusion protein is decanted. This crude extract was further purified as described below. 2.4.4. Affinity chromatography A 5.5 50 cm column (D x H, Bio-Rad) was filled with 15 ml of amylose resin (Bio-Rad). Before loading the crude extract, the column was washed with 8 volumes of Column buffer (1 M Tris– HCl; 0.2 M NaCl; 0.5 M EDTA; pH 7.4) and the crude extract was diluted with Column buffer to a final concentration of 2.5 mg/ml. The following steps were performed at 4 C. The column was loaded with the diluted extract (typically 800–1200 ml) at a flow rate of 2 ml/min and washed with 15 volumes of Column buffer. Finally, the fusion protein was eluted with Column Buffer supplemented with 20 mM maltose at a flow rate of 1 ml/min. 30 fractions of 3 ml were manually collected and the protein concentration was measured by means of the Bradford protein assay (Bradford, 1976). 2.4.5. Desalting, concentrating and SDS-PAGE The eluted fractions that contained the fusion protein were first pooled and then desalted (NaCl, maltose) and concentrated to a final concentration of about 2–4 mg/ml, using Centriprep Centrifugal Filter Devices (YM-30, Millipore). The crude extract was compared to the eluted fusion protein via SDS-PAGE (4–12% NuPage
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Bis–Tris Gel, Invitrogen). In addition a reference, MBP (Mr ¼ 42.7 kDa, New England Biolabs) was run in parallel with the different samples. Following electrophoresis, the SDS-gel was stained with Simply Blue Safe Stain (Invitrogen) for 1 h and bands were visualized after 1 h of rinsing the gel with water. 2.4.6. MALDI-TOF 1 ml of the desalted and concentrated fusion protein samples (cf. 2.4.5) was transferred to a stainless steel target plate and mixed with 1 ml of a saturated matrix and dried. MALDI-TOF mass spectrometry was preformed on a Reflex IV instrument (Burker Daltonics, GmbH, Bremen, Germany), equipped with a N2 laser (337 nm) and pulsed ion extraction accessory. The instrument was operating in a positive ion, reflectron mode and calibrated using a standard protein mixture (Burker Baltonic). Spectra were recorded within a mass range of 25 000–70 000 m/z and were the result of 5 20 laser shots. Laser intensity was optimized for best signalto-noise ratio and resolution. 2.5. Extraction of insect midgut enzymes and fungal peptidases Biologically active peptidases were obtained from midgut tissue by incubating dissected and cleaned midguts (n ¼ 5) during 1 h in Ringer’s solution (1 ml). Subsequently, midguts were removed and the solution containing the secreted enzymes was used for in vitro assays. 100 ml of a specific fungal-medium (Donatti et al., 2008) was inoculated with 1% of conidia stock (108 conidia per ml) of the fungus, B. bassiana. In order to induce secretion of cuticle degrading enzymes, autoclaved cuticle (5 mg/ml) of B. mori was added and the mixture was incubated for 72 h (28 C; 150 rpm). The secreted enzymes were separated from the fungal debris and insect cuticle by means of filtration (coffee filter, pore diameter: 200 mm). The crude extract was centrifuged several times until the supernatant was clear and ready to be tested in vitro. In addition, the supernatant containing the secreted enzymes was analyzed by means of SDS-PAGE. 2.6. In vitro peptidase/inhibitor activity assays The azocasein assay (Brock et al., 1982) was optimized depending on the experimental setup. Since casein is a general substrate, the peptidase activity of all peptidases present in the sample regardless of their mechanistic class and or specificity is measured. In general, 200 ml of (diluted) enzyme extract was added to 200 ml of 1% azocasein (Sigma) and incubated (32 C; 45 min). The reaction was terminated by addition of 150 ml of 10% trichloroacetic acid followed by 10 min cooling on ice. After centrifuging at 13 000 rpm for 10 min (at 4 C), 415 ml of the supernatant was centrifuged for a second time (13 000 rpm; 10 min). Then, 14 ml of NaOH (5 M) was added to 90 ml of the supernatant and absorbance was measured at alkaline pH (four technical replicates) at 405 nm. To measure the effect of PI, enzyme extract and recombinant PI (5–15 mM) and chemical PI, AEBSF (100 mM), [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, Sigma] were pre-incubated for 10 min (32 C). Then the assay was performed as described above. In all experiments, blanks (buffer instead of enzyme extract) were included and the peptidase activity and inhibitory activity (in %) were calculated. Peptidase activity ¼ Abssample Absblank Inhibitory activity (%) ¼ 100 [(AbsPI (Abscontrol Absblank)] 100
Absblank)/
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3. Results 3.1. Cloning and sequence analysis of BMPP-1 To determine the complete ORF encoding a Bombyx PP (designated as BMPP-1), a partial consensus sequence coding for the N-terminal part of BMPP-1 was derived from Bombyx ESTs (GenBank accession nos. AV401970, AU004695, AU003615, AU004115). Then, based on partially overlapping 30 ESTs a first set of primers (U1–D1) was designed to verify the complete ORF (Table 1 and Supplementary file 2). Using this primer set two fragments of different length (1.8 kb and 1.5 kb) were amplified by means of RT-PCR. Cloning and sequence analyses of these two cDNA fragments led to the identification of two variants of the BMPP-1 precursor; a long variant (BMPP-1b) with an extra internal fragment of approximately 0.3 kb as compared to a shorter a variant (BMPP-1a). Additional primer sets were derived to specifically amplify each of the two variants. In addition, a restriction site was added to the primer sets U3b/D3 and U4/D4 to allow for a directional ligation of the amplified fragments in an expression vector (see below). Sequence analysis of the different PCR products led to the unambiguous verification of the sequences of both precursors (Supplementary file 3). The BMPP-1a transcript contains an ORF of 1539 bp that codes for a precursor with eleven pacifastin-related inhibitor domains. The BMPP-1b-mRNA on the other hand has an ORF of 1848 bp encoding a longer variant with two additional PLDrelated domains 9 and 10 (Fig. 1). The difference in length between the two precursors is completely due to the extra region of 309 bp. Comparison of the 13 Bombyx PLD-like amino acid sequences confirmed the presence of the 6 conserved cysteine residues. In addition, the enzyme specificity of the 13 PLDs can be predicted based on the conserved position and the nature of the P1 residue (Fig. 1). 3.2. Gene structure of bmpp-1 An analysis of the bmpp-gene structure has indicated that the two different transcripts (BMPP-1a and BMPP-1b) result from alternative transcript splicing (Fig. 2). Comparing genomic sequences (GenBank accession number: AADK01011743, BAAB010153971 and AADK0 1002939) with the cloned cDNA sequences of BMPP-1a and BMPP1b showed that the gene is composed of seventeen exons. The long transcript (BMPP-1b) possesses two additional exons (12 and 13) as compared to the BMPP-1a-mRNA. These exons code for the inhibitor domains BMPD-9 and BMPD-10 (Fig. 2). The signal peptide as well as the thirteen PLD-related domains are each encoded by a single exon. In addition, three exons (2, 11 and 14) do not code for a PLD-related domain but correspond to three regions (87, 144 and 114 bp, respectively) that do not have a ‘pacifastin signature’. 3.3. Tissue distribution of BMPP-1 transcripts in the silkworm The relative abundance of two pacifastin-related precursor isoforms (BMPP-1a and BMPP-1b) in a variety of silkworm tissues, collected at two different time points (day 2 and day 6) of the last larval stage, was quantified by real-time RT-PCR analysis (Fig. 3). Statistical analysis of the three biological replicates by means of a non-parametric Mann–Whitney–Wilcoxon (MWW) test shows that the expression profiles of both BMPP-1 transcripts show significant differences between the different tissues, but are similar between day 2 and day 6. The BMPP-1a transcript levels show nonsignificant fluctuations between most tissues. One notable exception is the silk glands which contain significantly lower amounts of mRNA as compared to the other tissues (Fig. 3A). The BMPP-1b transcript on the other hand is mainly present in the head and in the trachea of larvae (6 days after penultimate ecdysis) and knows only a small expression in the other tissues (Fig. 3B).
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Fig. 1. Domain organization of Bombyx mori pacifastin-related precursor 1 (A) Bombyx mori pacifastin-related peptide domains (B). (A) The predicted signal sequence (white horizontal arrow) is followed by thirteen PLD-related domains [containing the conserved cystein pattern: grey and black blocks (Mer et al., 1994)]. The two black blocks correspond to domains 9 and 10 and are exclusive for the long variant BMPP-1b. The domains are separated by putative dibasic cleavage sites (black bars). As a result, full processing (black arrows) would result in the cleavage into eight peptide fragments composed of single or multi-domain inhibitor peptides. (B) The names of the two fragments (BMPP-N and BMPPC), the peptide domains (BMPD) and the conserved amino acid sequences are shown. The six conserved cystein residues (*) are shown in black. The diagram at the top of the figure illustrates the topography of the three disulfide bridges and a black arrowhead (;) indicates the P1-position within the grey marked reactive site. BMPD-9 and BMPD-10 (marked in black) belong exclusively to BMPP-1b.
3.4. Recombinant production of Bombyx PI In order to produce sufficient amounts of biologically active PI, a pMAL Protein Fusion and Purification System (New England Biolabs) was optimized. The pMAL expression system allows for the production of a gene product of interest (pacifastin-related peptides) attached to a fusion protein in bacteria (E. coli). In addition to the already available expression constructs for SGPI-1, SGPI2, SGPI-3 (Simonet et al., 2003b) new constructs were made for the N- and C-terminal regions of the Bombyx BMPP-1 precursor, referred to as BMPP-N and BMPP-C. To increase the productivity of
this expression system, a few specific changes were made to the protocol described by Simonet et al. (2003a,b) (cf. 2.4). Using this optimized procedure, a yield of 15–35 mg fusion protein per l bacterial culture was realised. The production and proper elution of the MBP-PI fusion proteins was verified via SDS-PAGE (Fig. 4) by comparing MBP (43 kDa) to the eluted, desalted and concentrated fusion proteins MBP-BMPP-N and MBP-BMPP-C (approximately 62 kDa and 65 kDa respectively). A more accurate estimation of the masses of both bands was obtained by means of MALDI-TOF and revealed that the molecular mass of MBP-BMPP-N is 61 500 Da and MBP-BMPP-C is 65 100 Da.
Fig. 2. Gene structure and alternative splicing of the bmpp-1 gene. The bmpp-1 gene structure consists of 17 exon regions (blocks). The first block (in white) symbolizes the exon that encodes the signal peptide of the PP. The grey and black blocks represent the exons that encode the 13 PLD-related domains (numbered from 1 to 13), while the shaded grey blocks stand for three exons that do not code for a specific PLD-related domain. The sequence of the last exon (domain number 13) contains a stop codon (pictured in red). Exons 12 and 13 (in black) encode the two domains (PLD-9 and PLD-10) that are unique for the longer BMPP-1b transcript. By connecting the black lines both scenarios of the differential bmpp-1 gene splicing are illustrated; the upper part represents the splicing scenario of the shorter BMPP-1a variant (excluding the two PLD-related domains 9 and 10). In the lower part of the figure the splicing scenario of the longer BMPP-1b PP is shown.
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Fig. 3. (Panels A and B): Graphic representation of relative BMPP-1a (A) and BMPP-1b (B) transcript levels measured at days 2 (black bars: -) and day 6 (grey bars: ,) of last instar silkworm larvae in different tissues; foregut (Fg), midgut (Mg), hindgut (Hg), gonads (Gon), head, ventral nerve cord (VNC), silk glands (SG), trachea (Trach) and fat body (Fb). Samples were analyzed in triplicate and the assays were repeated twice with independent (biological) replicates. The data represent mean values S.D., normalized relative to the endogenous control.
3.5. In vitro activity of produced Bombyx PI In addition to the produced pacifastin-like inhibitors (BMPP-C and BMPP-N), the inhibitory activity of a chemical inhibitor AEBSF was determined in parallel. AEBSF binds irreversibly to all serine peptidases allowing for a relative estimation of serine-type peptidase activity in samples (Fig. 5). To compare different enzyme
batches, assays were performed under standardized conditions, including identical incubation and temperature conditions and the use of enzyme dilutions with identical peptidase activities (DAbs405nm/min ¼ 0.30). The chemical inhibitor AEBSF is a strong inhibitor (>95%) of both types of serine peptidases (bovine trypsin and chymotrypsin). The Bombyx inhibitors BMPP-N and BMPP-C are potent inhibitors of both types of mammalian serine peptidases, with a slight preference for trypsin. In addition, the inhibitory activity and specificity of the produced PI was tested on midgut extracts of B. mori. All experiments were repeated at least twice (n 3) with independent midgut extracts. An extract of five Bombyx midguts was incubated with the two pacifastin-like PI and AEBSF. Based on the inhibitory activity of AEBSF the digestive enzymes in the midgut of Bombyx larvae are 90% serine peptidases. In contrast to the strong inhibition of mammalian serine peptidases, the inhibitory activity of the Bombyx PI (BMPP-C and BMPP-N) towards Bombyx digestive enzymes is weak (Fig. 5). The inhibitory activity of Bombyx pacifastin-like PI on the secreted peptidases of an entomopathogenic fungus, B. bassiana, was evaluated. According to molecular mass, two putative pathogen related enzymes (Pr) were identified. The 33 kDa band is likely to be a subtilisin-like peptidase (Pr1) while the second visualized protein-band (23 kDa) might be a trypsin-like peptidase (Pr2). The Pr1 (33 kDa) band is more pronounced than the Pr2 band (Fig. 6). In order to evaluate the possible role of pacifastin-like PI as inhibitors of these fungal cuticle degrading peptidases, their inhibitory activity on a B. bassiana extract was tested (Fig. 7). Only 40% of the peptidase activity of the extract can be assigned to serine peptidases, while no or very few cysteine peptidases are present. The produced BMPP-C and BMPP-N inhibit approximately 25% and 10% of this serine peptidase activity, respectively (Fig. 7).
4. Discussion 4.1. Domain and gene structure of BMPP-1
Fig. 4. SDS-PAGE gel of purified fusion proteins stained with Brilliant Blue under reducing conductions. In lane I, a molecular marker is shown (kDa). MBP-BMPP-N (0.1 mg; lane IV) and MBP-BMPP-C (0.1 mg; lane III) were compared to MBP (1.5 mg; lane II).
Although it is not uncommon that a PP contains multiple PLD-like domains, BMPP-1 is exceptional with as many as thirteen PLDs. Previous studies have shown that the enzyme specificity of locust PLD-like inhibitor peptides is mainly determined by the residue at
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Fig. 5. Effects of BMPI on bovine trypsin (in grey, 25 mg/ml), chymotrypsin (in black, 25 mg/ml) and on the digestive midgut peptidases of B. mori. For each PI the average inhibitory activity is presented (n ¼ 4) S.D.
the P1-position [nomenclature (Schechter and Berger, 1967)] (Boigegrain et al., 1992; Kellenberger et al., 1995; Hamdaoui et al., 1998; Malik et al., 1999; Roussel et al., 2001; Gaspari et al., 2002; Simonet et al., 2005). The P1 residue of chymotrypsin inhibitors corresponds to an aromatic and/or a bulky amino acid (Tyr, Phe, Leu
Fig. 6. SDS-PAGE gel of B. bassiana extract (1.95 mg) under reducing conductions and silver-stained. Two bands of different molecular mass correspond to proteins of 33 kDa and 23 kDa.
and Met), whereas trypsin inhibitors preferentially possess a positively charged residue (Arg and Lys) at their P1-position. Based on structural homology with pacifastin-related inhibitors from locusts, we can localize the reactive P1–P10 site of the Bombyx inhibitors between the last two C-terminal Cys residues. Accordingly, BMPD-2, -12 and -13 contain a Leu at the P1-position, suggesting that these domains will inhibit chymotrypsin-like enzymes. BMPD-4, -6 and -8–11 on the other hand are expected to be inhibitors of trypsin-like peptidases according to an Arg or Lys at their P1-position. The P1 location in BMPD-1 on the other hand is uncertain as this PLD-like inhibitor contains 5 instead of 4 residues between the last two Cys residues. Noteworthy is the ‘‘unusual’’ Asp (D) residue at the P1-position of the BMPI-3, -5 and -7 sequences. This ‘‘acidic’’ residue is also present in a locust pacifastin-like inhibitor (SGPI-7) but its inhibitory specificity is currently unknown. According to the presence of 8 putative dibasic cleavage sites, full processing of BMPP-1 would result in 8 different pacifastinrelated PI, with the number of PLDs varying between 1 and 3 (Fig. 1). The exact cleavage pattern of BMPP-1 into smaller inhibitors and the physiological relevance remains uncertain. However, based on the detection of several single domain PLD-like inhibitors in locust haemolymph and tissues (Hamdaoui et al., 1998), the processing of insects PPs suggests multiple functions for insect pacifastin members. Analysis of the bmpp-1 gene structure showed that this gene is composed of not less than seventeen exons. The first exon encodes the signal peptide and three exons correspond to very short ‘linker’ regions of which the function remains unclear. The long transcript (BMPP-1b) possesses two additional exons coding for the inhibitor domains BMPD-9 and BMPD-10. Remarkably, the thirteen PLDrelated domains are each encoded by a single exon, suggesting rapid exon duplication. In the one agpp-1 pacifastin gene of the mosquito Anopheles gambiae (Simonet et al., 2003a; Breugelmans et al., 2008b), the third exon encodes three PLD-like sequences, suggesting the occurrence of exon duplication followed by in-frame exon fusion. Based on the expression of five multidomain PPs in the locusts, S. gregaria, not only exon duplication, but also gene duplication seems to have occurred. It is obvious that analysis of other insect pacifastin gene structures is needed to study evolutionary aspects of the pacifastin gene family in detail. To avoid confusion we suggest the use of a ‘consensus’ terminology based on the previously
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Fig. 7. Inhibitory activity (percentage) of PI on the secreted peptidases of B. beauveria. Azocasein assay (AZO) of the recombinant produced pacifastin-like PI, AEBSF and Cystatin.
suggested nomenclature for the related precursors and inhibitor domains of this family (Simonet et al., 2002a), in which the pacifastin-related precursor encoding genes will be referred to as ‘‘gsppn’’ (‘‘genus species pacifastin-related precursor gene–number’’). 4.2. Tissue distribution of BMPP-1 transcripts in the silkworm Contrary to the well-studied expression profiles of locust pacifastin-related transcripts, no data are available on the expression of pacifastin-related transcripts in other insect species. Therefore, we analyzed the transcript levels of the BMPP-1 transcripts in different tissues of last instar larvae of the silkworm, B. mori. Comparing the quantitative RT-PCR data of both transcripts in different tissues reveals a different expression pattern. The high expression of the Bombyx BMPP-1b transcript in the head supports the idea of a possible function of pacifastin-related inhibitor peptides as regulators of neurological processes. In addition, a pacifastinrelated transcript (SGPP-2) and the encoded peptide SGPI-3 have previously been detected, respectively, in the brain and corpora cardiaca of the desert locust (Clynen et al., 2002; Simonet et al., 2004a). The shorter BMPP-1a transcript on the contrary is more equally expressed in several tissues. The broad tissue distribution of PP-transcripts has also been observed in locusts, supporting the idea that pacifastin-related peptides have multiple functions as regulators of different serine peptidase-dependent processes in insects. Like in the desert locust, BMPP pacifastin-related transcripts were detected in the fat body, foregut, hindgut and gonads. Contrary to locusts however, the Bombyx pacifastin-related transcripts are also expressed in midgut tissue. Furthermore, Bombyx pacifastin-like inhibitors are only weak inhibitors of endogenous midgut peptidases, whereas locust SGPI have been shown to be strong inhibitors of locust midgut enzymes (Boigegrain et al., 1992, 2000; Kellenberger et al., 1995, 2003; Malik et al., 1999; Patthy et al., 2002; Simonet et al., 2003b, 2005). Thus, despite the presence of BMPP transcripts in midgut tissue, a preventive function against leakage of endogenous digestive enzymes, as suggested previously (Eguchi, 1993), does not seem very likely. BMPP-1a and BMPP-1b transcripts were quantified at two different time points of last instar larvae; at day 2 these caterpillars are active and eating, while at day 6 they tend to stop eating, are less active and prepare for pupation. However, no significant differential expression was found between the two days, except for
a small difference in BMPP-1a expression in the hindgut and BMPP1b expression in the trachea, suggesting BMPP-1 is not involved with metabolic mechanisms in the silkworm larvae. The available genomic data and the detection of only two pacifastin-related precursor transcripts in the silkworm (compared to at least five transcripts in the desert locust) make this caterpillar an excellent candidate for future functional genomics research. 4.3. Recombinant production of Bombyx PI In order to produce sufficient amounts of biologically active PI, a pMAL Protein Fusion and Purification System (New England Biolabs) was optimized. The pMAL expression system allows for the production of a gene product of interest (pacifastin-related peptides) attached to a fusion protein in bacteria (E. coli). In addition to the already available expression constructs for SGPI-1, SGPI2, SGPI-3 (Simonet et al., 2003b) new constructs were made for the N- and C-terminal regions of the Bombyx BMPP-1 precursor, referred to as BMPP-N and BMPP-C. To increase the productivity of this expression system, a few specific changes were made to the protocol described by Simonet et al. (2003a,b). First, the TB-growth medium was replaced by the Overnight Express Instant TB Medium. Secondly, compared to the previously single inoculation, multi-staging was performed by means of 5% (v/v) inoculums in order to grow large cell culture volumes. Finally, the extraction procedure was improved by using a mild lysis buffer (BugBuster Protein Extraction Reagent, Novagen). Using this optimized procedure, a yield of 15–35 mg fusion protein per l bacterial culture was realised, this is a 15-fold increase compared to the procedure of Simonet et al. (2003a,b). 4.4. In vitro activity of produced Bombyx PI As no insect digestive enzymes are commercially available, bovine trypsin and chymotrypsin were used to test the inhibitory activity and specificity of the recombinant pacifastin-like inhibitors. Previous studies have shown that pacifastin-like inhibitors of trypsin-like peptidases according to their P1 residue (SGPI-1 and SGPI-5A) are weak inhibitors of bovine trypsin, while SGPI-2 and SGPI-3 are characterized as potent inhibitors of bovine chymotrypsin (Simonet et al., 2003b, 2004b).
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The inhibitory activity of a chemical inhibitor AEBSF and of the produced Bombyx pacifastin-like inhibitors was determined. As expected, AEBSF inhibits both bovine trypsin and chymotrypsin activity (95–98%). In addition, both produced Bombyx inhibitors can inhibit both types of mammalian serine peptidases (50–80%). The Bombyx inhibitor BMPP-C is composed of five PLDs (BMPD-9–13), two of which are predicted to possess trypsin specificity and three with chymotrypsin specificity (Fig. 1). BMPP-N is composed of four PLDs (BMPD-1–4) of which two (BMPD-1 and BMPD-3) have unknown specificity. Nevertheless, the measured serine peptidase inhibitory activity of BMPP-N can be assigned to the remaining two inhibitor domains (BMPD-2 and BMPD-4) that are predicted to possess trypsin and chymotrypsin specificity, respectively. In this experiment, recombinant Bombyx fusion proteins were used and therefore, since these may not necessarily exhibit an inhibitory activity identical to the natural protein, some caution is needed. The fact that SGPI-2 is a potent inhibitor of chymotrypsin is not contradictory to the present observation that the Bombyx fusion proteins have a relatively lower affinity for mammalian enzymes, nor is it an indication for a partial activity. In fact, several other pacifastin-like PI with confirmed folding (e.g. SGPI-1, LMPI-1, HI, .) were shown to be rather weak inhibitors of mammalian enzymes and some of these PI were tested in parallel with the Bombyx fusion proteins (thus under identical conditions), showing a very similar percentage of inhibition (typically between 50 and 80%). Despite their lower activity than SGPI-2 towards bovine chymotrypsin, one can however expect that these inhibitors are more potent inhibitors for (still unknown) endogenous target enzyme(s). Confirming to the alkaline pH in the lumen of lepidopteran midguts, the enzyme activity in the silkworm midgut is mainly the result of serine peptidases, since members of this peptidase family are distinguished by a neutral to alkaline pH optimum. In contrast to the strong inhibition of mammalian serine peptidases, the inhibitory activity of the Bombyx PI (BMPP-C and BMPP-N) towards Bombyx digestive enzymes is relatively weak regardless of their multiple inhibitor domains. Despite this weak inhibition, the combination of trypsin and chymotrypsin specific PLDs in BMPP-C and BMPP-N results in an inhibitory activity towards both types of serine peptidases (data not shown). In line with the differences in (mid)gut pH between mammals and caterpillars, the biochemical properties of the digestive peptidases might differ as well and influence the binding efficiency of the pacifastin-like PI. Compared to the species selectivity of locust core type I PLDs towards mammalian trypsin, the multidomain BMPIs are potent inhibitors of mammalian trypsin. This ability of BMPP-N and BMPP-C to inhibit mammalian trypsins might be the result of structural differences (i.e. core type interactions) compared to the locust core type I inhibitors. Based on these observations, it can be assumed that the digestive enzymes are not the endogenous target of pacifastin-like inhibitors in the silkworm. Therefore, despite the presence of BMPP transcripts in the midgut tissue, a protective function against leakage of digestive enzymes into the haemocoel is highly unlikely. The first barrier an entomopathogenic fungus has to overcome to invade the insect’s haemocoel is a protein and chitin containing solid matrix called the cuticle. The secretion of cuticle degrading peptidases is shown to be an important mechanism of entomopathogenic fungi to invade their host (Stleger, 1995). Hence, the presence of pacifastin-like inhibitors in hemolymph, trachea (when inhaled) and/or foregut and hindgut tissue (when ingested) and other tissues (i.e. fat body) might be a first line of defence against fungal peptidases. Therefore, we investigated the inhibitory activity of pacifastin-like PI on the secreted peptidases of an entomopathogenic fungus, B. bassiana. The extracted B. bassiana peptidases were separated on SDS-PAGE and according to molecular mass two
putative pathogen related enzymes (Pr) were identified. The subtilisin-like enzyme of B. bassiana (Pr1) has been identified (accession number: gb/AAC48979.1) (Joshi et al., 1995) as a protein with a (theoretical) molecular mass of 33 kDa, corresponding to the mass observed by SDS-PAGE (Fig. 4). This peptidase resembles the Pr1 of another entomopathogenic fungus, Metarhizium anisopliae, which is proven a potent cuticle degrading peptidase that is secreted during insect invasion (St Leger et al., 1992). Combined with the fact that (i) only secreted proteins were collected, (ii) this relatively pure extract displays proteolytic activity towards serine peptidases and (iii) chitinases can be excluded as they have a higher molecular mass, we are confident that the band of 33 kDa is indeed the subtilisin-like enzyme (Pr1). The trypsin-like enzyme (Pr2) on the other hand has not been identified in B. bassiana and a certain caution is needed. However, in a related fungus, M. anisopliae, the sequence of a Pr2 trypsin-like enzyme has been found and its proteolytic activity has been proven (accession number: embjCAA55477.1) (Smithson et al., 1995). Since the calculated molecular mass (23,020 kDa) coincides with the mass of the second band on SDS-PAGE (Fig. 4), the second band is likely to represent a trypsin-like (Pr2) fungal enzyme. It has been suggested that Pr1 and Pr2 are part of a cascade of peptidases that facilitates the degradation of host cuticle (St Leger et al., 1992). Pr2, which has been shown to be secreted in lower amounts than Pr1 by Donatti et al. (2008), is thought to complement the Pr1 subtilisinlike enzymes by opening up the proteins for further hydrolysis, assisting in penetration and providing peptides which can be utilized as nutrients (St Leger et al., 1992). In order to evaluate the possible role of pacifastin-like PI as inhibitors on these fungal cuticle degrading peptidases, their inhibitory activity on a B. bassiana extract was tested. Assuming that AEBSF inhibits 100% of the serine peptidase activity present in the fungal extract, BMPP-C was able to inhibit approximately 25% of all serine peptidase activity, while BMPP-N is a significantly weaker inhibitor of fungal peptidases. Based on (i) the observed higher level of secreted subtilisin-like enzymes (Pr1) compared to trypsin-like enzymes (Pr2) (Donatti et al., 2008) and (ii) the fact that none of the nine Bombyx PLDs possesses a P1 residue with a predicted affinity for subtilisin-like enzymes, the first three domains of BMPP-C might inhibit a relatively large fraction of the trypsin-like activity present in the fungal extract. Consequently, it cannot be excluded that pacifastin-like PI protect the organism against the enzymes secreted by fungal pathogens in vivo. However, in vitro analysis of the inhibitory activity of pacifastin-like inhibitors on purified Pr1 and Pr2 fungal enzymes will be needed to confirm these results. Acknowledgements The authors especially thank J. Puttemans and M. Chistriaens for technical assistance and Dr. B. Landuyt for the MALDI-TOF analysis. The authors gratefully acknowledge the IWT, the K.U. Leuven Research Foundation (GOA 2005/06), the Belgian Interuniversity Attraction Poles Programme (IUAP/PAI P6/14, Belgian Science Policy) and the FWO-Vlaanderen for financial support. Appendix. Supplementary information Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ibmb.2009.03.005. References Bloch, G., Robinson, G.E., 2001. Chronobiology – reversal of honeybee behavioural rhythms. Nature 410, 1048. Boigegrain, R.A., Mattras, H., Brehelin, M., Paroutaud, P., Coletti-Previero, M.A., 1992. Insect immunity: two proteinase inhibitors from hemolymph of Locusta migratoria. Biochem. Biophys. Res. Commun. 189, 790–793.
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