Comparative Biochemistry and Physiology, Part B 167 (2014) 59–64

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A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor Hu Wan a,1, Bo Yeon Kim a,1, Kwang Sik Lee a, Hyung Joo Yoon b, Kyung Yong Lee b, Byung Rae Jin a,⁎ a b

College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea Department of Agricultural Biology, National Academy of Agricultural Science, Suwon, Republic of Korea

a r t i c l e

i n f o

Article history: Received 12 August 2013 Received in revised form 14 October 2013 Accepted 14 October 2013 Available online 21 October 2013 Keywords: Antimicrobial activity Bumblebee Bombus ignitus Serine protease inhibitor Venom

a b s t r a c t Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki = 6.57 nM) and proteinase K (Ki = 7.11 nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Serine protease inhibitors are found in a wide variety of organisms, including insects and other arthropods. These inhibitors, which exhibit a disulfide-rich α/β fold structure and a P1 site, have been shown to inhibit trypsin and chymotrypsin (Laskowski and Kato, 1980). In arthropods, serine protease inhibitors play multiple roles in development and immunity (Kanost, 1999; Ligoxygakis et al., 2003; An and Kanost, 2010; Reichhart et al., 2011; Zhao et al., 2012). Mounting data indicate that serine protease inhibitors from arthropods can also target plasmin, elastases, and microbial serine proteases, in addition to trypsin and chymotrypsin (Choo et al., 2012; Kim et al., 2013a,b; Qiu et al., 2013; Wan et al., 2013a,b). Our previous studies have shown that a Kunitztype serine protease inhibitor in spiders can inhibit trypsin, chymotrypsin, plasmin, and elastase (Wan et al., 2013a), while a spider chymotrypsin inhibitor can target elastases and microbial serine proteases, such as subtilisin A and proteinase K (Wan et al., 2013b). In honeybees, chymotrypsin inhibitors have been reported to inhibit cathepsin G (Bania et al., 1999; Cierpicki et al., 2000) and elastase (Kim et al., 2013a). Furthermore, our recent study revealed that a Kazal-type serine protease inhibitor from honeybee venom not only inhibits subtilisin A and proteinase K but also exhibits antimicrobial activity against Gram-positive bacteria and fungi (Kim et al., 2013b). Similarly, several other serine protease inhibitors have been shown to exhibit ⁎ Corresponding author. Tel./fax: +82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 1 These authors contributed equally to this study. 1096-4959/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpb.2013.10.002

antimicrobial activity (Han et al., 2008; Augustin et al., 2009; Donpudsa et al., 2009; Li et al., 2009). Together with honeybees, bumblebees are the most abundant bee species in the world, and they are widely used in greenhouses to pollinate crops (Velthuis and van Doorn, 2006). We have previously shown that, similar to honeybee venom, bumblebee venom also contains Kunitztype serine protease inhibitors against trypsin and plasmin (Choo et al., 2012; Qiu et al., 2013); however, the functional roles of chymotrypsin inhibitors in bumblebee venom have not been elucidated. Moreover, the antimicrobial activity of serine protease inhibitors in bumblebee venom also has not been demonstrated. In this study, we discovered a novel serine protease inhibitor, BiVSPI, with antimicrobial activity in bumblebee (Bombus ignitus) venom. Here we describe the molecular characterization of this inhibitor and its activity against chymotrypsin, subtilisin A, and proteinase K. Furthermore, we describe the antimicrobial activity of BiVSPI against Gram-positive bacteria and entomopathogenic fungi.

2. Materials and methods 2.1. Bumblebees The B. ignitus (Hymenoptera: Apidae) bumblebee workers were supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea. The bumblebees were maintained at 28°C with 65% humidity in constant darkness, as described previously (Yoon et al., 2009).

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2.2. Gene cloning and sequence analysis A clone encoding BiVSPI was selected from a set of expressed sequence tags (ESTs) from a cDNA library that was constructed using whole bodies of B. ignitus (Choo et al., 2010b). Plasmid DNA was extracted using a Wizard Mini-Preparation kit (Promega, Madison, WI, USA). The cDNA sequence was analyzed using an ABI310 automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Sequenced cDNA was compared using the DNASIS and BLAST databanks (http://www.ncbi.nlm.nih.gov/BLAST). The signal peptide sequence was predicted by SignalP 4.0 software (http://www.cbs.dtu. dk/services/SignalP) (Nielsen et al., 1997). MacVector (ver. 6.5, Oxford Molecular Ltd., Oxford, UK) was used to align the predicted amino acid sequences of the serine protease inhibitor genes. Genomic DNA was extracted from the fat body tissue of a single B. ignitus worker bee using the Wizard Genomic DNA Purification Kit (Promega) and was subsequently used as a template for PCR. The following sequences of oligonucleotide primers were used for amplification: forward (1–21) 5′-ATGTCTCGTATCCTCTTTGTC-3′ and reverse (228–206) 5′-TTAACA CTTTTCTGGAAGAACGC-3′. The amplification primers were designed using the BiVSPI cDNA sequence. All the PCR products were verified by DNA sequencing. 2.3. RNA extraction and Northern blot analysis Total RNA was isolated from five different tissues (epidermis, muscle, midgut, fat body, and venom gland) of B. ignitus worker bees using a Total RNA Extraction Kit (Promega). Total RNA (5 μg/lane) was separated on a 1.0% formaldehyde agarose gel and then transferred onto a nylon blotting membrane (Schleicher & Schuell, Dassel, Germany). Hybridization was performed at 42 °C with the appropriate probe that was diluted in a hybridization buffer containing 5× SSC (0.75 M sodium chloride and 0.75 M sodium citrate), 5× Denhardt's solution (0.1% each of bovine serum albumin (BSA), Ficoll, and polyvinylpyrrolidone), 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA. BiVSPI cDNA was labeled with [α-32P] dCTP (Amersham Biosciences, Piscataway, NJ, USA) using the Prime-It II Random Primer Labeling kit (Stratagene, La Jolla, CA, USA), and labeled cDNA was used as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2× SSC at 65 °C and then exposed to autoradiography films. 2.4. Protein expression and purification Recombinant BiVSPI was produced in the Spodoptera frugiperda (Sf9) insect cell line using the Autographa californica nucleopolyhedrovirus (AcNPV) expression system as previously reported (Choo et al., 2012; Qiu et al., 2013). BiVSPI cDNA was PCR-amplified from pBluescript-BiVSPI using the forward primer 5′-GGATCCATGTCTCGTATCCTCTTTG-3′ and the reverse primer 5′-CTCGAGTTAATGATGATGATGATGATGTTAACACTT TTCTGGAAGAAC-3′. The reverse primer was engineered to include the His-tag sequence. The PCR cycling conditions were as follows: 94 °C for 3 min, 30 cycles of amplification (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min), and 72 °C for 5 min. PCR products were sequenced using the BigDye Terminator Cycle Sequencing Kit and an automated DNA sequencer (Perkin-Elmer Applied Biosystems). The isolated BiVSPI fragment was inserted into the pBacPAK8 vector (Clontech, Palo Alto, CA, USA) to generate an expression vector under the control of the AcNPV polyhedrin promoter. For expression experiments, 500 ng of the construct (pBacPAK8-BiVSPI) and 100 ng of AcNPV viral DNA (Je et al., 2001) were co-transfected into 1.0–1.5 × 106 Sf9 cells for 5 h using the Lipofectin transfection reagent (Gibco BRL, Gaithersburg, MD, USA). Transfected cells were cultured at 27 °C for 5 days in a TC100 medium (Gibco BRL), supplemented with 10% fetal bovine serum (FBS, Gibco BRL). Recombinant baculoviruses were propagated in Sf9 cells that were cultured in TC100 medium at 27 °C. The recombinant proteins

were purified using the MagneHis™ Protein Purification System (Promega). Recombinant BiVSPI was identified by 14% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. A polyclonal antibody against BiVSPI was then produced in BALB/c mice using the recombinant protein (Qiu et al., 2011), and antibody activity was tested by Western blot. All Western blot analyses were performed using an enhanced chemiluminescence Western blotting system (Amersham Biosciences) with anti-His or anti-BiVSPI primary antibodies and horseradish peroxidase-conjugated anti-mouse IgG secondary antibodies at 1:5000 (v/v) dilution. Protein concentrations were determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). 2.5. Serine protease inhibition assay Serine protease inhibition assays were performed as previously described (Choo et al., 2012; Kim et al., 2013a,b; Qiu et al., 2013; Wan et al., 2013a,b). Briefly, 20 nM bovine trypsin (Sigma-Aldrich, St. Louis, MO, USA), 20 nM bovine α-chymotrypsin (Sigma), 20 nM subtilisin A from Bacillus licheniformis (Sigma), or 20 nM proteinase K from Engyodontium album (Sigma) was incubated in 100 mM Tris–HCl (pH 8.0) containing 20 mM CaCl2 and 0.05% Triton X-100 with increasing amounts of BiVSPI at 37 °C for 30 min. The residual enzyme activity was determined at 405 nm or 410 nm using the following substrates: 0.5 mM Nα-benzoyl-DL-arginine p-nitroanilide-hydrochloride (BApNA, Sigma) for trypsin and 0.5 mM succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, Sigma) for α-chymotrypsin, subtilisin A, and proteinase K. To assess the antifibrinolytic and anti-elastolytic activity of BiVSPI, 20 nM porcine pancreatic elastase (Sigma), human plasmin (Sigma), human thrombin (Sigma), human tissue plasminogen activator (tPA; Sigma), or bovine factor Xa (Novagen, Darmstadt, Germany) was incubated with increasing amounts of BiVSPI at 37 °C for 30 min in a 50 mM Tris–HCl buffer (pH 7.4). The residual enzymatic activity was determined at 405 nm using 0.5 mM of the substrate S4760 (Sigma) for elastase, S-2238 (Chromogenix, Mölndal, Sweden) for thrombin, S-2251 (Chromogenix) for plasmin, S-2288 (Chromogenix) for tPA, or S2222 (Chromogenix) for factor Xa (Choo et al., 2010b, 2012; Kim et al., 2013a,b; Qiu et al., 2013; Wan et al., 2013a,b). The initial reaction rate was determined by calculating the slope of the linear portion of the kinetic curve. An inhibitory effect was expressed as the percent reduction of the initial hydrolysis rate. The reaction rate in the absence of inhibitor was defined as 100%, and the inhibitor concentration that decreased the rate of hydrolysis by 50% (IC50) was also determined. The value of the inhibition constant (Ki) was calculated using the equation Ki = IC50 / (1 + S/Km) (Sinauridze et al., 2011). 2.6. Microbial binding assay Microbial binding assays were performed as previously described (You et al., 2010; Kim et al., 2013b). Bacillus subtilis, Bacillus thuringiensis, and Escherichia coli were grown in a Luria-Bertani medium, and Beauveria bassiana was grown in potato dextrose broth. When the cultures reached an OD600 of 0.4, 4 ml of each bacterial or fungal culture was harvested, washed in PBS, and resuspended in 40 μl of recombinant BiVSPI (0.8 μg). Following incubation at room temperature for 10 min, the suspensions were centrifuged, and the pellets were washed and resuspended in 40 μl of PBS. Samples of the pellets and supernatants were subjected to 15% SDS-PAGE and Western blot analysis using an antiserum probe against the 6× His tag as described above. 2.7. Immunofluorescence staining As described for the microbial binding assay, B. thuringiensis, B. subtilis, E. coli, and B. bassiana were harvested, washed three times with PBS, and resuspended in 40 μl of recombinant BiVSPI (0.8 μg). After a 10 min incubation at room temperature, the bacteria and fungi

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Fig. 1. Cloning of the BiVSPI gene. (A) The nucleotide sequence of BiVSPI cDNA and the deduced amino acid sequence (GenBank accession no. KF500100). The start codon (ATG) is boxed, and the stop codon is indicated with an asterisk. The putative polyadenylation signal is double-underlined, and the predicted signal sequence is underlined. (B) The genomic structure of the BiVSPI gene (GenBank accession no. KF500101), which was inferred from an analysis of the BiVSPI cDNA, is shown, and the numbers indicate the location in the genomic sequence. (C) The alignment of the amino acid sequences between BiVSPI and other known serine protease inhibitors is shown, and the characteristic cysteine residues are indicated by solid circles. The P1 position is marked with an asterisk. The sources of the aligned sequences were B. ignitus (this study, GenBank accession no. KF500100), B. terrestris (AFX62368), B. impatiens (XP_003484766), Megachile rotundata (XP_003708657), Apis florea (XP_003696076), A. mellifera (XP_001120243), and A. cerana (JX899417). The BiVSPI sequence was used as a reference for the identity/similarity (Id/Si) values.

were fixed in acetone (−20°C) for 2min and air-dried. The bacteria and fungi were washed three times in PBS and then pre-incubated in PBS containing 2% BSA at room temperature for 20 min. After another wash with PBS, the bacteria and fungi were incubated for 1 h with anti-His tag mouse polyclonal antiserum that was diluted 1:500 (v/v) in PBS containing 1% BSA. The bacteria and fungi were washed twice with PBS for 10 min each and then incubated for 1 h with fluorescein-

conjugated goat anti-mouse secondary antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) that was diluted 1:400 (v/v) in PBS containing 1% BSA. After five successive washes in PBS for 25 min each, the bacteria and fungi were wet-mounted. BiVSPI localization within bacterial and fungal cells was visualized using laser scanning confocal microscopy (Carl Zeiss LSM 510, Zeiss) as previously described (You et al., 2010; Kim et al., 2013b).

Fig. 2. Expression of BiVSPI. (A) The expression of BiVSPI in B. ignitus worker bees is shown. Total RNA was isolated from the epidermis, muscle, midgut, fat body, and venom gland of B. ignitus worker bees. RNA was separated by 1.0% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with radiolabeled BiVSPI cDNA (lower panel). BiVSPI transcripts are indicated with an arrow. The ethidium bromide-stained RNA gel shows uniform loading (upper panel). (B) SDS-PAGE (left) and Western blot analysis (right) of purified recombinant BiVSPI that was expressed in baculovirus-infected Sf9 insect cells. Recombinant BiVSPI was identified using an anti-His-tag antibody. (C) Western blot detection of BiVSPI in the venom gland and venom of B. ignitus worker bees.

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protease inhibitor genes. We found one EST sequence that contains the full-length sequence of a candidate gene, which we named BiVSPI (GenBank accession number KF500100). The BiVSPI gene consists of three exons encoding 75 amino acids (Fig. 1A and B). The signal peptide sequence was determined using the SignalP program. The BiVSPI protein contains a 20-amino acid signal peptide and a 55-amino acid mature peptide (Fig. 1A). An analysis of the predicted BiVSPI amino acid sequence revealed similarities to members of other bee serine protease inhibitor families, including ten conserved cysteine residues and a methionine at position P1 (Fig. 1C). These features suggested that BiVSPI might be structurally and functionally similar to other chymotrypsin inhibitors. To confirm that BiVSPI is a B. ignitus-derived serine protease inhibitor, we examined the expression pattern of BiVSPI in B. ignitus worker bees. Northern blot analyses were performed using total RNA from the epidermis, muscle, midgut, fat body, and venom gland of B. ignitus. The results revealed that the BiVSPI gene is constitutively expressed in the venom gland and fat body (Fig. 2A). To further characterize BiVSPI, we expressed recombinant BiVSPI with a 6× His tag as an 8 kDa peptide in baculovirus-infected insect cells (Fig. 2B). We then examined the expression pattern of BiVSPI to confirm that it is a component of B. ignitus venom. Western blot analyses using the BiVSPI polyclonal antibody demonstrated the presence of BiVSPI in both the venom gland and the venom (Fig. 2C), suggesting that BiVSPI is a serine protease inhibitor that is expressed in the venom gland and stored in the venom sac of B. ignitus. 3.2. BiVSPI inhibits microbial serine proteases Fig. 3. Enzyme inhibition by BiVSPI. (A) Trypsin or chymotrypsin was incubated with increasing amounts of BiVSPI, and the residual enzymatic activity was then determined (n = 3). (B) The inhibitory activity of BiVSPI against microbial serine proteases is shown. Subtilisin A or proteinase K was incubated with increasing amounts of BiVSPI, and the residual enzyme activity was determined (n = 3).

2.8. Antimicrobial activity assay The antimicrobial activity of purified recombinant BiVSPI was assayed as previously described (Choo et al., 2010a; You et al., 2010; Kim et al., 2013b) against the Gram-positive bacteria B. thuringiensis and B. subtilis and the Gram-negative bacterium E. coli using a liquid growth inhibition assay. A total of 200 μl of inoculum (105 cfu/ml) was added to each well of a 96-well plate containing serial dilutions of recombinant BiVSPI (equal volumes of PBS were used as a control). The 96-well plates were incubated at 37 °C for 24 h with shaking at 220 rpm. Bacterial growth inhibition was determined by measuring the absorbance at 595 nm. The results were expressed as the mean values from three independent replicates. The minimal inhibitory concentration (MIC) for the antibacterial assay was defined as the lowest concentration that caused a 50% inhibition of bacterial growth (Choo et al., 2010a). Recombinant BiVSPI was also tested for antifungal activity against the fungus B. bassiana using a liquid growth inhibition assay. A total of 200 μL of inoculum (2 × 104 conidia/ml) was added to each well of a 96-well plate containing serial dilutions of BiVSPI (or an equal volume of PBS as a control). The 96-well plates were incubated at 22 °C for 48 h with shaking at 220 rpm. Fungal growth inhibition was determined by measuring the absorbance at 595 nm, and the results were expressed as the mean values of three independent replicates. The IC50 values for antifungal activity are expressed as the concentration of BiVSPI required to inhibit fungal growth by 50% (Choo et al., 2010a).

We first investigated the inhibitory effects of recombinant BiVSPI against trypsin and chymotrypsin. BiVSPI inhibited chymotrypsin with an IC50 of 19.56 nM and a Ki of 15.24 nM, but it did not exhibit any inhibitory activity against trypsin (Fig. 3A and Table 1). Antifibrinolytic and anti-elastolytic activities have been documented for several other serine protease inhibitors (Choo et al., 2012; Kim et al., 2013a; Qiu et al., 2013). However, we found that BiVSPI had no detectable effects on the activity of plasmin, tPA, thrombin, factor Xa, or elastase (data not shown), indicating that BiVSPI may not be an antifibrinolytic or anti-elastolytic factor. We then demonstrated that BiVSPI also acts as an inhibitor of microbial serine proteases. BiVSPI blocked the activity of both subtilisin A (IC50 =6.57 nM; Ki = 6.83nM) and proteinase K (IC50 =7.11 nM; Ki = 7.02 nM) (Fig. 3B and Table 1). 3.3. BiVSPI exhibits antimicrobial activity Our last finding suggested that BiVSPI may also exhibit antimicrobial activity. Therefore, we next examined the microbial binding ability of BiVSPI. Western blot analyses revealed that BiVSPI could bind to the Gram-positive bacteria B. subtilis and B. thuringiensis as well as the entomopathogenic fungus B. bassiana but not to the Gramnegative bacterium E. coli (Fig. 4A). Consistent with these data, immunofluorescence staining revealed that BiVSPI was localized to the cell walls of B. subtilis, B. thuringiensis, and B. bassiana (Fig. 4B). We next assessed whether the binding of BiVSPI to live bacterial and fungal cells was correlated with growth inhibition. To address this issue, Table 1 The inhibitory activities of BiVSPI against chymotrypsin and microbial serine proteases.

3. Results

Enzyme

Concentration (nM)a

IC50 (nM)

Ratiob

Ki (nM)

3.1. BiVSPI is a serine protease inhibitor in bumblebee venom

Chymotrypsin Subtilisin A Proteinase K

20 20 20

19.56 6.57 7.11

0.98 0.33 0.35

15.24 6.83 7.02

To identify novel serine protease inhibitors in bumblebee venom, we searched a B. ignites cDNA library for ESTs that potentially contain serine

a b

The concentration of enzyme in this experiment. The molar ratio of the IC50 and the enzyme concentration.

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Fig. 4. Microbial binding of recombinant BiVSPI. (A) Western blot analysis of BiVSPI microbial binding activity is shown. Live B. subtilis, B. thuringiensis, E. coli, or B. bassiana was incubated with BiVSPI for 10 min. Bound BiVSPI (P) was separated from free BiVSPI in the supernatant (S) via centrifugation. The samples were analyzed by Western blot with an anti-His antibody. (B) Immunofluorescence staining was performed to visualize the binding of BiVSPI to live bacterial and fungal cells. B. subtilis, B. thuringiensis, E. coli, or B. bassiana were incubated with BiVSPI for 10 min. BiVSPI (green) was bound to B. subtilis, B. thuringiensis, and B. bassiana but not to E. coli cells. Merged confocal images are shown in the third column. The scale bar corresponds to 5 μm.

we determined the antimicrobial activity of BiVSPI against B. subtilis, B. thuringiensis, E. coli, and B. bassiana. Consistent with the microbial binding results, recombinant BiVSPI blocked the proliferation of B. subtilis (MIC50 = 29.45 μM), B. thuringiensis (MIC50 = 91.03 μM), and B. bassiana (IC50 = 30.09 μM) (Table 2). These results indicate that BiVSPI functions as an antimicrobial factor.

4. Discussion Several serine protease inhibitors in bees have been identified and characterized. For example, honeybee chymotrypsin inhibitors have been shown to block the activity of cathepsin G (Bania et al., 1999; Cierpicki et al., 2000) and elastase (Kim et al., 2013a). We recently described a Kazal-type serine protease inhibitor from honeybee venom that inhibits subtilisin A and proteinase K (Kim et al., 2013b). We have also identified Kunitz-type serine protease inhibitors in bumblebee

venom that inhibit trypsin and plasmin (Choo et al., 2012; Qiu et al., 2013). However, no chymotrypsin inhibitors have been functionally characterized in bumblebee venom. In this study, we identified the first chymotrypsin inhibitor in bumblebee venom that also acts as a microbial serine protease inhibitor. We found that BiVSPI exhibits many features Table 2 The antimicrobial activity of BiVSPI against bacteria and fungus. Microorganism

MIC50 (μM)

Gram-negative bacterium

B. subtilis B. thuringiensis E. coli

29.45 91.03 ND

Fungus

B. bassiana

30.09

Gram-positive bacteria

IC50 (μM)

ND, not detected.

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that are similar to those of other chymotrypsin inhibitors, including a cysteine-rich structure and a P1 residue (Bania et al., 1999; Cierpicki et al., 2000; Qiu et al., 2012; Kim et al., 2013a). Sequence analysis revealed that BiVSPI shares protein sequence identity with other bee chymotrypsin inhibitors in Bombus terrestris (98% protein sequence identity) and Bombus impatiens (97% protein sequence identity). Serine protease inhibitors possess a P1 site, which corresponds to the specificities of their cognate enzymes (Laskowski and Kato, 1980; Župunski et al., 2003; Yuan et al., 2008). Generally, chymotrypsin inhibitors possess a Leu, Met, Phe, Tyr, Trp, or Asn P1 residue (Laskowski and Kato, 1980; Cierpicki et al., 2000; Chang et al., 2001; Kim et al., 2013a,b). We found that BiVSPI possesses ten cysteine residues and a Met–Glu pair at the P1–P1´ residues, which are also present in the chymotrypsin inhibitors from B. terrestris and B. impatiens bumblebees. Taken together, these data suggest that BiVSPI is a member of the chymotrypsin inhibitor family. BiVSPI was found to be expressed in the venom gland and present in the venom, suggesting that it is a component of bee venom. We expressed recombinant BiVSPI (8kDa) in baculovirus-infected insect cells and found that it exhibits inhibitory activity against chymotrypsin but not trypsin. These data further confirmed that BiVSPI is a chymotrypsin inhibitor in bumblebee venom. In addition to the inhibition of trypsin and/or chymotrypsin, several serine protease inhibitors from insects have been shown to target plasmin, thrombin, elastase, subtilisin A, or proteinase K (Mende et al., 1999, 2004; Choo et al., 2012; Qiu et al., 2013; Kim et al., 2013a,b; Wan et al., 2013a,b). BiVSPI has no detectable effect on plasmin, factor Xa, thrombin, tPA, or elastase, but it does inhibit subtilisin A and proteinase K. Our findings are similar to those of a previous study in which a Kazal-type serine protease inhibitor from the venom of the honeybee Apis cerana also inhibited microbial serine proteases (Kim et al., 2013b). Collectively, our results demonstrate that BiVSPI acts as a microbial serine protease inhibitor. Some serine protease inhibitors that can block microbial serine proteases have also been reported to exhibit antimicrobial activity (Han et al., 2008; Augustin et al., 2009; Donpudsa et al., 2009; Li et al., 2009; Kim et al., 2013b; Wan et al., 2013b). The results of our binding assays and antimicrobial activity assays showed that BiVSPI is also an antimicrobial factor. BiVSPI was found to bind directly to live B. thuringiensis, B. subtilis, and B. bassiana but not to E. coli. In addition, the binding affinity of BiVSPI was inversely correlated with bacterial and fungal growth. These findings revealed that BiVSPI has both antibacterial activity against Gram-positive bacteria and antifungal activity against entomopathogenic fungi. However, the exact mechanisms of BiVSPI-mediated antimicrobial activity need to be further elucidated. In summary, we have shown that BiVSPI is a chymotrypsin inhibitor in bumblebee (B. ignitus) venom that acts as a microbial serine protease inhibitor. BiVSPI likely functions as an antimicrobial factor that displays antibacterial activity against Gram-positive bacteria and antifungal activity. Given that BiVSPI does not exhibit antifibrinolytic activity, a property of the Kunitz-type serine protease inhibitor in B. ignitus venom (Choo et al., 2012), our results suggest that BiVSPI is a novel microbial serine protease inhibitor. Taken together, this study provides new insight into the functional roles of bee venom serine protease inhibitors. Acknowledgments This work was supported by the Dong-A University Research Fund. References An, C., Kanost, M.R., 2010. Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6. Insect Biochem. Mol. Biol. 40, 683–689. Augustin, R., Siebert, S., Bosch, T.C., 2009. Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra's innate immune system. Dev. Comp. Immunol. 33, 830–837.

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A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the ve...
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