Open Access

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

  • Kohei Kazuma1,
  • Kenji Ando1,
  • Ken-ichi Nihei2,
  • Xiaoyu Wang1,
  • Marisa Rangel3, 4,
  • Marcia Regina Franzolin5,
  • Kanami Mori-Yasumoto6,
  • Setsuko Sekita6,
  • Makoto Kadowaki1,
  • Motoyoshi Satake7 and
  • Katsuhiro Konno1Email author
Journal of Venomous Animals and Toxins including Tropical Diseases201723:40

https://doi.org/10.1186/s40409-017-0130-y

Received: 13 March 2017

Accepted: 18 August 2017

Published: 29 August 2017

Abstract

Background

Among the hymenopteran insect venoms, those from social wasps and bees – such as honeybee, hornets and paper wasps – have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS.

Methods

A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated.

Results

On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes.

Conclusions

LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Keywords

Peptidomic analysis LC-ESI-MS Solitary bee Venom Linear cationic α-helical peptide

Background

Among the hymenopteran insects, the venoms from social wasps and bees – including honeybees, hornets and paper wasps – have been well documented [1, 2]. Their venoms are composed of a number of peptides and proteins and are used for defending their nests and themselves from predators. In contrast, venoms from solitary wasps and bees still require further research. In recent years, we have studied venoms from solitary wasps from Japan and found peptide neurotoxins, antimicrobial and cytolytic peptides and bradykinin-related peptides [3]. However, venoms from solitary bees have never been studied until quite recently.

The first study on solitary bee venoms was published only in 2008 about the European solitary bee Melecta albifrons [4]. A novel peptide, melectin, was isolated and characterized. Melectin has similar characteristics to those of melittin and mastoparan from the honeybee and hornet venoms. It is rich in hydrophobic and basic amino acids, amphipathic properties, and shows antimicrobial, mast cell degranulating and hemolytic activities. Accordingly, this peptide belongs to linear cationic α-helical peptides. Since then, studies describing similar solitary bee venom peptides have appeared: osmin [5], panurgine-1 [6], macropin [7], codesane [8], and HYL [9] (Table 1).
Table 1

Solitary bee venom peptides

Melectin

GFLSILKKVLPKVMAHMK-NH2

Osmin

GFLSALKKYLPIVLKHV-NH2

Panurgine-1

LNWGAILKHIIK-NH2

Macropin

GFGMALKLLKKVL-NH2

Codesane

GMASLLAKVLPHVVKLIK-NH2

HYL

GIMSSLMKKLAAHIAK-NH2

Xylopin

GFVALLKKLPLILKHLH-NH2

Xylopinin

GFVALLKKLPLILKHLP-NH2

These studies describe only the isolation and characterization of major peptides, which comprise a few components of the venom. However, such venoms consist of a complex mixture of many constituents, which cooperatively act for the venom toxicity and biological functionality. Accordingly, in order to know the exact nature of a venom, the chemical characterization of whole components may be important. In this viewpoint, we investigated the peptide component profile of the venom of Xylocopa appendiculata circumvolans, a solitary bee inhabiting in Japan, by peptidomic analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) [5, 10]. Furthermore, we isolated two major peptides, designated xylopin and xylopinin, and found that they belong to linear cationic α-helical peptides. Biological characterization of xylopin revealed that it is an antimicrobial and cytolytic peptide.

Methods

LC-ESI-MS

The crude venom was analyzed with a LC (Accela 600 Pump, Thermo Scientific) connected with ESI-FTMS (LTQ Orbitrap XL, Thermo Scientific). About 10% of crude venom from a single specimen diluted in 10 μL of water was subjected to reversed-phase HPLC using CAPCELL PAK C18 UG 120, 1.5 × 150 mm (Shiseido Co., Ltd., Japan) with linear gradient from 5% to 65% CH3CN/H2O/0.1% formic acid at a flow rate of 200 μL/min over 20 min at 25 °C. ESI-FTMS was operated by Xcalibar software (Thermo Scientific) as: capillary voltage, + 4.6 kV; capillary temp., 350 °C; sheath and aux gas flow, 50 and 30, respectively (arbitrary units). MS/MS spectra were obtained by data dependent MS/MS mode (two most intense peaks by HCD) and the obtained spectra were manually analyzed to give peptide sequences, which were confirmed by MS-Product in ProteinProspector program (http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct).

MALDI-TOF MS

MALDI-TOF MS spectra were acquired on an Autoflex TOF/TOF mass spectrometer (Bruker Daltonics, Japan) equipped with 337 nm pulsed nitrogen laser under reflector mode. The accelerating voltage was 20 kV. Matrix, α-cyano-4-hydroxycinnamic acid (Aldrich), was prepared at a concentration of 10 mg/mL in 1:1 CH3CN/ 0.1%TFA. External calibration was performed with [Ile7]-angiotensin III (m/z 897.51, monoisotopic, Sigma) and human ACTH fragment 18–39 (m/z 2465.19, monoisotopic, Sigma). The sample solution (0.5 μL) dropped onto the MALDI sample plate was added to the matrix solution (0.5 μL) and allowed to dry at room temperature. For TOF/TOF measurement, argon was used as a collision gas and ions were accelerated at 19 kV. The series of b and y ions were afforded, which enabled identification of whole amino acid sequence by manual analysis.

Purification

Female bees of Xylocopa appendiculata circumvolans were collected at Kami-ichi, Toyama in Japan. The venom sacs from five individuals were dissected immediately after collection and extracted with 1:1 acetonitrile-water containing 0.1% TFA (CH3CN/H2O/0.1% TFA), and lyophilized.

The lyophilized extracts were subjected to reversed-phase HPLC (Shimadzu Corp., Japan) using CAPCELL PAK C18, 6 × 150 mm (Shiseido Co., Ltd., Japan) with a linear gradient from 5% to 65% CH3CN/H2O/0.1% TFA at a flow rate of 1 mL/min over 30 min (Fig. 1). This process released xylopin and xylopinin eluted at 25.1 min and 26.0 min, respectively.
Fig. 1

LC-ESI-MS profile of crude venom extracts of Xylocopa appendiculata circumvolans. About 10% of crude venom extract of a single specimen was subjected to reverse-phase HPLC using CAPCELL PAK C18 (1.5 × 150 mm) with linear gradient of 5–65% CH3CN/H2O/0.1% formic acid over 20 min at flow rate of 200 μL/min. a UV absorption by PDA. b Total ion current (TIC). Numbers in B show “virtual” fraction number as in Tables 2 to 6

Amino acid sequencing

Automated Edman degradation was performed by a gas-phase protein sequencer PPSQ-10 (Shimadzu Corp., Japan).

Peptide synthesis

Peptides were synthesized on an automated PSSM-8 peptide synthesizer (Shimadzu Corp., Japan) by stepwise solid-phase method using N-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. All the resins and Fmoc-L-amino acids were purchased from HiPep Laboratories (Kyoto, Japan). Cleavage of the peptide from the resin was achieved by treatment with a mixture of TFA/H2O/triisopropylsilane (TIS) (95:2.5:2.5) at room temperature for 2 h. After removal of the resin by filtration and washing twice with TFA, the combined filtrate was added dropwise to diethyl ether at 0 °C and then centrifuged at 3000 rpm for 10 min. Thus, obtained crude synthetic peptide was purified by semipreparative reverse-phase HPLC using CAPCELL PAK C18, 10 × 250 mm with isocratic elution of 40–60% CH3CN/H2O/0.1% TFA at a flow rate of 3 mL/min. The homogeneity and the sequence were confirmed by MALDI-TOF MS [m/z 1939.1 (M + H)+] and analytical HPLC (co-eluted with natural peptide by using CAPCELL PAK C18, 6 × 150 mm with isocratic elution of 45% CH3CN/H2O/0.1% TFA at a flow rate of 1 mL/min).

Antimicrobial activity (determination of minimal inhibitory concentration, MIC)

The microorganisms used in this study were: Staphylococcus aureus ATCC 25923; Micrococcus luteus ATCC 10240; Bacillus subtilis ATCC 6633; clinical isolates of: Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium; Escherichia coli ATCC 25922; clinical isolates of: Shigella boydii, Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis, Morganella morgannii; Pseudomonas aeruginosa ATCC 27853; Stenotrophomonas maltophilia ATCC 13637; Acinetobacter baumanii/calcoaceticus (clinical isolate); Saccharomyces cerevisae and Candida albicans ATCC 90112.

The MICs of the tested peptide were determined in the following form: 50 μL of bacterial suspension (106 CFU/mL) in each well of a 96-well microtitre plates were incubated at 37 °C for 18 h with various concentration of 50 μL of the peptide solution, resulting in a final volume of 100 μL with 104 CFU/well, according CLSI [11]. Following incubation, microbial growth was measured by monitoring the optical density (OD) increase at 595 nm in an ELISA reader (Multiskan® EX Thermo Fisher Scientific, EUA). The results were expressed as inhibition percentage of OD against a control (microorganisms in the absence of peptide). In addition, the lowest concentration of peptide at which there is no visible growth after overnight incubation was observed.

Mast cell degranulating activity

The ability of the peptides to induce mast cell degranulation was investigated in vitro using the protocol of quantification of the granular enzyme β-hexosaminidase released in the supernatants of PT18 cells (a connective tissue-type mast cell model) and RBL-2H3 cells (a mucosal-type mast cell model), according to Ortega et al. [12]. For this, 4 × 106 PT18 cells or 1.2 × 105 RBL-2H3 cells (200 μL) were incubated in the presence of the peptides for 30 min in Tyrode’s solution at 37 °C/5% CO2. After this, the cells were centrifuged and the supernatants were collected. The cells incubated only with the Tyrode’s solution were lysed with 200 μL of 0.5% Triton X-100 (Sigma-Aldrich) solution to evaluate the total enzyme content. From each experimental sample to be assayed, four aliquots (10 μL) of the supernatant were taken to separate microwell plates. To these samples, 90 μL of the substrate solution containing 1.3 mg/mL of p-nitrophenyl-N-acetyl-β-D-glucosamine (Sigma Chemical Co.) in 0.1 M citrate, pH 4.5, was added and the plates incubated for 12 h at 37 °C. The reactions were stopped by addition of 100 μL of 0.2 M glycine solution, pH 10.7, and the optical density determined at 405 nm in an ELISA reader (Labsystems Multiskan Ex). The extent of secretion was expressed as the net percentage of the total β-hexosaminidase activity in the supernatant of unstimulated cells. The results represent the mean of quadruplicate tests ± standard deviation (SD).

Hemolytic activity

The use of mice in this assay was in agreement with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and was approved by the Ethical Committee for Animal Research of Butantan Institute (protocol no. 459/08).

To evaluate the pore-forming interaction of the peptide with biological murine membranes, a hemolytic assay was performed. A 4% suspension of mouse erythrocytes (ES) was prepared as previously described [13, 14]. Different concentrations of the peptide were incubated with the ES at room temperature (±22 °C) in an ELISA plate (96 wells) for 1 h and centrifuged (1000×g for 5 min). The hemolytic activity of the supernatant was measured by the absorbance at 540 nm using the absorbance of the Krebs-Henseleit physiological solution (in mM: NaCl, 113; KH2PO4, 1.2; KCl, 4; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; and glucose, 11.1), which was the vehicle for the peptide, as a blank. Total hemolysis was obtained with 1% Triton X-100, and the percentage of hemolysis was calculated relative to this value.

Leishmanicidal activity

Medium 199 was used for the cultivation of promastigote forms of Leishmania major (MHOM/SU/73/5ASKH). Promastigotes were cultured in the medium [supplemented with heat-inactivated (56 °C for 30 min) fetal bovine serum (10%)] at 27 °C, in a 5% CO2 atmosphere in an incubator [15].

The leishmanicidal effects of the peptides were assessed using the improved 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay) method as follows. Cultured promastigotes were seeded at 4 × 105/50 mL of the medium per well in 96-well microplates. Then, 50 mL of different concentrations of test compounds dissolved in a mixture of DMSO and the medium were added to each well. Each concentration was tested in triplicate. The microplate was incubated at 27 °C in 5% CO2 for 48 h. TetraColor ONE (10 mL) a mixture of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H–tetrazolium,monosodium salt and 1-methoxy-5-methylphenazinium methosulfate was added to each well and the plates were incubated at 27 °C for 6 h. Optical density values (test wavelength 450 nm; reference wavelength 630 nm) were measured using a microplate reader (Thermo BioAnalysis Japan Co., Ltd.). The values of 50% inhibitory concentration of the peptides were estimated from the dose-response curve.

Channel-like incorporation in mimetic lipid bilayers

The experiments were performed with the automated Patch-Clamp device Port-a-Patch (Nanion Technologies, Germany), using borosilicate glass chips NPC-1 with aperture diameter of approximately 10 μm. The resistance of the apertures was approximately 1 MΩ in 500 mM KCl solution. Current signals resulting from pore formation were amplified by EPC-10 amplifier (Heka Elektronik, Lambrecht, Germany) and recorded in computer after conversion performed by an analogical/digital interface ITC-1600. The system was computer controlled by the PatchControl™ software (Nanion) [16, 17].

Symmetrical solutions of 150 or 500 mM KCl with 5 mM Tris were used. Asolectin (Sigma), a negatively charged mixture of lipids, was used to form artificial membranes. Asolectin was dissolved in n-decane at a concentration of 2 mg/mL. The bilayers were painted onto the aperture of the chip using disposable polypropylene pipet tips. Measurements of the capacitive currents evoked by control voltage pulses and increase in the membrane resistance indicated the formation of bilayers. After the formation of a lipid bilayer (Rm > 1 GΩ), xylopin diluted with Milli-Q water at a 10 μM concentration was added to the cis side of the chip (top) to observe the single channel activity. The volume of peptide solution was never superior to 10% of the solution at the cis side. Voltage pulses were applied at the trans side of the chip (bottom). Usually, single channel activity started approximately 10 min after adding the peptide, as monitored by a constant Vhold of −100 mV or 100 mV. Pore conductance of incorporated channels was determined under positive and negative voltage pulses (Vhold). The experiments were performed at room temperature (~22 °C). The data were analyzed by PatchMaster and Matlab softwares.

Results

On-line mass fingerprinting

LC-ESI-MS profile is shown in Fig. 1. The volume of peptide solution never exceeded 10% of the amount of crude venom from a single specimen, which is sufficient for LC-ESI-MS analysis (mass fingerprinting and peptide sequencing). On-line mass fingerprint was prepared from TIC by “virtual fractionation”, collecting MS spectra from a certain range of retention time, and then, the molecular mass was analyzed in each fraction. The results are summarized in Table 2. A total of 124 molecular mass peaks were found from 18 virtual fractions. The low molecular mass components (m/z 100–300) are free amino acids, biogenic amines and nucleic acids (data not shown) and those of m/z range from 500 to 4000 should be peptides, in particular, m/z from 500 to 2000 accounts for 60%, implying that a majority of components in this venom are relatively small peptides.
Table 2

Mass fingerprint of crude venom from X. appendiculata circumvolans

Fraction no.

Retention time (min)

[M + H]+ m/z

1

1.0–1.5

116.071, 175.119, 184.073, 348.071, 381.080, 405.236, 441.101

2

1.5–2.0

132.102, 268.104, 322.077, 377.058, 733.323, 759.499

3

2.0–3.0

284.099, 373.281, 437.051, 469.277, 654.357, 817.504, 947.484, 1002.508

4

3.0–4.2

182.117, 431.214, 598.428, 937.390, 1368.747

5

4.2–5.0

623.423, 646.423, 1517.714, 2064.031

6

5.0–6.0

930.588, 969.653, 1165.500, 1189.459, 1338.913, 1306.915, 1715.808, 2807.314

7

6.0–6.4

322.176, 393.214, 961.656, 1210.815, 1451.994, 1925.941

8

6.4–7.0

714.306, 1211.799, 1268.821, 1336.563, 1510.001, 3225.624, 3243.642

9

7.0–8.0

637.346, 715.291, 793.482, 838.421, 875.527, 1082.720, 1178.861, 1337.549, 1565.078, 1636.116, 2064.036, 3400.643

10

8.0–9.0

690.456, 747.477, 908.509, 933.515, 937.678, 1249.897, 1427.606, 1608.000, 1626.012, 1694.121, 2065.019, 3187.725, 3245.730

11

9.0–10.0

379.114, 506.298, 696.502, 761.492, 988.656, 1021.593, 1085.709, 1366.821, 1384.832, 1882.253, 3187.723, 3245.728

12

10.0–10.8

370.199, 941.598, 1690.115, 1735.185, 1939.274

13

10.8–11.4

619.381, 926.118, 1553.055, 1783.363, 1997.276

14

11.4–12.0

1311.877, 1803.199, 1899.267, 2087.312, 2139.353, 2236.641, 3087.760, 3256.821

15

12.0–12.7

680.314, 704.272, 1424.961, 1956.252, 2121.345, 2197.359, 2281.379, 2648.565

16

12.7–13.2

2095.280, 2113.292

17

13.2–13.6

662.303, 1316.450, 2077.267, 2153.287, 2171.297

18

13.6–15.0

1709.063, 3860.509, 4015.525

Peptide sequencing by MS/MS analysis

Data dependent MS/MS measurement afforded MS/MS spectra from 79 peptide molecules. Manual sequence analysis of these MS/MS spectra revealed the full sequence of 58 peptides, and the rest of the 21 peptides were only partially sequenced (data not shown). The analyzed full sequences are shown in Table 3.
Table 3

Peptide sequences analyzed from MS/MS spectra

Fraction no.

[M + H]+

Sequence

Fraction no.

[M + H]+

Sequence

1

405.236

HLH-NH2

9

637.346

FAFPR

793.482

FLVSSLK

838.421

SNFAFPR

875.527

GFVALLKK

1082.720

LPLILKHLH-NH2

1178.861

LLKKLPLILK

1337.549

DGLDEYEPEDR

1565.078

LLKKLPLILKHLH-NH2

1636.116

ALLKKLPLILKHLH-NH2

2

759.499

ILKHLH-NH2

10

690.456

FVALLK

747.477

GFVLKK

908.509

DFLVSSLK

937.678

LKKLPLIL

1249.897

ALLKKLPLILK

1694.121

ALLKKLPLILKHLHG

3

373.281

ILK

11

506.298

GFVAL

469.277

HVLT

 

696.502

PLKLI

817.504

ILKHLHG

 

988.656

GFVALLKKL

947.489

EMKSVEPK

 

1021.593

LDFLVSSLK

1085.709

GFVALLKKLP

1882.253

FVALLKKLPLILKH

4

431.214

SVEP

12

1690.115

GFVALLKKLPLILKH

598.428

LKKLP

1735.185

VALLKKLPLILKHLH-NH2

937.390

EYEPEDR

1939.274

GFVALLKKLPLILKHLH-NH2

5

623.423

LILKH

13

619.381

GFVALL

646.413

LVSSLK

1553.055

GFVALLKKLPLILK

1997.276

GFVALLKKLPLILKHLKG

6

930.588

LILKHLHG

14

1311.877

GFVALLKKLPLI

969.653

PLILKHLH-NH2

1803.199

GFVALLKKLPLILKHL

1165.500

LDEYEPEDR

1899.267

GFVALLKKLPLILKHLP-NH2

1338.913

KKLPLILKHLH-NH2

2139.353

EAGFVALLKKLPLILKHLH-NH2

1396.915

KKLPLILKHLHG

  

7

322.176

GFV

15

1424.961

GFVALLKKLPLIL

393.214

GFVA

961.656

KLPLILKH

1210.815

KLPLILKHLH-NH2

1451.994

LKKLPLILKHLH-NH2

8

1211.799

KLPLILIKHLH

   

1268.821

KLPLILKHLHG

1336.563

NGLDEYEPEDR

1500.001

LKKLPLILKHLHG

These sequences can be classified according to homology and similarity. Most of them are related to the major peptide xylopin (mentioned below). As shown in Table 4, most of them are truncated peptides from both N- and C-terminus, in other words, they have a partial structure of xylopin. Seemingly, these truncated peptides are cleavage products of xylopin in some way, but it is not sure whether they are originally contained in the venom or not. Table 5 summarizes the peptides that have a similar partial sequence to xylopin as well, but no amidated C-terminus and have G (glycine) at the C-terminus instead. They are clearly the precursors of amidated C-terminus counterparts because the C-terminal amidation (post-translational modification) takes place by oxidation-hydrolysis of C-terminal G (glycine) residue.
Table 4

Peptides related to xylopin

Fraction no.

[M + H]+

Sequence

7

322.176

GFV

7

393.214

GFVA

11

506.502

GFVAL

13

619.381

GFVALL

10

747.477

GFVALLK

9

875.527

GFVALLKK

11

988.656

GFVALLKKL

11

1085.709

GFVALLKKLP

14

1311.877

GFVALLKKLPLI

15

1424.961

GFVALLKKLPLIL

13

1553.055

GFVALLKKLPLILK

12

1690.115

GFVALLKKLPLILKH

14

1803.199

GFVALLKKLPLILKHL

3

373.281

ILK

5

623.423

LILKH

7

961.656

KLPLILKH

8

1211.799

KLPLILKHLH

9

1178.861

LLKKLPLILK

10

690.456

FVALLK

10

1249.897

ALLKKLPLILK

10

937.678

LKKLPLILK

11

696.502

LPLILK

1

405.236

HLH-NH2

2

759.499

ILKHLH-NH2

6

969.653

PLILKHLH-NH2

9

1082.720

LPLILKHLH-NH2

7

1210.815

KLPLILKHLH-NH2

6

1338.913

KKLPLILKHLH-NH2

7

1451.994

LKKLPLILKHLH-NH2

9

1565.078

LLKKLPLILKHLH-NH2

9

1636.116

ALLKKLPLILKHLH-NH2

12

1735.185

VALLKKLPLILKHLH-NH2

11

1882.253

FVALLKKLPLILKHLH-NH2

14

1899.267

GFVALLKKLPLILKHLP-NH2 a

12

1939.274

GFVALLKKLPLILKHLH-NH2 b

19

2139.353

EAGFVALLKKLPLILKHLH-NH2

aXylopinin, bxylopin

Table 5

Peptides without amidated C-terminus

Fraction no.

[M + H] +

Sequence

3

817.504

ILKHLHG

6

930.588

LILKHLHG

8

1268.821

KLPLILKHLHG

6

1396.915

KKLPLILKHLHG

8

1510.001

LKKLPLILKHLHG

10

1694.121

ALLKKLPLILKHLHG

13

1997.276

GFVALLKKLPLILKHLHG

The rest of the peptides in this venom may be new peptides as summarized in Table 6. All these have no homology to any known peptides.
Table 6

Unknown peptides

Fraction no.

[M + H]+

Sequence

3

469.277

HVLT

3

654.357

EVLSAH-NH2

4

431.214

SVEP

3

947.484

EMKSVEPK

9

637.346

FAFPR

9

838.421

SNFAFPR

5

646.413

LVSSLK

9

793.482

FLVSSLK

10

908.509

DFLVSSLK

11

1021.593

LDFLVSSLK

4

937.390

EYEPEDR

6

1165.500

LDEYEPEDR

8

1336.563

NGLDEYEPEDR

9

1337.549

DGLDEYEPEDR

Purification and sequence determination of major peptides

Two major peptides, called xylopin and xylopinin, were purified by reversed-phase HPLC (Fig. 2). The primary sequence of xylopin was determined by Edman degradation as GFVALLKKLPLILKHLH, which corresponded to a peptide component with m/z 1939.274 (M + H)+ in the crude venom, and accordingly, the C-terminus is amidated. The solid-phase synthesis of this peptide and the HPLC comparison of the synthetic specimen with the natural peptide finally corroborated the sequence.
Fig. 2

Fractionation of venom extracts of Xylocopa appendiculata circumvolans by reverse-phase HPLC using CAPCELL PAK C18 (6 × 150 mm) with linear gradient of 5–65% CH3CN/H2O/0.1% TFA over 30 min at flow rate of 1 mL/min. UV absorption was monitored at 215 nm

The sequence of xylopinin was determined by MALDI-TOF/TOF analysis as GFVALLKKLPLILKHLP-NH2, in which L and I were differentiated by w and d ions, and corresponded to the peptide with m/z 1899.267 (M + H)+ in the crude venom.

The chemical features of xylopin and xylopinin, rich in hydrophobic and basic amino acids with no disulfide bond, are characteristic of linear cationic cytolytic peptides [18]. The known solitary bee venom peptides, melectin and osmin, can be included in this type of peptides, and are highly homologous to these new peptides. This class of peptides has been known to adopt an amphipathic α-helical conformation, showing an amphiphilic character under appropriate conditions [1922], and the amphipaticity of peptides has been considered essential for their biological activities [23]. In fact, if the helical wheel projection of xylopin and xylopinin sequences were drawn, amphipathic α-helical conformations would be depicted as in Fig. 3. Based on this view, all the hydrophilic amino acid residues, S, H and K, are located on one side, whereas the hydrophobic amino acid residues, A, F, I, L and V are on the other side of the helix.
Fig. 3

Helical wheel projection of the sequence of xylopin and xylopinin. In this view through the helix axis, the hydrophilic His (H) and Lys (K) residues are located on one side and the hydrophobic Ala (A), Phe (F), Ile (I) and Leu (L) residues on the other side of the helix

Biological activities

Biological activities of xylopin were evaluated by using synthetic specimen. The mast cell degranulation, hemolysis, antimicrobial and antiprotozoan (leishmanicidal) activities were tested because these are characteristic biological activities for these types of peptide.

Mast cell degranulation activity on RBL-2H3 cells was similar to mastoparan at low concentrations (<30 μM), whereas at higher concentrations (100 μM), it was more potent than mastoparan (Fig. 4). Antimicrobial activity can be considered strong and of broad spectrum, with MICs from 1.9 to 15 μM. The peptide showed the lowest MIC values against gram-positive bacteria, with exception of S. aureus ATCC25923 and Enterococcus spp., and presented potent activities against yeasts (Table 7). Hemolytic activity against mouse erythrocytes, however, was low, reaching only 30% at the highest concentration of 1 mM. Xylopin showed significant leishmanicidal activity with an IC50 of 25 μM against Leishmania major.
Fig. 4

The degranulation in RBL-2H3 cells (a mucosal-type mast cell model) measured by the β-hexosaminidase release, basal and after treatment with xylopin, the novel venom peptide from the solitary bee Xylocopa appendiculata circumvolans. Concentrations are in μM and data represent the mean from two to four independent experiments

Table 7

Minimum inhibitory concentration (MIC) of xylopin

Microorganism

MIC (μM)

Gram-positive

 Staphylococcus aureus ATCC 25923

15.0

 Micrococcus luteus ATCC 10240

1.9

 Bacillus subtilis ATCC 6633

3.75

 Streptococcus pyogenes (CS)

3.75

 Streptococcus agalactiae (CS)

3.75

 Enterococcus. faecalis (CS)

15.0

Gram-negative

 Escherichia coli ATCC 25922

3.75

 Pseudomonas aeruginosa ATCC 27853

7.5

 Stenotrophomonas maltophilia ATCC 13637

3.75

 Shigella boydii (CS)

3.75

 Klebsiella pneumoniae (CS)

7.5

 Serratia marcescens (CS)

7.5

 Enterobacter cloacae (CS)

7.5

 Proteus mirabilis (CS)

>30

 Morganella morgannii (CS)

30

 Acinetobacter baumanii/calcoaceticus (CS)

3.75

Yeast

 Candida albicans ATCC 90112

7.5

 Sacharomyces cerevisae

3.75

Channel-like incorporation in mimetic lipid bilayers

Xylopin induced pore formation in painted asolectin artificial lipid bilayers at 1 μM concentration. The apertures occurred when voltage was clamped at either positive or negative values. Pores of different conductance levels (from 45 to 260 pS in a 150 mM KCl solution, Vhold ± 140 mV; and from ~75 to 175 pS at Vhold ± 100 mV in a 500 mM KCl solution) were recorded in our experiments (Fig. 5).
Fig. 5

Representative recordings of single channel incorporation in asolectin artificial lipid bilayers induced by xylopin at 1 μM concentration. a Vhold = + 140 mV, pore conductances = 63 and 105 pS. b Vhold = −140 mV, pore conductance = 143 and 259 pS. c Vhold was set at +100 mV for 5 s and was switched to −100 mV for the remaining 5 s, pore conductance = 175 pS. Solutions: a and b 150 mM KCl, and c 500 mM KCl (symmetrical). Arrows indicate channel apertures or closings. Four independent experiments were performed

Discussion

In this study, we have analyzed all the components in the crude venom of Xylocopa appendiculata circumvolans, a solitary bee inhabiting from Japan, by using LC-ESI-MS and MS/MS. It revealed that this venom contained 124 components and most of them are small peptides. The peptide sequences were further analyzed by manual analysis of their MS/MS spectra, which led to the determination of whole sequence of 58 peptides. However, most of them are related to the major peptide xylopin, having a truncated partial sequence of xylopin. Therefore, these peptides may come from cleavage of xylopin in some way, but it is not clear whether they are originally contained in the venom or not.

Most notably, these results were obtained by using only 10% of the amount of a single venom content. Among the hymenopteran insect venoms, solitary bee venom has not been extensively studied yet. One of the reasons for this may come from the difficulty of collecting a sufficient amount of venom for chemical analysis, which is due to the insect solitary life style. However, as shown in this study, the remarkable progress of mass spectrometry in sensitivity made it possible to perform this type of peptidomic analysis with so minute amount of venom. It is particularly advantageous not only for solitary bee venom but also for solitary wasp venom, and the studies along this line are in progress in our laboratory.

In addition to peptidomic analysis, we have purified and characterized the major peptide components, xylopin and xylopinin, by the conventional method. The chemical and biological characteristics of xylopin are similar to the known solitary bee venom peptides – melectin and osmin – and, accordingly, this novel peptide belongs to the linear cationic α-helical peptide group. Xylopin presented broad-spectrum antimicrobial activity, with very low hemolytic activity. Xylopin has also a Pro10 residue in the sequence, in a similar position as both melectin and osmin that present a Pro11. According to Cerovsky et al. [4], the Pro11 residue conferred to this peptide selectivity to the antimicrobial activity, as well as low hemolytic activity.

The pore formation by xylopin in artificial lipid bilayers was confirmed through electrical measurements. This is the first report of solitary bee venom peptides inducing pore formation in artificial lipid bilayers. Asolectin was employed because it is negatively charged and has shown to be a good membrane model for this class of peptide in previous studies [14, 24]. Conductance of the pores formed by xylopin was bigger than the conductance of the pores formed by the eumenitin-R and F, and EMP-ER and -EF from solitary wasp venoms [14]. Additionally, large conductance pores (>500 pS) were not observed in the presence of xylopin, similarly with the eumenine mastoparan peptides EMP-ER and -EF [14], probably due to their amidated C-terminal, that would prevent cluster formation constituted by several units of the peptide.

Conclusions

LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed the component profile of this venom, which mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized by the conventional technique. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Abbreviations

CS: 

Clinical sample

ES: 

Erythrocytes

ESI-FTMS: 

Electrospray ionization-Fourier transformed mass spectrometry

FTMS: 

Fourier transformed mass spectrometry

HPLC: 

High performance liquid chromatography

LC: 

Liquid chromatographty

LC-ESI-MS: 

Liquid chromatography-elctrospray ionization-mass spectrometry

MALDI-TOF MS: 

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry

MIC: 

Minimal inhibitory concentration

MS: 

Mass spectrometry

MS/MS: 

Tandem mass spectrometry

OD: 

Optical density

SD: 

Standard deviation

TIC: 

Total ion current

TOF/TOF: 

Time-of-flight/time-of-flight

Declarations

Acknowledgments

KK thanks the support from JSPS KAKENHI (grant no. 15 K07805). MR thanks the support from FAPESP (grant no. 2008/00173-4) and CNPq (grant no. 473645/2012-2).

Funding

A part of this work was supported by the JSPS KAKENHI grant number 15 K07805, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Part of this work was supported by FAPESP (grant no. 2008/00173–4) and CNPq (473,645/2012–2).

Authors’ contributions

KK (corresponding author) designed this work, prepared this manuscript and performed MS/MS analysis of the peptide sequence. KK (first author) contributed to LC-ESI-MS data acquisition and analysis. KA collected and extracted the bee samples, performed HPLC analysis and peptide synthesis. KN performed MALDI-TOF/MS analysis. XW and MK performed mast cell degranulation assay. MR and MRF designed and performed antimicrobial, hemolytic and and pore forming assay. KMY, SS and SM contributed to leishmanicidal assay. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The use of mice in hemolytic assays was in agreement with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and was approved by the Ethical Committee for Animal Research of Butantan Institute (protocol no. 459/08).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Natural Medicine, University of Toyama
(2)
Faculty of Agriculture, Utsunomiya University
(3)
Immunopathology Laboratory, Butantan Institute
(4)
Department of Physiological Sciences, Institute of Biological Sciences, University of Brasília
(5)
Bacteriology Laboratory, Butantan Institute
(6)
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University
(7)
Laboratory of Plant Resources for Medicine, Showa Pharmaceutical University

References

  1. Banks BCE, Shipolini RA. Chemistry and pharmacology of honey-bee venom. In: Piek T, editor. Venoms of the hymenoptera. London: Academic; 1986. p. 329–416.View ArticleGoogle Scholar
  2. Nakajima T. Pharmacological biochemistry of vespid venoms. In: Piek T, editor. Venoms of the hymenoptera. London: Academic; 1986. p. 309–27.View ArticleGoogle Scholar
  3. Konno K, Kazuma K, Nihei K. Peptide toxins in solitary wasp venoms. Toxins (Basel). 2016;8(4):114.View ArticleGoogle Scholar
  4. Čeřovský V, Hovorka O, CvaČka J, Voburka Z, Bednárová L, BoroviČková L, et al. Melectin: a novel antimicrobial peptide from the venom of the cleptoparasitic bee Melecta albifrons. Chembiochem. 2008;9(17):2815–21.View ArticlePubMedGoogle Scholar
  5. Stöcklin R, Favreau P, Thai R, Pflugfelder J, Bulet P, Mebs D. Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (hymenoptera: Megachilidae). Toxicon. 2010;55(1):20–7.View ArticlePubMedGoogle Scholar
  6. Čujová S, Slaninová J, Monincová L, FuČík V, Bednárová L, Štokrová J, et al. Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (hymenoptera: Andrenidae). Amino Acids. 2013;45(1):143–57.View ArticlePubMedGoogle Scholar
  7. Monincová L, Veverka V, Slaninová J, Buděšínský M, Fučík V, Bednárová L, et al. Structure-activity study of macropin, a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (hymenoptera: Melittidae). J Pept Sci. 2014;20(6):375–84.View ArticlePubMedGoogle Scholar
  8. Čujová S, Bednárová L, Slaninová J, Straka J, Čeřovský V. Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells. J Pept Sci. 2014;20(11):885–95.View ArticlePubMedGoogle Scholar
  9. NeŠuta O, Hexnerová R, BuděŠínský M, Slaninová J, Bednárová L, Hadravová R, et al. Antimicrobial peptide from the wild bee Hylaeus signatus venom and its analogues: structure-activity study and synergistic effect with antibiotics. J Nat Prod. 2016;79(4):1073–83.View ArticlePubMedGoogle Scholar
  10. Favreau F, Menin L, Michalet S, Perret F, Cheneval O, Stöcklin M, et al. Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. Toxicon. 2006;47(6):676–87.View ArticlePubMedGoogle Scholar
  11. CLSI. Performance standards for antimicrobial susceptibility testing. Twenty-second informational supplement, National Committee of clinical laboratory standards. CLSI M100, S22, Ed. 22. 2011; 31(1).Google Scholar
  12. Ortega E, Schneider H, Pecht I. Possible interactions between the Fc epsilon receptor and a novel mast cell function-associated antigen. Int Immunol. 1991;3(4):333–42.View ArticlePubMedGoogle Scholar
  13. Rangel M, Malpezzi ELA, Susini SMM, de Freitas JC. Hemolytic activity in extracts of the diatom Nitzschia. Toxicon. 1997;35(2):305–9.View ArticlePubMedGoogle Scholar
  14. Rangel M, Cabrera MPS, Kazuma K, Ando K, Wang X, Kato M, et al. Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps. Toxicon. 2011;57(7–8):1081–92.View ArticlePubMedGoogle Scholar
  15. Takahashi M, Fuchino H, Satake M, Agatsuma Y, Sekita S. In vitro screening of leishmanicidal activity of Myanmar timber extracts. Biol Pharm Bull. 2004;27(6):921–5.View ArticlePubMedGoogle Scholar
  16. Fertig N, Blick RH, Behrends JC. Whole cell patch clamp recording performed on a planar glass chip. Biophys J. 2002;82(6):3056–62.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Sondermann M, George M, Fertig N, Behrends JC. High-resolution electrophysiology on a chip: transient dynamics of alamethicin channel formation. Biochim Biophys Acta. 2006;1758(4):545–51.View ArticlePubMedGoogle Scholar
  18. Kuhn-Nentwig L. Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci. 2003;60(12):2651–68.View ArticlePubMedGoogle Scholar
  19. Wakamatsu K, Okada A, Miyazawa T, Ohya M, Higashijima T. Membrane-bound conformation of Mastoparan-X, a G-protein-activating peptide. Biochemistry. 1992;31(24):5654–60.View ArticlePubMedGoogle Scholar
  20. Hori Y, Demura M, Iwadate M, Ulrich AS, Niidome T, Aoyagi H, et al. Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers. Eur J Biochem. 2001;268(2):302–9.View ArticlePubMedGoogle Scholar
  21. Sforça ML, Oyama S Jr, Canduri F, Lorenzi CCB, Pertinhez TA, Konno K, et al. How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp. Biochemistry. 2004;43(19):5608–17.View ArticlePubMedGoogle Scholar
  22. Todokoro Y, Yumen I, Fukushima K, Kang SW, Park JS, Kohno T, et al. Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR. Biophys J. 2006;91(4):1368–79.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Wimley WC. Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol. 2010;5(10):905–17.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Silva JC, Neto LM, Neves RC, Goncalves JC, Trentini MM, Mucury-Filho R, et al. Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, hymenoptera). Int J Antimicrob Agents. 2017;49(2):167–75.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2017