Bee Venom Peptide Research Articles

Structural Determinants of Peptide Nanopore Formation.

We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.

Efficacy of the bee-venom antimicrobial peptide Osmin against sensitive and carbapenem-resistant Klebsiella pneumoniae strains

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae strains causes severe problems in the treatment of bacterial infections owing to limited treatment options. Especially, carbapenem-resistant Klebsiella pneumoniae (CRKP) is rapidly spreading worldwide and is emerging as a new cause of drug-resistant healthcare-associated infections. CRKP also has been announced by the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) as one of the most pressing antibiotic resistance threats. Antimicrobial peptides (AMPs) are drawing considerable attention as ideal antibiotic alternative candidates to combat MDR bacterial infections. In a previous study, Osmin is composed of 17 amino acids and is isolated from solitary bee (Osmia rufa) venom. Herein, we evaluated the potential of Osmin to be used against drug-resistant K. pneumoniae as an alternative to conventional antibiotics. Osmin exhibited significant antimicrobial and anti-biofilm activity and lower toxicity than melittin, a well-known bee venom peptide. Additionally, we confirmed that it possesses a bactericidal mechanism that rapidly destroys bacterial membranes. Osmin was relatively more stable than melittin under the influence of various environmental factors and unlike conventional antibiotics, it exhibited a low bacterial resistance risk. During in vivo tests, Osmin reduced bacterial growth and the expression of pro-inflammatory cytokines and fibrosis-related genes in mice with CRKP-induced sepsis. Overall, our results indicate a high potential for Osmin to be used as a valuable therapeutic agent against drug-resistant K. pneumoniae infections.

Lasioglossin-1 peptide inhibits binding of spike protein of SARS-CoV-2 to ACE2 receptor: an in silico approach of some bee venom peptides

ABSTRACT The coronavirus prevalent, initiated by the SARS-CoV-2 virus, has induced a global health crisis, recently. Coronavirus can bind to ACE2 receptors on the surface of human cells and infect them through the RBD domain of spike protein. Bee venom peptides have anti-inflammatory effects and were proposed for coronavirus cure. To investigate this proposal, 11 peptides from bee venom were selected and 3D structures of them were prepared. Each peptide was simulated for 100 ns molecular dynamics simulation and 22 conformations were extracted from the last 20 ns of MD simulation. Then All 220 conformations were docked to the RBD domain of spike protein by haddock server and the best complexes for each peptide were chosen for another 100 ns MD simulation. Then MM/PBSA binding free energy calculations were done. The results revealed that the Lasioglossin-1 peptide (VNWKKVLGKIIKVAK) had better interaction with the RBD domain of spike protein than other peptides and can reduce virus entrance to human cells. This peptide can be a potential drug for coronavirus. Also, to find a probable better peptide, two mutations were created in this peptide, and previous procedures were repeated and it determined that none of the mutated peptides were better than native Lasioglossin-1.

Transcriptomic Responses Underlying the High Virulence of Black Queen Cell Virus and Sacbrood Virus following a Change in Their Mode of Transmission in Honey Bees (Apis mellifera).

Over the last two decades, honey bees (Apis mellifera) have suffered high rates of colony losses that have been attributed to a variety of factors, chief among which are viral pathogens, such as deformed wing virus (DWV), whose virulence has increased because of vector-based transmission by the invasive, ectoparasitic varroa mite (Varroa destructor). A shift in the experimental mode of transmission of the black queen cell virus (BQCV) and sacbrood virus (SBV) from fecal/food-oral (direct horizontal) to vector-mediated (indirect horizontal) transmission also results in high virulence and viral titers in pupal and adult honey bees. Agricultural pesticides represent another factor that acts independently or in interaction with pathogens, and they are also thought to cause colony loss. Understanding the molecular mechanisms underlying the higher virulence following a vector-based mode of transmission provides deeper insight into honey bee colony losses, as does determining whether or not host-pathogen interactions are modulated by exposure to pesticides. Through an experimental design with controlled laboratory, we investigated the effects of the modes of transmission of BQCV and SBV (feeding vs. vector-mediated via injection) alone or in combination with chronic exposure to sublethal and field-realistic concentrations of flupyradifurone (FPF), a novel agricultural insecticide, on honey bee survival and transcription responses by using high-throughput RNA sequencing (RNA-seq) analysis. Co-exposure to viruses via feeding (VF) or injection (VI) and FPF insecticide had no statistically significant interactive effect on their survival compared to, respectively, VF or VI treatments alone. Transcriptomic analysis revealed a distinct difference in the gene expression profiles of bees inoculated with viruses via injection (VI) and exposed to FPF insecticide (VI+FPF). The number of differentially expressed genes (DEGs) at log2 (fold-change) > 2.0 in VI bees (136 genes) or/and VI+FPF insecticide (282 genes) was very high compared to that of VF bees (8 genes) or the VF+FPF insecticide treatment (15 genes). Of these DEGs, the expression in VI and VI+FPF bees of some immune-related genes, such as those for antimicrobial peptides, Ago2, and Dicer, was induced. In short, several genes encoding odorant binding proteins, chemosensory proteins, odor receptors, honey bee venom peptides, and vitellogenin were downregulated in VI and VI+FPF bees. Given the importance of these suppressed genes in honey bees' innate immunity, eicosanoid biosynthesis, and olfactory associative function, their inhibition because of the change in the mode of infection with BQCV and SBV to vector-mediated transmission (injection into haemocoel) could explain the high virulence observed in these viruses when they were experimentally injected into hosts. These changes may help explain why other viruses, such as DWV, represent such a threat to colony survival when transmitted by varroa mites.

Identification of novel Bioactivities from Bee venom to target TNF-α for cancer therapy

Cancer is one of the major public health problems globally which arises due to uncontrolled cellular proliferation. Tumor necrosis factor alpha is a member of the TNF/TNFR cytokine superfamily. Currently, the venom from various sources have been widely used in the treatment of cancer. The bioactive present in bee venom has been reported to have potential antimicrobial, anti-inflammatory, and anticancer activity which has drawn the attention to identify the novel inhibitor against TNF-α. Bee venom has been reported to target ovarian, breast, prostate and malignant hepatocellular carcinoma. TNF-α is involved in the maintenance and homeostasis of the immune system, inflammation, and host defense. The oncogenic protein TNF-α plays a critical role in the development of various cancers including renal, lung, liver, prostate, bladder, and breast cancer. TNF-α enhances cancer cell growth, proliferation, invasion, and metastasis, as well as tumor angiogenesis. Due to the high prevalence and mortality, TNF-α associated cancers have remained a significant health problem globally. TNF acts biologically by activating certain signaling pathways such as nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). Various toxins are being studied as alternatives for cancer treatments, and bee venom and its active components are drawing attention as potential anticancer agents. The present study identifies novel anticancer peptides that target the oncoprotein against life-threatening cancer. Docking calculations indicate that anticancer peptides, namely Melittin, Phospholipase A2, Tertiapin, and Hyaluronidase bind TNF-α respectively with the lowest binding affinity. Interestingly, Mast cell degranulating (MCD) and Apamin have the highest binding affinity with TNF-α in comparison with the above four peptides. The two lead compounds namely MCD and Apamin have the highest docking score -1253.4 and -1067.8 respectively. The present study reveals that the bee venom peptides namely MCD and Apamin interact with TNF-α associated cancer for targeted therapy of cancer. These predicted anticancer peptides are valuable candidates for in vitro or in vivo peptide therapeutic drug studies against the TNF-α associated cancers.

Time-resolved solid state NMR of biomolecular processes with millisecond time resolution

We review recent efforts to develop and apply an experimental approach to the structural characterization of transient intermediate states in biomolecular processes that involve large changes in molecular conformation or assembly state. This approach depends on solid state nuclear magnetic resonance (ssNMR) measurements that are performed at very low temperatures, typically 25–30 K, with signal enhancements from dynamic nuclear polarization (DNP). This approach also involves novel technology for initiating the process of interest, either by rapid mixing of two solutions or by a rapid inverse temperature jump, and for rapid freezing to trap intermediate states. Initiation by rapid mixing or an inverse temperature jump can be accomplished in approximately-one millisecond. Freezing can be accomplished in approximately 100 microseconds. Thus, millisecond time resolution can be achieved. Recent applications to the process by which the biologically essential calcium sensor protein calmodulin forms a complex with one of its target proteins and the process by which the bee venom peptide melittin converts from an unstructured monomeric state to a helical, tetrameric state after a rapid change in pH or temperature are described briefly. Future applications of millisecond time-resolved ssNMR are also discussed briefly.

Factors driving the compositional diversity of Apis mellifera bee venom from a Corymbia calophylla (marri) ecosystem, Southwestern Australia.

Bee venom (BV) is the most valuable product harvested from honeybees ($30 - $300 USD per gram) but marginally produced in apiculture. Though widely studied and used in alternative medicine, recent efforts in BV research have focused on its therapeutic and cosmetic applications, for the treatment of degenerative and infectious diseases. The protein and peptide composition of BV is integral to its bioactivity, yet little research has investigated the ecological factors influencing the qualitative and quantitative variations in the BV composition. Bee venom from Apis mellifera ligustica (Apidae), collected over one flowering season of Corymbia calophylla (Myrtaceae; marri) was characterized to test if the protein composition and amount of BV variation between sites is influenced by i) ecological factors (temperature, relative humidity, flowering index and stage, nectar production); ii) management (nutritional supply and movement of hives); and/or iii) behavioural factors. BV samples from 25 hives across a 200 km-latitudinal range in Southwestern Australia were collected using stimulatory devices. We studied the protein composition of BV by mass spectrometry, using a bottom-up proteomics approach. Peptide identification utilised sequence homology to the A. mellifera reference genome, assembling a BV peptide profile representative of 99 proteins, including a number of previously uncharacterised BV proteins. Among ecological factors, BV weight and protein diversity varied by temperature and marri flowering stage but not by index, this latter suggesting that inter and intra-year flowering index should be further explored to better appreciate this influence. Site influenced BV protein diversity and weight difference in two sites. Bee behavioural response to the stimulator device impacted both the protein profile and weight, whereas management factors did not. Continued research using a combination of proteomics, and bio-ecological approaches is recommended to further understand causes of BV variation in order to standardise and improve the harvest practice and product quality attributes.

Antidepressive effect of an inward rectifier K+ channel blocker peptide, tertiapin-RQ.

Renal outer medullary K+ channel, ROMK (Kir1.1, kcnj1) is expressed in the kidney and brain, but its role in the central nervous system remains unknown. Recent studies suggested an involvement of the ROMK channel in mental diseases. Tertiapin (TPN) is a European honey bee venom peptide and is reported to selectively block the ROMK channel. Here, we have chemically synthesized a series of mutated TPN peptides, including TPN-I8R and -M13Q (TPN-RQ), reported previously, and examined their blocking activity on the ROMK channel. Among 71 peptides tested, TPN-RQ was found to block the ROMK channel most effectively. Whole-cell patch-clamp recordings showed the essential roles of two disulfide bonds and the circular structure for the blockade activity. To examine the central role, we injected TPN-RQ intracerebroventricularly and examined the effects on depression- and anxiety-like behaviors in mice. TPN-RQ showed an antidepressive effect in tail-suspension and forced swim tests. The injection of TPN-RQ also enhanced the anxiety-like behavior in the elevated plus-maze and light/dark box tests and impaired spontaneous motor activities in balance beam and wheel running tests. Administration of TPM-RQ suppressed the anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in antidepressant-related nuclei, e.g. the dorsal raphe nucleus and locus coeruleus. TPN-RQ may exert its antidepressive effects through a different mechanism from current drugs.

Effects of Glycosylation and d-Amino Acid Substitution on the Antitumor and Antibacterial Activities of Bee Venom Peptide HYL.

Glycosylation is a promising strategy for modulating the physicochemical properties of peptides. However, the influence of glycosylation on the biological activities of peptides remains unknown. Here, we chose the bee venom peptide HYL as a model peptide and 12 different monosaccharides as model sugars to study the effects of glycosylation site, number, and monosaccharide structure on the biochemical properties, activities, and cellular selectivities of HYL derivatives. Some analogues of HYL showed improvement not only in cell selectivity and proteolytic stability but also in antitumor and antimicrobial activity. Moreover, we found that the helicity of glycopeptides can affect its antitumor activity and proteolytic stability, and the α-linked d-monosaccharides can effectively improve the antitumor activity of HYL. Therefore, it is possible to design peptides with improved properties by varying the number, structure, and position of monosaccharides. What's more, the glycopeptides HYL-31 and HYL-33 show a promising prospect for antitumor and antimicrobial drugs development, respectively. In addition, we found that the d-lysine substitution strategy can significantly improve the proteolytic stability of HYL. Our new approach provides a reference or guidance for the research of novel antitumor and antimicrobial peptide drugs.

The Effect of Bee Venom Peptides Melittin, Tertiapin, and Apamin on the Human Erythrocytes Ghosts: A Preliminary Study

Red blood cells (RBCs) are the most abundant cells in the human blood that have been extensively studied under morphology, ultrastructure, biochemical and molecular functions. Therefore, RBCs are excellent cell models in the study of biologically active compounds like drugs and toxins on the structure and function of the cell membrane. The aim of the present study was to explore erythrocyte ghost’s proteome to identify changes occurring under the influence of three bee venom peptides-melittin, tertiapin, and apamin. We conducted preliminary experiments on the erythrocyte ghosts incubated with these peptides at their non-hemolytic concentrations. Such preparations were analyzed using liquid chromatography coupled with tandem mass spectrometry. It was found that when higher concentrations of melittin and apamin were used, fewer proteins were identified. Moreover, the results clearly indicated that apamin demonstrates the greatest influence on the RBCs ghosts proteome. Interestingly, the data also suggest that tertiapin exerted a stabilizing effect on the erythrocyte membrane. The experiments carried out show the great potential of proteomic research in the projects focused on the toxin’s properties as membrane active agents. However, to determine the specificity of the effect of selected bee venom peptides on the erythrocyte ghosts, further proteomic research should be focused on the quantitative analysis.

Characterization of L382 and S383 in the βDELSEED‐motif of ATP synthase

BackgroundAntibiotic resistance is rising on an alarming rate. Finding alternative ways to combat microbial infections is a matter of human survival. Selective inhibition of microbial ATP synthase has the potential to eradicate antibiotic resistant microbes. ATP synthase is the chief source of ATP production in almost all organisms. Antimicrobial properties of some venom peptides are connected to the binding and inhibition of ATP synthase. Peptides bind at the bDELSEED‐motif of E. coli ATP synthase and inhibit Escherichia coli ATP synthase to variable degrees. In order to understand the selective binding and inhibition of microbial ATP synthase it is essential to fully elucidate the binding site. Currently our lab is studying the role of Leu‐382 and Ser‐383 of the bDELSEED‐motif in peptide binding.MethodGrowth properties of wild type, null, and bDELSEED‐motif mutant E. coli strains were checked on minimal media with 3% glucose media and succinate media with 1/4th case amino acids. Wild type and mutant membrane bound F1Fo enzymes were isolated by growing on minimal media to late log phase. Cells were harvested in super centrifuge, French Pressed, and finally membrane‐bound ATP synthase isolated by ultracentrifugation. Purity of isolated protein was validated by western blot using anti‐F1‐β antibody. Biochemical inhibition of wild type and mutant ATP synthase was validated by NBD‐Cl. βLeu‐382 and βSer‐383 mutants were inhibited by honey bee venom peptide melittin. Time kinetics for full enzyme inhibition was determined along with reversibility of enzyme inhibition.ResultsOur growth results show that mutant strains grew between wild type and null strain on limiting glucose. Growth on succinate media corroborate the growth on limiting glucose. Melittin inhibited mutant enzymes to variable degrees. Moreover, we found that loss of βLeu‐382 and βSer‐383 residues caused reduced level of peptide binding at βDELSEED‐motif.ConclusionsWe conclude that residue Leu‐382 and Ser‐383 of βDELSEED‐motif are essential for proper orientation and binding of peptides at the peptide‐binding site. Moreover, βLeu‐382 and βSer‐383 play important role in maintenance of the βDELSEED‐motif.Support or Funding InformationThis work was supported by the KCOM Biomedical Sciences Graduate Program Grant Award Fund # 851‐044 to AS and ZA.

The membrane effects of melittin on gastric and colorectal cancer.

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6–24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 μg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.

Identification of Aethina tumida Kir Channels as Putative Targets of the Bee Venom Peptide Tertiapin Using Structure-Based Virtual Screening Methods.

Venoms are comprised of diverse mixtures of proteins, peptides, and small molecules. Identifying individual venom components and their target(s) with mechanism of action is now attainable to understand comprehensively the effectiveness of venom cocktails and how they collectively function in the defense and predation of an organism. Here, structure-based computational methods were used with bioinformatics tools to screen and identify potential biological targets of tertiapin (TPN), a venom peptide from Apis mellifera (European honey bee). The small hive beetle (Aethina tumida (A. tumida)) is a natural predator of the honey bee colony and was found to possess multiple inwardly rectifying K+ (Kir) channel subunit genes from a genomic BLAST search analysis. Structure-based virtual screening of homology modelled A. tumida Kir (atKir) channels found TPN to interact with a docking profile and interface “footprint” equivalent to known TPN-sensitive mammalian Kir channels. The results support the hypothesis that atKir channels, and perhaps other insect Kir channels, are natural biological targets of TPN that help defend the bee colony from infestations by blocking K+ transport via atKir channels. From these in silico findings, this hypothesis can now be subsequently tested in vitro by validating atKir channel block as well as in vivo TPN toxicity towards A. tumida. This study highlights the utility and potential benefits of screening in virtual space for venom peptide interactions and their biological targets, which otherwise would not be feasible.

Site-Specific Characterization of Intermediates in Folding-Tetramerization of Melittin by the Rapid Mix/Freeze Method and Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) NMR at Low Temperature (25K)

Protein folding study has been regarded very important for fundamental understanding of protein structures and functions. Although significant progresses have been made, the characterization of the kinetic transition pathways and intermediate states has been challenged and limited by either time or spatial resolutions. In this presentation, I will introduce a newly developed rapid mixing/freezing method for the sensitivity-enhanced solid-state NMR study at low temperature (25 K) by using the dynamic nuclear polarization (DNP) method. Our rapid mixer/freezer device can mix two solutions to introduce the folding within a couple of milliseconds and rapidly freeze at liquid nitrogen temperature (80K) within few microseconds to trap the intermediate states during the folding transition. In this study, the folding and tetramerization of a small bee venom peptide, melittin was investigated. Melittin, selectively isotopically 13C and 15N labeled to monitor the folding and the possible dimerization, underwent rapid pH change and was freeze-trapped at different time points (2, 4, 9, 30 ms). The low temperature MAS-DNP NMR spectra of frozen samples captured at varying transition time points revealed the site-specific changes of the simultaneous folding and oligomeric transitions (monomer-dimer-tetramer). The detail information of the mixer/freezer design, and the characterization of melittin intermediates and its pathway will be discussed in the presentation.

Inhibition of KACh channels by the bee venom peptide tertiapin-Q rescues inherited cardiac conduction defects and sino-atrial bradycardia and atrioventricular block in models of congenital dysfunction

Pacemaker activity of the sino-atrial node (SAN) generates the heart rate. SAN dysfunction leads to sick sinus syndrome (SSS) characterized by abnormally low heart rate (HR). The only currently available therapy for SSS is electronic pacemaker implantation. Mice lacking SAN L-type Ca v 1.3 Ca 2+ channels (Ca v 1.3 −/− ) present symptoms similar to those observed of SSS in humans especially SAN bradycardia and atrioventricular (AV) block. Cardiac voltage-gated Na+ channels (Nav) are essential for SAN impulse conduction through the AV node and the working myocardium. Mutations in the α-subunit of the cardiac Nav1.5 channel isoform are associated with inherited dysfunction of heart impulse conduction. We recently showed that genetic ablation of muscarinic-gated K + channels (I KAch ) rescues bradycardia of Ca v 1.3 −/− mice. We tested pharmacologic inhibition of I KAch by the peptide tertiapine-Q to rescue SSS and conduction dysfunction in Ca v 1.3 −/− mice having concurrent ablation of Ca v 1.3 and T-type Ca v 3.1 channels (Ca v 1.3 −/− /Ca v 3.1 −/− ) and Nav1.5 ± mice, which present ECG profiles similar to individuals carrying congenital bradycardia and conduction defects. We employed telemetric ECG recordings of SAN activity, HR and AV dysfunction in mice before and after administration of different doses of tertiapin-Q. Tertiapin-Q significantly improves the HR of Ca v 1.3 −/− (19%), Ca v 1.3 −/− /Ca v 3.1 −/− (23%) from doses of 0.1 to 5 mg/kg. HRs of tertiapine-Q-treated mice were similar to those recorded in untreated wild-type mice. Tertiapin-Q also improved cardiac conduction of Nav1.5 ± mice by 24%. Pharmacological inhibition of I KAch by tertiapin-Q prevents SAN dysfunction and improves conduction in three models of congenital bradycardia and impulse conduction defects suggesting the possibility of I KACh pharmacologic targeting to manage SAN and conduction disease.

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

BackgroundAmong 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.MethodsA 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.ResultsOn-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.ConclusionsLC-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.

220 - Inhibition of KACh channels by the bee venom peptide tertiapin-Q rescues sino-atrial bradycardia and atrioventricular block in models of congenital sino-atrial dysfunction

220 - Inhibition of KACh channels by the bee venom peptide tertiapin-Q rescues sino-atrial bradycardia and atrioventricular block in models of congenital sino-atrial dysfunction

Synthetic secapin bee venom peptide exerts an anti-microbial effect but not a cytotoxic or inflammatory response

Our previous study demonstrated that the secapin peptide from the venom of the Asiatic honeybee (Apis cerana, AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. In the present study, we investigated the anti-microbial activity and cytotoxicity of a synthetic AcSecapin-1 peptide. Seven synthetic AcSecapin-1 peptides (AcSecapin-S1 to AcSecapin-S7) were synthesized based on the peptide sequence of AcSecapin-1. AcSecapin-S1, which consists of the 25-amino acid sequence identical to that of the mature AcSecapin-1 peptide, exhibited the highest anti-microbial activity against bacteria and fungi. This was followed by AcSecapin-S6, which was missing 10 N-terminal amino acids and 6 C-terminal amino acids from AcSecapin-S1. Furthermore, AcSecapin-S1 was not cytotoxic and did not activate macrophages. Taken together, our data demonstrated that a synthetic AcSecapin-1 peptide could exhibit anti-microbial activity without producing a cytotoxic or inflammatory response, suggesting that this synthetic AcSecapin-1 peptide can be used as an anti-microbial agent for biomedical applications.

Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response.

Application of high performance liquid chromatography-tandem mass spectrometry for detection of melittin, a characteristic peptide of bee venom

Bee venom is widely used for the manufacturing of cosmetics and pharmaceuticals. A procedure for the detection of melittin, a characteristic component of bee venom, based on high performance liquid chromatography with tandem mass spectrometric detection is reported. Conditions of chromatography-mass spectrometric detection of melittin are selected, and a solid-phase extraction-based procedure for melittin isolation from preparations containing bee venom is developed. The procedure proposed is successfully tested on both raw venom samples and cosmetic and pharmaceutical preparations containing natural bee venom.