Expression profiles of venom components in some social hymenopteran species over different post-capture periods.

To explore and compare the expression patterns of venom components depending on post-capture periods, venom gland-specific transcriptome and proteome analyses were conducted for five model hymenopteran species at a series of time points after capture. Venom gland-specific genes with signal sequences were considered as putative venom component genes. Expression patterns of venom gland-specific genes in all the social wasps and bees examined varied considerably depending on the post-capture period. Higher numbers of venom genes exhibited a decreasing expression pattern than an increasing pattern as the capture period increased. For example, genes encoding most of the allergens (dipeptidyl peptidase 4, endocuticle structural glycoprotein, odorant-binding protein, phospholipase A1, A2, B1, serine protease, serine protease inhibitor and venom allergen 5), pain-producing factor (mast cell degranulating peptide), and paralyzing factor (neprilysin) commonly exhibited decreasing expression patterns in all of the hymenopteran species tested, except for some of the major venom genes in Apis mellifera and Bombus ignitus, which showed an increasing pattern. These findings indicate species- or group-specific variations in the expression patterns of major venom genes. Taken together, flash freezing in liquid nitrogen immediately after capture was determined to be the best way to obtain the most natural expression profiles of venom components in social wasp species, thus, enabling a better understanding of the toxic potential of venom in wasp sting accidents. This study provides guidance for establishing optimal protocols for venom gland isolation and venom extraction from wasps and bees that can ensure the most naturally represented venom composition.

Lipolytic Activity of a Carboxylesterase from Bumblebee (Bombus ignitus) Venom.

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme's role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

Alteration of Bumblebee Venom Composition toward Higher Elevation.

Venomous animals use venom, a complex biofluid composed of unique mixtures of proteins and peptides, for either predation or defense. Bumblebees, which occur in various habitats due to their unique thermoregulatory properties, mainly use venom for defense. Herein, we conducted an exploratory analysis of the venom composition of a bumblebee species (Bombus pascuorum) along an elevation gradient in the western Swiss Alps using shot-gun proteomic approaches to assess whether their defense mechanism varies along the gradient. The gradient was characterized by high temperatures and low humidity at low elevations and low temperatures and high humidity at high elevations. Venom composition is changing along the elevation gradient, with proteomic variation in the abundances of pain-inducing and allergenic proteins. In particular, the abundance of phospholipase A2-like, the main component of bumblebee venom, gradually decreases toward higher elevation (lower temperature), suggesting venom alteration and thus a decrease in bumblebee defense towards harsher environments. Larger datasets may complement this study to validate the observed novel trends.

Potential Application of Venom Proteins in Designing of Medicines for Treating Human Neurodegenerative Disorders.

BACKGROUND: Neurodegenerative disorder are persistently increasing and relentlessly affecting the individuals, families and society as whole. Regrettably these disorders are resistant to the available drugs, the outcomes are only palliative while the side effects of the therapy harm the patient compliance as well as treatment. Drugs from venomous source have been considered as an effective alternative for such types of disorders, particularly neurodegenerative diseases. Due to emerging advancement in the field of proteomics, genomics and molecular biology, characterization and screening of these novel compounds become more assessable. CONCLUSION: In this reverence, the present study reviews the current consideration of the mode of action and the future prediction concerning the use of novel compounds isolated from arthropods and other venomous animals in the treatment of major neurodegenerative diseases such as Parkinson disease, Alzheimer disease, Multiple Sclerosis, Epilepsy and Amyotrophic Lateral Sclerosis.

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.

Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.

Honeybee venom proteome profile of queens and winter bees as determined by a mass spectrometric approach.

Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.

The genomes of two key bumblebee species with primitive eusocial organization.

BACKGROUND: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. RESULTS: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. CONCLUSIONS: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.

Expression and anticancer activity analysis of recombinant human uPA1­43-melittin.

The present study is focused on expression of a target fusion protein which can be used in ovarian cancer target therapy. It aimed to construct human urokinase-type plasminogen activator (uPA)(1-43)-melittin eukaryotic expression vector to express recombinant human uPA(1-43)-melittin (rhuPA(1-43)-melittin) in P. pastoris and to detect its anticancer effects on ovarian cancer cells. The DNA sequences that encode uPA1-43 amino acids and melittin were synthesized according to its native amino acid sequences and consequently inserted into pPICZαC vector. Then uPA1-43-melittin -pPICZαC was transformed into P. pastoris X-33, and rhuPA(1-43)-melittin was expressed by methonal inducing. The bioactivities of recombinant fusion protein were detected with inhibition effects on growth of ovarian cancer cells, cell cycle detection and TUNEL assay. The results of DNA sequence analysis of the recombinant vector uPA(1-43)-melittin -pPICZαC demonstrated that the DNA encoding human uPA 1-43 amino acids and 1-26 amino acids of melittin was correctly inserted into the pPICZαC vector. After being induced by methonal, fusion protein with molecular weight 7.6 kDa was observed on the basis of SDS-PAGE and western blot analysis. The recombinant protein was able to suppress growth of SKOV3, induce cell cycle arrest and apoptosis of SKOV3 cells. The fusion protein does not have any obvious toxicity on normal tissues. RhuPA(1-43)-melittin was successfully expressed in P. pastoris. Taking uPA(1-43) amino acids specifically binding to uPAR as targeted part of fusion protein, and making use of antitumor activity of melittin, the recombinant fusion protein it was able to inhibit growth of ovarian tumors and to be applied for effective targeted treatment.

Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed.

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic ß-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.

Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland.

BACKGROUND: Honeybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment. RESULTS: Of the 43 proteins identified in GV, < 40% were venom toxins, and >60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified. CONCLUSIONS: Our data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom.

The importance of tissue specificity for RNA-seq: highlighting the errors of composite structure extractions.

BACKGROUND: A composite biological structure, such as an insect head or abdomen, contains many internal structures with distinct functions. Composite structures are often used in RNA-seq studies, though it is unclear how expression of the same gene in different tissues and structures within the same structure affects the measurement (or even utility) of the resulting patterns of gene expression. Here we determine how complex composite tissue structure affects measures of gene expression using RNA-seq. RESULTS: We focus on two structures in the honey bee (the sting gland and digestive tract) both contained within one larger structure, the whole abdomen. For each of the three structures, we used RNA-seq to identify differentially expressed genes between two developmental stages, nurse bees and foragers. Based on RNA-seq for each structure-specific extraction, we found that RNA-seq with composite structures leads to many false negatives (genes strongly differentially expressed in particular structures which are not found to be differentially expressed within the composite structure). We also found a significant number of genes with one pattern of differential expression in the tissue-specific extraction, and the opposite in the composite extraction, suggesting multiple signals from such genes within the composite structure. We found these patterns for different classes of genes including transcription factors. CONCLUSIONS: Many RNA-seq studies currently use composite extractions, and even whole insect extractions, when tissue and structure specific extractions are possible. This is due to the logistical difficultly of micro-dissection and unawareness of the potential errors associated with composite extractions. The present study suggests that RNA-seq studies of composite structures are prone to false negatives and difficult to interpret positive signals for genes with variable patterns of local expression. In general, our results suggest that RNA-seq on large composite structures should be avoided unless it is possible to demonstrate that the effects shown here do not exist for the genes of interest.

Conduction and block of inward rectifier K+ channels: predicted structure of a potent blocker of Kir2.1.

Dysfunction of Kir2.1, thought to be the major component of inward currents, I(K1), in the heart, has been linked to various channelopathies, such as short Q-T syndrome. Unfortunately, currently no known blockers of Kir2.x channels exist. In contrast, Kir1.1b, predominantly expressed in the kidney, is potently blocked by an oxidation-resistant mutant of the honey bee toxin tertiapin (tertiapin-Q). Using various computational tools, we show that both channels are closed by a hydrophobic gating mechanism and inward rectification occurs in the absence of divalent cations and polyamines. We then demonstrate that tertiapin-Q binds to the external vestibule of Kir1.1b and Kir2.1 with K(d) values of 11.6 nM and 131 µM, respectively. We find that a single mutation of tertiapin-Q increases the binding affinity for Kir2.1 by 5 orders of magnitude (K(d) = 0.7 nM). This potent blocker of Kir2.1 may serve as a structural template from which potent compounds for the treatment of various diseases mediated by this channel subfamily, such as cardiac arrhythmia, can be developed.

Molecular cloning and antifibrinolytic activity of a serine protease inhibitor from bumblebee (Bombus terrestris) venom.

Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A(2) (PLA(2)), serine proteases, and serine protease inhibitors. In this study, we identified a bumblebee (Bombus terrestris) venom serine protease inhibitor (Bt-KTI) that acts as a plasmin inhibitor. Bt-KTI consists of a 58-amino acid mature peptide that displays features consistent with snake venom Kunitz-type inhibitors, including six conserved cysteine residues and a P1 site. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. The recombinant peptide demonstrated properties similar to Kunitz-type trypsin inhibitors. Bt-KTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, Bt-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. These findings demonstrate the antifibrinolytic role of Bt-KTI as a plasmin inhibitor.

Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants.

BACKGROUND: Specific IgE (sIgE) antibodies to both bee and wasp venom can be due to a sensitivity to both insect venoms or due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: Investigating whether a basophil activation test (BAT) with both venoms as well as with bromelain and horseradish peroxidase (HRP) or recombinant allergen-based IgE testing can improve the diagnostic procedure. METHODS: Twenty-two Hymenoptera-venom allergic patients with sIgE antibodies to both bee and wasp venom were studied. sIgE antibodies to MUXF3 CCD, bromelain, HRP, rApi m 1, and rVes v 5 were determined, and a BAT (Flow2 CAST) with venom extracts, bromelain, and HRP was performed. Further recombinant allergen-based IgE testing was done by using an ELISA, if required. The reactivity of basophils was calculated from the insect venom concentration at half-maximum stimulation. RESULTS: Double positivity/double negativity/single positivity to rApi m 1 and rVes v 5 was seen in 12/1/9 patients. Further recombinant allergen-based IgE testing in the last ones revealed positive results to the other venom in all cases except one. BAT was double positive/double negative/single positive in 6/2/14 patients. Four patients with negative results in sIgE antibodies to CCDs had positive results in BAT. BAT with bromelain/HRP showed a sensitivity of 50%/81% and a specificity of 91%/90%. CONCLUSION: Component-resolved IgE testing elucidates the pattern of double positivity, showing a majority of true double sensitizations independent of CCD sensitization. BAT seems to add more information about the culprit insect even if the true clinical relevance of BAT is not completely determined because of ethical limitations on diagnostic sting challenges. BAT with HRP is a good method to determine sensitivity to CCDs.

Location and reduction of icarapin antigenicity by site specific coupling to polyethylene glycol.

Icarapin is a bee venom protein found to induce IgE-mediated allergic reaction. In this study, icarapin of Asian honey bee was cloned and sequenced. By in silico screening, S198 was found to be the potential antigenic site. This site was changed to cysteine and coupled with PEG5K. Compared to the wild type icarapin and the S198C variant, PEGylated S198C variant induced lower level of IgG and IgE antibodies in mice, showing that it is indeed located in an antigenic site. Our work may be generalized to other proteins for the discovery of antigenic sites and the reduction of antigenicity.

Fibrin(ogen)olytic activity of bumblebee venom serine protease.

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.

Api m 10, a genuine A. mellifera venom allergen, is clinically relevant but underrepresented in therapeutic extracts.

BACKGROUND: Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. METHODS: Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. RESULTS: Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. CONCLUSION: Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy.

Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell.

Bee venom phospholipase A(2) (BvPLA(2)) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA(2) (AccPLA(2)) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA(2) fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA(2) fusion protein was cross reactive with the anti-AmPLA(2) (BvPLA(2) of the European honeybee, Apis mellifera) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA(2) in Western blot analysis. The PLA(2) activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 micromol/(min.mg). In summary, the recombinant AccPLA(2) protein, a native BvPLA(2)-like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA(2) in the pharmaceutical industry.