Identification of Venom Components from Aculeate Hymenoptera and Characterization of Their Properties

Abstract

To identify the venom components and their expression patterns of some Aculeata bees/wasps, venom gland-specific transcriptome analysis was conducted. FPKM values were normalized with the average of the transcription level of reference gene (-tubulin). Common components in both solitary and social wasp venoms include hyaluronidase, phospholipase A2, metalloendopeptidase, etc. Although it has been expected that more diverse bioactive components with the functions of prey inactivation and physiology manipulation are present in solitary wasps, the information on venom compositions of solitary wasps obtained in this study was not sufficient to generalize this notion. Nevertheless, some neurotoxic peptides (e.g., pompilidotoxin and dendrotoxin-like peptide) and proteins (e.g., insulin-like peptide binding protein) appear to be specific to solitary wasp venom. In contrast, several proteins, such as venom allergen 5 protein, venom acid phosphatase, and various phospholipases, appear to be relatively more abdundant in social wasp venom. In the venom gland transcriptome of bumblebees, major allergens or pain producing factors were barely identified, implying that bumblebee venoms are relatively less toxic than those of social or solitary wasps. Finally, putative functions of main venom components and their application are also discussed. <br/> To investigate the differences in venom properties and toxicities between Vespa crabro and V. analis, the transcriptomic profiles of venom glands, along with the venom components, were analyzed and compared. A total of 35 venom-specific genes were identified in both venom gland transcriptomes, but their transcriptional profiles were different between V. crabro and V. analis. In addition, the major venom components were identified and confirmed by mass spectroscopy. In general, most major venom genes were more abundantly expressed in V. crabro, whereas some minor venom genes exhibited higher transcription rates in V. analis, including muscle LIM protein, troponin, paramyosin, calponin, etc. Our findings reveal that the overall venom components of V. crabro and V. analis are similar, but that their expression profiles and levels are considerably different. The comparison of venom gland transcriptomes suggests that V. crabro likely produces venom with more highly enriched major venom components, which has potentially higher toxicity compared with V. analis venom.<br/> To examine the differences in the potential toxicity and bioactivity of mastoparans, vespid chemotactic peptides, vespakinins and bombolitins of V. crabro, V. analis, Parapolybia varia, V. mandarina, Bombus ardens, B. consobrinus, B. terrestris and B. ussurensis, differential toxicological and pharmacological activities of synthesized venom peptides were investigated.<br/> The hemolytic, antimicrobial, and antitumor activities of synthesized V. analis mastoparan were higher than those of V. crabro mastoparan. These differential bioactivities are likely due to the amino acid sequence differences in the mature peptides.<br/> PvVCP, PvVespk, and VmVespk showed little to low hemolytic activities. Only VmVCP showed hemolytic activity at a high concentration. Among the four peptides tested, VmVCP showed both anti-microbial and anti-fungal activities, whereas PvVCP showed only anti-fungal activity to Candida albicans. Interestingly, PvVCP showed significantly stronger anti-tumor activities to two ovarian cancer cell lines compared with VmVCP. Vespks only showed anti-tumor activity to SK-OV-3 cells but not to NIH-OVCAR-3 cells.<br/> Among the four bombolitins tested, bombolitin T showed the highest hemolytic and anti-tumor activities. All bombolitins exhibited strong anti-microbial and anti-fungal activities, and bombolitin A specifically possessed the highest anti-microbial activity against the Gram-negative bacteria Escherichia coli.<br/> To design selective anti-tumor peptides with reduced hemolytic activity, bombolitin T was chosen as a model to be modified by amino acid substitution. The lowest hemolytic activity was observed with the addition of a hydrophobic amino acid (Ile14) to the C-terminal end as well as the substitution of Ser10 and Leu12 with a hydrophobic amino acid (Leu) and positively charged amino acid (Lys), respectively. This finding implies that the balanced substitution of hydrophobic and positively charged amino acids could affect the cytotoxicity. This study provides new information on the properties of anti-tumor peptides in the venoms of bumblebees and the basic approaches for peptide design tools for the reduction of cytotoxicity.<br/> <br/> Key words: venom gland, venom peptide, transcriptional profiles, RNA sequencing, circular dichroism, biological acitivity, principal component analysis, peptide substitution<br/>