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Research article - Vol. 23, 2017 |
Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata
1 Graduate School of Material Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan.
2 Graduate School of Science, Department of Biology & Geosciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan.
3 Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan.
4 Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan.
5 Research Institute for Chemical Process Technology, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
6 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
ABSTRACT
Background
Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet.
Methods
The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests.
Results
Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom.
Conclusion
We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.
Key words: Xylocopa appendiculata; Carpenter bee; Venom peptides; Solitary bee; Mass spectrometry analysis
Funding
This work was supported by JSPS KAKENHI, grant number 15 K01814.
Received: January 27, 2017.
Accepted: May 9, 2017.
Revised: May 23, 2017.
Correspondence: shinada@sci.osaka-cu.ac.jp