Abstract
BackgroundMass 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.MethodsThe 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.ResultsThree 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.ConclusionWe 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.
Highlights
Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner
The significant biological effects of the crude venom has been suggested, the biologically active peptides of the carpenter bee venom including that of X. appendiculata has not been isolated yet;
MALDI-TOF MS and Tandem mass spectrometry (MS/MS) analysis Matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) analysis was performed by UltraFlex speed (Bruker Daltonics, Germany)
Summary
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). In conjunction with our continuous research program on the isolation and biological study of the Hymenoptera venom substances, we were interested in the venom of the Japanese carpenter solitary bee Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). We considered that the target venom is a challenging sample because of the following reasons: the crude venom of carpenter bees showed significant biological effects such as lethal activities in a small bird and mice [15]; the sting of Xylocopa virginica and Xylocopa vioracea seems to be as painful in humans as are honeybee stings [15]; the significant biological effects of the crude venom has been suggested, the biologically active peptides of the carpenter bee venom including that of X. appendiculata has not been isolated yet; it is difficult to collect X. appendiculata because of their solitary lives; and only a small amount of the venom substances is available due to the fact that the venom sac of X. appendiculata is smaller than those of honeybees and vespid wasps (Fig. 1)
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