Enzymatic characterization and proteomic profiling of venoms from the medically important Androctonus species Alhussin M.A. Megaly, Masahiro Miyashita, Abdulaziz R. Alqahtani, Mohammed Abdel-Wahab Journal of Genetic Engineering and Biotechnology, 2025 Egyptian scorpions of the Androctonus genus (family Buthidae) produce life-threatening stings owing to their neurotoxic venom. However, the composition and enzymatic activities of their venoms remain poorly understood: We used electrophoresis to analyze the protein components of venoms collected from three Androctonus species: Androctonus amoreuxi , Androctonus australis , and Androctonus bicolor . Mass spectrometric analysis was performed to characterize the peptides present in these venoms. The phospholipase A 2 (PLA 2 ), hyaluronidase, and protease activities of the venoms were examined to gauge their potential contribution to venom toxicity. Finally, the antibacterial and hemolytic activities of the venoms were evaluated. The electrophoretic profiles of the three venoms showed features specific to each species, with distinct protein bands observed at 75, 74, 67, 48, 46, 40, and 28 kDa, along with a notable band above the 15-kDa mark. Liquid chromatography/mass spectrometry analyses were used to detect the presence of 369, 324, and 351 components in with molecular masses in the range of 500–10,000 Da in the venoms of A. amoreuxi , A. australis , and A. bicolor , respectively. Disulfide-rich peptides (three disulfide bridges) were abundant, but peptides without disulfide bonds were also detected in all venom samples. All three venoms exhibited hyaluronidase activities, whereas protease and PLA 2 activities were either weak (at 1 µg and 10 µg) or undetectable, even at higher concentrations (up to 20 µg). All assays were performed using venoms standardized by dry weight to ensure consistent protein quantities. Crude venoms of A. amoreuxi and A. australis showed antibacterial activity against E. coli and B. subtilis (5–10 μg), whereas A. bicolor required 10 μg. Hydrophobic fractions (40–55 min) of A. australis alone retained this activity. This work furthers our knowledge of the enzymatic and peptide composition of Androctonus venoms, unveiling their potential in drug delivery enhancement and other biomedical applications. These findings will inform the development of better strategies for the treatment and prevention of scorpion envenomation.
Structural Determination of Bl-3, an Insecticidal Peptide from the Buthacus leptochelys Scorpion Venom Ryo Shimase, Yusuke Yoshimoto, Alhussin Mohamed Abdelhakeem Megaly, Mohammed Abdel-Wahab, Yoshiaki Nakagawa, Masahiro Miyashita Mass Spectrometry, 2025 Scorpion venoms contain a variety of peptides that exhibit toxicity toward insects or mammals by acting on ion channels.We previously isolated four insecticidal peptides (Bl-1, Bl-2, Bl-3, and Bl-4) from the venom of Buthacus leptochelys.Among these, the complete amino acid sequence of Bl-1 was determined, whereas only N-terminal partial sequences were obtained for the others.In the present study, we determined the complete sequence of Bl-3 through de novo sequencing of enzymatically digested fragments.The discrimination between Leu and Ile was achieved based on side-chain fragmentation observed under high-energy CID conditions.Bl-3 was identified as a 65-residue peptide containing four disulfide bonds.During the sequencing analysis, deamidation of the Asn residue at position 30 was observed, which is likely to have occurred after the purification step.Sequence comparison revealed that Bl-3 shares high similarity with -toxins that act on sodium channels and exhibit non-selective toxicity toward both insects and mammals.These findings suggest that Bl-3 is likely to exert non-selective toxicity through a mechanism similar to that of -toxins.
Molecular Diversity of Linear Peptides Revealed by Transcriptomic Analysis of the Venom Gland of the Spider Lycosa poonaensis Alhussin Mohamed Abdelhakeem Megaly, Masahiro Miyashita, Mohammed Abdel-Wahab, Yoshiaki Nakagawa, Hisashi Miyagawa Toxins, 2022 Spider venom is a complex mixture of bioactive components. Previously, we identified two linear peptides in Lycosa poonaensis venom using mass spectrometric analysis and predicted the presence of more linear peptides therein. In this study, a transcriptomic analysis of the L. poonaensis venom gland was conducted to identify other undetermined linear peptides in the venom. The results identified 87 contigs encoding peptides and proteins in the venom that were similar to those in other spider venoms. The number of contigs identified as neurotoxins was the highest, and 15 contigs encoding 17 linear peptide sequences were identified. Seven peptides that were representative of each family were chemically synthesized, and their biological activities were evaluated. All peptides showed significant antibacterial activity against Gram-positive and Gram-negative bacteria, although their selectivity for bacterial species differed. All peptides also exhibited paralytic activity against crickets, but none showed hemolytic activity. The secondary structure analysis based on the circular dichroism spectroscopy showed that all these peptides adopt an amphiphilic α-helical structure. Their activities appear to depend on the net charge, the arrangement of basic and acidic residues, and the hydrophobicity of the peptides.
Characterization of 2 linear peptides without disulfide bridges from the venom of the spider Lycosa poonaensis (Lycosidae) Alhussin M A Megaly, Yusuke Yoshimoto, Yugo Tsunoda, Masahiro Miyashita, Mohammed Abdel-Wahab, Yoshiaki Nakagawa, Hisashi Miyagawa Bioscience Biotechnology and Biochemistry, 2021 Spider venom is a complex mixture of bioactive components, in which peptides play an important role by showing neurotoxicity or cytotoxicity. Disulfide-rich peptides are major components in the venom, but linear peptides without disulfide bridges are also present and often show antimicrobial activity. In this study, we analyzed the venom of the spider Lycosa poonaensis (Lycosidae) to find novel antimicrobial peptides using mass spectrometry. The result revealed that 120 out of 401 detected components were nondisulfide-bridged peptides. From them, the sequence of 2 peptides (lyp2370 and lyp1987) were determined by MS/MS analysis. The biological activity test revealed that lyp2370 has only weak antibacterial activity. On the other hand, lyp1987, which is identical to M-lycotoxin-Ls3b from the Lycosa singoriensi venom, showed significant antibacterial activity. The weak activity of lyp2370 was found to be due to the presence of a Glu residue on the hydrophilic face of its amphipathic α-helical structure.
A Fluorescent Compound from the Exuviae of the Scorpion, Liocheles australasiae Yusuke Yoshimoto, Masato Tanaka, Masahiro Miyashita, Mohammed Abdel-Wahab, Alhussin M. A. Megaly, Yoshiaki Nakagawa, Hisashi Miyagawa Journal of Natural Products, 2020 Most scorpions fluoresce under UV light. To date, two types of fluorescent compounds have been identified in scorpions, but it has been assumed that other unknown compounds may be responsible for the fluorescence. In this study, we isolated a fluorescent compound from the exuviae of the scorpion Liocheles australasiae identified as a macrocyclic diphthalate ester with a molecular mass of 496.2 Da. The same compound was also detected in extracts from four other scorpion species. This suggests that this compound is shared by multiple scorpion species, although its contribution to the cuticle fluorescence may be relatively small.
