Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components

Abstract

We previously identified 92 toxin-like peptides and proteins, including pilosulin-like peptides 1⁻6 from the predatory ant Odontomachus monticola, by transcriptome analysis. Here, to further characterize venom components, we analyzed the venom and venom sac extract by ESI-MS/MS with or without trypsin digestion and reducing agent. As the low-molecular-mass components, we found amino acids (leucine/isoleucine, phenylalanine, and tryptophan) and biogenic amines (histamine and tyramine) in the venom and venom sac extract. As the higher molecular mass components, we found peptides and proteins such as pilosulin-like peptides, phospholipase A₂s, hyaluronidase, venom dipeptidyl peptidases, conotoxin-like peptide, and icarapin-like peptide. In addition to pilosulin-like peptides 1⁻6, we found three novel pilosulin-like peptides that were overlooked by transcriptome analysis. Moreover, pilosulin-like peptides 1⁻6 were chemically synthesized, and some of them displayed antimicrobial, hemolytic, and histamine-releasing activities.

Keywords: ant; mass spectrometry analysis; pilosulin-like peptide; venom.

Figure 1

Low-molecular-mass components of O. monticola venom and venom sac extract as analyzed by LC-ESI-MS. (A) Selected ion chromatograms of total ion chromatogram, (B) leucine/isoleucine, (C) phenylalanine, (D) tryptophan, (E) histamine, and (F) tyramine. The observed and calculated m/z values are shown with the corresponding structural formulae.

Figure 2

Multiple alignment, identity matrix, and phylogenic analysis of pilosulin-like peptides. (A) The amino acid sequences of melittin and pilosulin-like peptides were aligned with ClustalW in Lasergene 12 (DNASTAR, Madison, WI, USA) and manually modified. Arrows indicate the putative processing and modification sites for signal peptidase, dipeptidyl peptidase, amidatinglyase, and carboxypeptidase. Proline and alanine residues in the spacer region between the signal and mature peptides of pilosulin-related peptides are highlighted in yellow. Nucleotide sequences for pilosulin-like peptides 7, 8, and 9 were assigned DDBJ/EMBL/GenBank Accession Numbers LC416796–LC416798, respectively. (B) Percentage amino acid sequence identities between melittin and pilosulin-like peptides are shown. (C) The alignment of pilosulin-like peptides, pilosulin 1, and melittin precursors by ClustalV in Lasergene 12 was used to construct a phylogenic tree using the neighbor-joining (NJ) method. The phylogenic tree rooted with the amino acid sequence of melittin. The numbers above the branches indicate the percentage of 1000 bootstrap replicates.

Figure 3

High-molecular-mass components of O. monticola venom and venom sac extract as analyzed by LC-ESI-MS. (A) The patterns of the total ion current of O. monticola venom and venom sac extract under nonreducing and (B) reducing conditions are shown. Peaks containing pilosulin-like peptides are labeled by arrows.

Figure 4

Dimer formation of pilosulin-like peptides 4 and 7. Monomers of pilosulin-like peptides 4 and 7 were connected by a disulfide bridge at the amino acid position 21. Unique amino acid residues in pilosulin-like peptides 4 and 7 are highlighted in yellow and red, respectively. C* indicates S-(carbamoylmethyl)-L-cysteine.

Figure 5

Amino acid sequences and helical wheel projection of pilosulin-like peptides 2, 3, 4, 6, and 7. (A) Amino acid sequences of mature pilosulin-like peptides. The cationic amphipathic helix regions in pilosulin-like peptides (PLP) 2, 3, 4, 6, and 7 predicted by HeliQuest are highlighted in red. (B) Helical wheel projections of pilosulin-like peptides 2, 3, 4, 6, and 7 drawn by HeliQuest. Nonpolar amino acids (F, I, L, M, V, and W), basic amino acids (K), acidic amino acids (D), small polar amino acids (A and G), aromatic polar amino acids (H), hydroxyl-containing polar amino acids (S and T), amide-containing polar amino acids (N), and proline (P) are highlighted by yellow, blue, red, gray, sky-blue, purple, pink, and green color, respectively.