Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes

Abstract

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

Fig. 1.

RP-HPLC chromatograms of DVEs from Bothrops cotiara (BC), B. fonsecai (BF), and B. jararaca (BJ) obtained by solid phase extraction.

Fig. 2.

CID MS/MS spectra of de novo sequenced peptides. A, Spectrum of the 39 amino acid peptide [I/L]TEPVV[I/L]NFFAGEYTA[Q/K]AHGW[I/L]DST[I/L][Q/K]SRDAARDVNRAS from a LAAO found in the venom of BC at m/z 862.2 and charge +5. The spectrum was smoothed and deisotoped to singly-charged states and after interpretation, ion series were labeled by the module BioLynx (MassLynx 4.1, Waters, Milford, MA) using a mass window of ± 1.0 Da because of a m/z shift of 0.2 from theoretical value; B, Spectrum of the peptide HHDHHAAVGGGGGGGGGGA (Bot.ja pHpG-1) found in the venom of BJ at m/z 522.3 and charge +3. The spectrum was smoothed and deisotoped to singly charged states and after interpretation, ion series were labeled by the module BioLynx using a mass window of ± 0.3 Da.

Fig. 3.

Heat maps of amino acid cleavage frequencies in positions P4-P4′ for all identified peptides in the venoms of BC, BF, and BJ venoms analyzed in the absence (A) or in the presence of proteinase inhibitors (B). Heat maps of the difference in amino acid cleavage frequencies in positions P4-P4′ for all identified peptides in the venoms of BC, BF and BJ analyzed in the presence and in the absence of proteinase inhibitors (C).

Fig. 4.

CID MS/MS spectra of peptides identified by automated search. A, Spectrum of peptide HLEKNKGLFSKDY from the pro-domain of bothropasin found in the venom of BF at m/z 527.0 and charge +3; B, Spectrum of the 37 amino acid peptide SVNVDASLANLEVWSKKDLIKVEKDSSKTLTSFGEWR from the SVMP insularinase-A found in the venom of BJ at m/z 836.9 and charge +5. The spectra were smoothed and deisotoped to singly-charged states and ion series were labeled by the module BioLynx using a mass window of ±0.3 Da.

Fig. 5.

Peptides identified from the best LAAO hit (Bp-LAAO from B. pauloensis) in the venoms of BC, BF and BJ were superimposed in the crystallographic structure of Cr-LAAO (47). Structure parts shown in yellow indicate the homologous or identical peptides identified in the venoms. A, C, and E, are from the venoms of BC, BF, and BJ analyzed in the absence of proteinase inhibitors, respectively; B, D, and F are from the venoms of BC, BF, and BJ analyzed in the presence of proteinase inhibitors, respectively.

Fig. 6.

Evaluation of BK hypotensive effects in anesthetized Wistar rats before and after the administration of BPPs. Intravenous bolus injection of each BPP was made at the dose of 60 nmol. A, BPP-10c (n = 4); B, BPP-10d (n = 5); C, BPP-10e (n = 4); D, BPP-10f (n = 5); E, BPP-11f (n = 5) and F, BPP-11h (n = 4). Data are expressed as mean ± S.E. * p < 0.05; ** p < 0.01 and *** p < 0.001 compared with the values obtained with 0.5 μg of BK before peptide injection.