Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Abstract

Background: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities.

Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database.

Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%).

Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.

Keywords: ACE inhibitors; Tityus serrulatus; enzymes; proteases; proteome; scorpion venom.

Figure 1.. A: Chromatographic profile of Tityus serrulatus venom in FPLC system with CMC-52 column. Fractionation of Ts venom (50 mg) was performed in a FPLC system with CMC-52 column, equilibrated with buffer A (50 mM ammonium bicarbonate, pH 7.8), under a flow rate of 0.5 mL/min and 25 °C. Sample (2 mL) was initially eluted with buffer A, followed by a linear concentration gradient (0 to 100%) of buffer B (0.6 M ammonium bicarbonate, pH 7.8), represented by the green line. Volume collected per tube: 4.0 mL. B: Electrophoretic profile of the fractions from the CMC-52 chromatography in 16.5% Tricine-SDS-PAGE. Gel was stained with PlusOne Coomassie Blue PhastGel^® R-350 and destained with 10% acetic acid (v/v).

Figure 2.. A: Trypsin and chymotrypsin activity assay in the presence of fraction I in two concentrations (1.90 mg/mL and 5.65 mg/mL). The absorbance was determined at 410 nm. *p<0.05 Fr I (5.65 mg/mL) + Trypsin + substrate vs Trypsin + substrate; #p<0.05 Fr I (1.90 mg/mL) + Trypsin + substrate vs Trypsin + substrate. B: Proteolytic activity of Fraction I (10 μL, 47 μg/μL) over azocasein in the absence and presence of inhibitors of metalloprotease (EDTA) and serine protease (PMSF). The absorbance was determined at 450 nm. *p<0.05 Fr I + EDTA and Fr I + PMSF vs Fr I. C: Inhibition assay of ACE activity by Fraction I (48.4 mg/mL). Reactions were read at 492 nm. *p<0.05 ACE + Fr I vs ACE. C-: negative control; Fr: Fraction I. Values are expressed as mean ± SD.

Figure 3.. Relative distribution of venom protein classes in Fraction I from Ts venom determined by shotgun-proteomics. AMPs: antimicrobial peptides; CRISPs: cysteine-rich secretory proteins; Other enzymes: lysozyme, chymotrypsin and peptidylglycine alpha-amidating monooxygenase.

Figure 4.. De novo sequencing of the CRISP identified in the Fraction I using the JAW07031.1 (putative cysteine-rich protein from Tityus serrulatus) as a template.