Article pubs.acs.org/jpr
Moving Pieces in a Venomic Puzzle: Unveiling Post-translationally Modified Toxins from Tityus serrulatus Thiago Verano-Braga,*,†,§ Alexandre A. A. Dutra,§,‡ Ileana R. León,† Marcella N. Melo-Braga,† Peter Roepstorff,† Adriano M. C. Pimenta,‡ and Frank Kjeldsen† †
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil
‡
S Supporting Information *
ABSTRACT: Besides being a public health problem, scorpion venoms have a potential biotechnological application since they contain peptides that may be used as drug leads and/or to reveal novel pharmacological targets. A comprehensive Tityus serrulatus venom proteome study with emphasis on the phosphoproteome and N-glycoproteome was performed to improve our knowledge on the molecular diversity of the proteinaceous toxins. We combined two peptide identification methodologies, i.e., database search and de novo sequencing, to achieve a more comprehensive overview of the molecular diversity of the venoms. A total of 147 proteins were identified, including neurotoxins, enzymes, bradykinin-potentiating peptides, and molecules with antimicrobial and diuretic activities. Among those, three proteins were found to be phosphorylated, and one N-glycosylated. Finally, cleavage of toxin polypeptide chains seems to be a common post-translational modification in the venom since 80% of the identified molecules were, in fact, products of toxins proteolysis. KEYWORDS: Tityus serrulatus, venomics, venom proteome, top-down, bottom-up, PEAKS, post-translational modifications, PTMs, scorpion venom, animal venom parthenogenetic reproduction.1 Scorpion sting is therefore not an unusual event in this country, where in 2010 a total of 50,126 cases (26.3 stings per 100,000 inhabitants), leading to 88 deaths (0.2% mortality), were reported by the Brazilian Ministry of Health.2 Aside from their associated public health problems, scorpion venoms have a huge potential for biotechnological applications (e.g., prospective new drug leads),3,4 and toxins can be useful molecular tools to investigate ion channels structures and functions.5,6 Scorpion venoms are a complex mixture of nucleotides, lipids, biogenic amines, enzymes, and peptides, including disulfidebridged peptides (DBPs) that modulate ion channels activities7−9 and non-disulfide-bridged peptides (NDBPs) with reported vascular10−12 or antimicrobial13−15 activities. Classically, the molecular diversity of scorpion toxins has been assessed by using a function-to-structure approach, which relies on the ability of the toxins to induce conspicuous pharmacological effects.16 This approach led to the identification of a large number of lethal scorpion toxins and provided knowledge of their structures and biological activities.17,18 However, toxins without any apparent toxicity might not be identified using the above-mentioned approach. Only in the beginning of the past
1. INTRODUCTION Tityus serrulatus belongs to the Buthidae scorpion family and is found mainly in the highly populated Southeastern region of Brazil (Figure 1). This species is well adapted to urban environments and spreads fast as an outcome of its
Figure 1. Geographical distribution. Tityus serrulatus, also known as the Brazilian yellow scorpion, is found mainly in the southeastern region of Brazil. The specimens used in this study were collected at Belo Horizonte (19°55′ S and 43°56′ W), the capital city of the Minas Gerais State. © XXXX American Chemical Society
Received: April 5, 2013
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dx.doi.org/10.1021/pr4003068 | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
10 min at room temperature (RT). The sample was transferred to a 10-kDa cutoff Microcon YM-10 filter unit (Millipore, Billerica, MA) and centrifuged at 14,000 × g for 30 min at RT. The filter was washed twice with 50 mM TEAB, and the flowthrough containing scorpion toxins (
