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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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Pectic substances are components of the middle lamella of the plant cell wall and are composed of partially methyl-esterified galacturonic acid residues linked by α-1,4 glycosidic bonds. These substances are naturally degraded by pectinases, such as the endopolygalacturonase (PG), which is responsible for the reaction of hydrolysis of α- (1,4) bonds of pectin. Pectinases and other enzymes responsible for cell wall degradation (PCWDEs) have been extensively studied in plants, bacteria, fungi and insects. As a defense strategy, the plants try to inhibit their enzymatic activity by producing inhibitors of PG, the PGIPs (Polygalacturonase Inhibitor Protein). PGIPs have a structure formed by leucine-rich repeats (LRRs), which are arranged to form two β-sheets, being that β1-sheet contains residues crucial for interaction with PGs. The high level of PGIP expression does not prevent infection, however, it significantly limits colonization of the tissue by the invading organism. Among the most studied PGIPs are polygalacturonase inhibitors from the bean (Phaseolus vulgaris), which consist of a family of 4 clustered genes (pvpgip1, pvpgi2, pvpgi3 and pvpgi4). Due to the application of pectinases in several industrial fields and to the importance of these inhibitors, the PvPGIP2 and PvPGIP3, inhibitors of the beans were recombinantly produced in Pichia pastoris, which were subjected to functional assays. Partial inhibition of Aspergillus niger pectinase activity against recombinant proteins PvPGIP2 and PvPGIP3 were observed. In addition, PvPGIP2 partially inhibited the activity of the Neosartorya glaba´s fungus pectinase for the first time reported in the literature. The recombinant proteins were also tested against pectinase of the insect Sphenophorus levis (Sl-EndoPG), but the activity of the same was not reduced. As substâncias pécticas são componentes da lamela média da parede celular vegetal e são compostas de resíduos de ácido galacturônico, parcialmente metil-esterificados e ligados por ligações α-1,4 glicosídicas. Estas substâncias são naturalmente degradadas por pectinases, como a endopoligalacturonase (PG), que é responsável pela reação de hidrólise de ligações α-(1,4) da pectina. As pectinases e outras enzimas responsáveis pela degradação da parede celular das plantas (PCWDEs) têm sido extensivamente estudadas em plantas, bactérias, fungos e insetos. Como estratégia de defesa, as plantas tentam inibir a atividade enzimática dessas enzimas, produzindo inibidores de PG, os PGIPs (Polygalacturonase Inhibitor Proteins). Os PGIPs possuem uma estrutura formada por repetições ricas em leucina (LRRs), que são organizadas para formar duas folhas β, sendo que a folha β1 contem resíduos cruciais para interação com as PGs. O alto nível de expressão dos PGIPs não previne a infecção, porém, limita significativamente a colonização do tecido pelo organismo invasor. Dentre os PGIPs mais estudados encontram-se os inibidores de poligalacturonase oriundos do feijão (Phaseolus vulgaris), os quais consistem em uma família de 4 genes agrupados (pvpgip1, pvpgi2, pvpgi3 e pvpgi4). Devido a aplicação de pectinases em diversos campos industriais e à importância desses inibidores, foram produzidos, neste trabalho, de forma recombinante em Pichia pastoris, os inibidores PvPGIP2 e PvPGIP3 do feijão, os quais foram submetidos a ensaios funcionais. Foi observada a inibição parcial da atividade da pectinase de Aspergillus niger pelas proteínas recombinantes PvPGIP2 e PvPGIP3. Além disso a PvPGIP2 inibiu parcialmente a atividade da pectinase do fungo Neosartorya glaba, pela primeira vez relatado na literatura. As proteínas recombinantes também foram testadas frente à pectinase do inseto Sphenophorus levis (Sl-EndoPG), porém a atividade da mesma não foi reduzida. CAPES: 133186/2017-2

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.

Tityus serrulatus (Ts) is the main scorpion species of medical importance in Brazil. Ts venom is composed of several compounds such as mucus, inorganic salts, lipids, amines, nucleotides, enzymes, kallikrein inhibitor, natriuretic peptide, proteins with high molecular mass, peptides, free amino acids and neurotoxins. Neurotoxins are considered the most responsible for the envenoming syndrome due to their pharmacological action on ion channels such as voltage-gated sodium (Nav) and potassium (Kv) channels. The major goal of this review is to present important advances in Ts envenoming research, correlating both the crude Ts venom and isolated toxins with alterations observed in all human systems. The most remarkable event lies in the Ts induced massive releasing of neurotransmitters influencing, directly or indirectly, the entire body. Ts venom proved to extremely affect nervous and muscular systems, to modulate the immune system, to induce cardiac disorders, to cause pulmonary edema, to decrease urinary flow and to alter endocrine, exocrine, reproductive, integumentary, skeletal and digestive functions. Therefore, Ts venom possesses toxins affecting all anatomic systems, making it a lethal cocktail. However, its low lethality may be due to the low venom mass injected, to the different venom compositions, the body characteristics and health conditions of the victim and the local of Ts sting. Furthermore, we also described the different treatments employed during envenoming cases. In particular, throughout the review, an effort will be made to provide information from an extensive documented studies concerning Ts venom in vitro, in animals and in humans (a total of 151 references).