Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family

Accepted Manuscript Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family Sebastian Estrada-Gómez, Lyz Gomez-Rave, Leidy Vargas-Muñoz, Arie van der Meijden PII:

S0041-0101(17)30054-5

DOI:

10.1016/j.toxicon.2017.02.007

Reference:

TOXCON 5563

To appear in:

Toxicon

Received Date: 2 December 2016 Revised Date:

8 February 2017

Accepted Date: 11 February 2017

Please cite this article as: Estrada-Gómez, S., Gomez-Rave, L., Vargas-Muñoz, L., van der Meijden, A., Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family, Toxicon (2017), doi: 10.1016/j.toxicon.2017.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family.

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Characterization of six different scorpion venoms.

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Sebastian Estrada-Gómez1,2 *, Lyz Gomez-Rave 1,3, Leidy Vargas-Muñoz 4, Arie van der Meijden 5.

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1. Programa de Ofidismo/Escorpionismo - Serpentario, Universidad de Antioquia UdeA,

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Carrera 53 No 61-30, Medellín, Zip code 050010, Antioquia, Colombia.

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2. Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia UdeA, calle

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70 No 52-21, Medellín, Zip code 050010, Antioquia, Colombia. E-Mail:

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[email protected]

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3. Maestría Bioquímica Clínica, Facultad de Ciencias de la Salud, Colegio Mayor de Antioquia,

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Medellín, Zip code 050040, Antioquia, Colombia. E-mail: [email protected] 4. Facultad de Medicina, Universidad Cooperativa de Colombia, Calle 50 A No 41-20

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Medellín, Zip code 050012, Antioquia, Colombia. E-Mail:

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[email protected]

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5. CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Campus Agrário de Vairão, P-4485-661 Vila do Conde, Portugal. E-mail:

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[email protected]

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* Corresponding autor: Sebastian Estrada-Gómez, [email protected]

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Abstract

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The objective of this study was to characterize a selection of six different scorpion venoms using

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biological and biochemical methods, including a preliminary MS/MS and a post-translational

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modifications analysis. Despite the diversity of scorpion species of medical importance in Africa 1

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and Colombia, the venoms of these arachnids have been poorly studied in these two regions. We

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report the biochemical, electrophoretic, chromatographic profile, internal peptide sequences with

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a post-translational modification report, and a preliminary antitumor activity of five different

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scorpions of the Buthidae family, Androctonus amoreuxi, Babycurus jacksoni, Grosphus grandidieri,

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Hottentotta gentili and Tityus fuhrmanni, and one of the Scorpionidae family Pandinus imperator.

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No L-amino oxidase activity was detected in the evaluated venoms. Proteolytic activity using

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azocasein was detected only in G. grandidieri and P. imperator, indicating the possible presence of

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metalloproteinases in these two venoms. Proteolytic activity using NOBA was detected in all

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venoms indicating the presence of serine-proteinases. Phospholipase A2 activity was detected in

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the venoms of P. imperator, G. grandidieri, H. gentili and A. amoreuxi, with P. imperator venom

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being the most active. All venoms analyzed contained defensin-like proteins, alpha toxins,

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metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity,

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among others. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a dose-

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dependent cytotoxic activity over MCF-7 cells. Only two isolated RP-HPLC fractions from P.

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imperator and T. fuhrmanni showed cytotoxic activity over MCF-7. No cytotoxic activity was found

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in the venoms from A. amoreuxi, B. jacksoni, and H. gentili.

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Keywords: Grosphus, Babycurus, Hottentotta, Tityus, metalloproteinases, phospholipases,

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antitumor, MCF-7, post-translational modifications.

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1. Introduction.

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Understanding the composition and mode of action of scorpion venom is important for both the

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treatment of victims of scorpionism, as well as the potential applications of the compounds of

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scorpion venom for antimicrobial and anti-cancer applications. For scorpion species in several

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geographic areas, such knowledge is sorely lacking.

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Although there is not any official report from minister of health, scorpions accidents in Colombia

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have been rising every year, with deaths reported in localities where the most lethal scorpions

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(family Buthidae) can be found, such as the Magdalena River in the states of Antioquia (one this

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year), Cundinamarca, Huila, Tolima and Valle del Cauca (1). One of the best epidemiology reports

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comes from the Toxicology Information Center from the Universidad Nacional de Colombia

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(CIGITOX). This report is based on telephonic medical inquiries where they receive around 90

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accidents consults every year (1). Although Tityus fuhrmanni is not the most lethal Buthidae

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scorpion distributed in Colombia, this scorpion can be found in densely populated areas, like the

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Aburra Valley (Medellín), inhabited by more than 3 million people. Hospitals from this city have

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attended T. fuhrmanni accidents on newborns, children and adults. Unfortunately the venom of

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this important scorpion was hitherto not characterized.

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According to Chippaux and Goiffon, the genera Androctonus and Hottentotta are among the most

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deadly or potentially deadly scorpions in Africa (2). There are no any epidemiological reports

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about G. grandidieri in Madagascar. Pandinus imperator is mainly distributed in the mainland of

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western Africa. Although this scorpion is one of the largest species in the world, the venom is not

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very dangerous for humans (2). The characterization of all these venoms is still incipient, except

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the venom from A. amoreuxi and P. imperator, were many different toxins have been reported 3

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and characterized. Hottentotta gentili and G. grandidieri venoms have to date not been studied at

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all.

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77 Scorpion venoms have evolved over 400 million years into complex libraries of toxins affecting

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different systems, with a remarkable affinity for the nervous system. The variety of protein

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(peptides, proteins and enzymes) and non-protein compounds (salts, neurotransmitters, etc.)

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make these venoms a promising target to identify molecules with antibacterial, antifungal,

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antiviral, antimalarial and anticancer activities (3-5), and a novel potential source for the design of

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new drugs (6, 7). The most active molecules displaying such activities are peptides showing both

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non-disulphide bridge (NDBP) or disulphide bridge (DBP) structures, presenting linear motifs or

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inhibitory cysteine knots respectively. Disulfide-rich peptides are the major molecules in this

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venom, containing around 30 to 70 amino acids residues and three or four disulfide bridges (4, 8-

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15). These toxins’ major targets are the ion channels in the nervous system, like sodium (Nav),

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potassium (Kv), chlorine (Cl) or calcium (Cav) channels, blocking or gating the channel mechanism

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and thereby exhibiting a neurotoxic activity. The non-disulfide rich compounds were discovered

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recently. The main characteristics of these molecules are the lack of disulfide bridges, the diverse

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sequences, the hemolytic and antibacterial activity, and the relatively low molecular mass (1-4

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kDa). Most of these peptides possess an amphipathic α-helical structure like those reported for

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different cationic antimicrobial molecules (4, 8-15). Post-translational modifications (PTM) play a

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key role in functional proteins like the mentioned above, giving higher complexity and peptide

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diversity of venoms. Despite the importance of this feature, there is not enough literature of these

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relevant modifications described for scorpion venoms.

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Anticancer peptides (ACPs) can be classified into two groups; those affecting microbial and cancer

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cells or those affecting microbial and cancer cells plus healthy mammalian cells (16). Although the

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mechanism of action of ACPs is still controversial, their effects occur either by membranolytic or

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non-membranolytic mechanisms (16, 17). Membranolytic mechanism are related to linear NDBP

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peptides showing an amphipathic moiety, most of them containing cationic and hydrophobic

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residues capable of interacting with microbial- or cancer cell membranes. This interaction is due to

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the increased anionicity of the cytoplasmic membrane of these cells, disrupting membranes and

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altering membrane permeability (5, 16). Non-membranolytic mechanisms are related to the

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interaction of ACPs with membrane proteins, altering their function, which is similar to the

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mechanism affecting ionic channels. Until today, only one snake Zn-metalloproteinase from

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Porthidium nasutum was reported to induce specific apoptosis activity in JurKat (T-cell acute

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lymphocytic leukemia) and K562 (Chronic myeloid leukemia) cells (Bonilla 2016).

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Different venoms and toxins have been reported affecting MCF-7 adenocarcinoma mammary cells,

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such as maurocalcine from Scorpio maurus (18) and BmK-CBP from Mesobuthus martensii. The

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latter is a serine protease that recognizes and binds to the membrane of these cells (19). Two

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linear peptides from Tityus serrulatus have been reported to affect these cancer cell lines (20).

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Here we report the biological and biochemical activity, as well as the electrophoretic and

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chromatographic profile, MS sequence together with a PTM report, and a potential antitumor

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activity of the complete venoms and fractions of Androctonus amoreuxi (Buthidae), Babycurus

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jacksoni (Buthidae), Hottentotta gentili (Buthidae), Grosphus grandidieri (Buthidae), Tityus

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fuhrmanni (Buthidae), and Pandinus imperator (Scorpionidae). No previous report regarding 5

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venom characterization or their biological activities have been published for the species Babycurus

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jacksoni, Hottentotta gentili, Grosphus grandidieri and Tityus fuhrmanni.

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123 2. Materials and Methods.

125 2.1. Venom extraction.

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Androctonus amoreuxi from Egypt, Babycurus jacksoni from Tanzania, Grosphus grandidieri from

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Madagascar, Hottentotta gentili from Morocco and Pandinus imperator from Togo, Africa and

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Tityus fuhrmanni from Colombia, specifically from the Antioquia province (North-west Andean

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region) were kept in captivity with water ad libitum and food offered every 2-3 weeks in a

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polyphagic diet of arthropods. Venom extraction as carried out using electro-stimulation. Metal

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electrodes, impregnated with a saline solution, were carefully positioned on the metasoma and a

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block signal with an amplitude of 18V at 40-60Hz was applied twice with an interval of 5 sec using

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a custom-made electro-stimulator (model 01). Collected venom was transferred to dry low-protein

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binding vials, lyophilized and stored at -20°C until use. These procedures were in accordance with

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the ethical principles in animal research adopted by the World Health Organization for the

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characterization of venoms (21, 22). These procedure were analyzed by the Comité Institucional

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para el Cuidado y Uso de animales - CICUA. After each extraction, all animals were kept alive in

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captivity.

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2.2. Electrophoretic profile.

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All electrophoretic profiles of all crude venoms were analyzed using 12% sodium dodecyl sulfate

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polyacrylamide gels (SDS-PAGE) according to Laemmli (23), and stained with Coomassie blue R-

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250. Venoms were loaded at a concentration of 1.5 mg/ml and a final volume of 20 µl. Molecular

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weights were estimated using standard low rank markers (Bio-Rad).

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One milligram of whole venom was dissolved in 200 μL of solution A (0.1% TFA in water) and

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centrifuged at 3500 g. The supernatant was then applied to a reverse-phase RESTEK C18 column

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(250×4.6 mm), and separated on a Shimadzu Prominence HPLC. Proteins were eluted by a gradient

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towards solution B (0.1% TFA in acetonitrile) as follows: 5% B for 5 min, 5–15% B over 10 min, 15–

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45% B over 60 min, and 45–70% B over 12 min at a flow rate of 1.0 mL/min (24). The

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chromatographic run was monitored at 215 nm and fractions were collected, lyophilized and

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stored until used.

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PLA2 activity of all venoms was measured using the assay described by Cho and Kézdy (25) and

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Holzer and Mackessy (26), modified for 96-well plates. The standard assay mixture contained 200

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μL of buffer (10 mMTris–HCl, 10 mM CaCl2, 100 mMNaCl, pH 8.0), 20 μL of substrate NOBA, 20 μL

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of water and 20 μL of the venom at a concentration of 5 µg/μL, or the RP-HPLC collected fractions

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(positive in the indirect hemolytic assay), in a final volume of 260 μL. The mixture was incubated

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for 60 min at 37 °C, and the absorbance was recorded at 405 nm. Each test was performed in

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triplicate.

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2.5. Proteolytic activity. Azocasein (Sigma–Aldrich, St. Louis, MO) was used as substrate to measure the proteolytic activity

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of all venoms, and to determine the presence of metalloproteases, according to Wang et al, (27)

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with some modifications. Briefly, 5 and 2.5 μg of the venom were dissolved in 20 μl of 25 mM Tris

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(0.15 M NaCl, 5 mM CaCl2), pH 7.4. These solutions were incubated with a solution of azocasein at

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a concentration of 10 mg/ml, previously diluted in the same buffer. After 90 min of incubation at

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37 °C, the reaction was stopped by adding 200 μl of trichloroacetic acid. Samples were then

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centrifuged at 360 g for 5 min. Supernatant (100 μl) was mixed with an equal volume of 0.5 M

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NaOH, and the absorbance was measured at 450 nm. Results are shown as units of proteolytic

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activity, which correspond to the amount of enzyme that induces a change in absorbance of 0.2.

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Each test was performed in triplicate.

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A second proteolytic activity assay was performed on all venoms to determine the presence of

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serine protease enzymes following the method described by Patiño et al (28). The enzymatic

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activity was measured using the synthetic substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide

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(BapNA). The standard assay mixture contained 50 μL of buffer (10 mM Tris–HCl, 10 mM CaCl2,

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100 mM NaCl, pH 8.0), 200 μL of substrate, 10 μL of water or enzyme in a final volume of 260 μL.

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After the addition of 2.5 µg/µl of each venom, the mixture was incubated for up to 40 min at 37

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°C, with the absorbance at 405 nm being recorded at 10 min intervals. Each test was performed in

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triplicate.

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2.6. LAAO activity. The assay was performed in triplicate, using 96-well microplates. Ten microliters of the enzyme

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solution (1 µg/µL) were added per well with 90 µL of the substrate solution to start the reaction.

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The standard reaction mixture consisted of 250 µM of L-leucine, 2 mM o-phenylenediamine (OPD),

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0.8 U/mL horseradish peroxidase and BSLAAO in a total volume of 100 µL of 50 mMTris–HCl buffer

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(pH 8) per well. After incubation at 37° C for 60 min the reaction was stopped by adding 50 µL of

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2M H2SO4. As negative control, distilled water was used. The absorbance of the reaction mixture

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was recorded at 492 nm using a microplate reader (Awareness Technology, FL, USA) (29). The

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enzyme activity was expressed as the increase of absorbance after the incubation time. All assays

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were performed in triplicate.

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2.7. Peptide mass determination by High-Resolution LC-MS.

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We selected from the RP-HPLC chromatograms peaks with the best intensity and resolution.

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Unfortunately, we did not have enough H. gentili sample for the preliminary MS/MS analysis. The

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only exception were the venoms from P. imperator and T. fuhrmanni. In these two venoms, we

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analyzed the fractions enhancing cytotoxic activity over MCF-7 cells. In all cases, the selected dried

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fractions were digested and submitted into the MS/MS equipment.

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2.7.1. Sample Digestions.

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Sequence grade Lys-C/Trypsin (Promega) was used to enzymatically digest the venom samples.

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The samples were reduced and alkylated. All digestions were carried out in the Barocycler

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NEP2320 (PBI) at 50°C under 20 kpsi for 2 hours. Digested samples were cleaned over C18 spin

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columns (Nest Group) and dried. Resulting pellets were resuspended in 97% purified H2O/3%

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ACN/0.1% formic acid (FA). A volume of 5 µL was used for nano LC-MS/MS analysis.

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210 2.7.2. LC-MS/MS.

Fractions were run on a nano Eksigent 425 HPLC system coupled to the Triple TOF 5600 plus

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(Sciex, Framingham, MA). The method used for analysis was 120 minutes at 300 nL/minute over

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the cHiPLC nanoflex system. The trap column was a Nano cHiPLC 200 µm x0.5 mm ChromXP C18-

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CL 3 µm x 120 Å followed by the analytical column, the Nano cHiPLC 75 µm x 15 cm ChromXP C18-

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CL 5 µm x 120 Å. The sample was injected into the Triple TOF 5600 and through the Nanospray III

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source equipped with an emission tip (New Objective). Peptides from the digestion were eluted

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from the columns using a mobile phase A of purified H2O/0.1% formic acid (FA) and a mobile

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phase B of ACN/0.1 % FA. With a flow rate of 0.3 µl/min, the method started at 95% A for 1

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minute followed by a gradient of 5% B to 35% B in 90 minutes and from 35% B to 80% B in 2

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minutes. Eighty percent of B was held for 5 minutes before being brought to 5% B and held for

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20 minutes. PTM are reported for each containing peptide.

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2.7.3. Data analysis.

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The data acquisition was performed monitoring 50 precursor ions at 250 ms/scan. Mascot

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Daemon v.2.4.0 (Matrix Science) was used for database searches against the different databases.

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Some fragments were downloaded from UniProt or NCBI website . Samples were run in the

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Bindley Bioscience Center at Purdue University.

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2.8. Antibiotic activity. All venoms were analyzed. Antibiotic susceptibility tests were performed as proposed by Bauer et

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al. (30), and the Clinical and Laboratory Standards Institute (CLSI) guidelines, with some

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modifications. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were grown

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on Mueller-Hinton agar (MH) and then suspended in 5ml of MH sterile broth. Turbidity was

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measured at 600nm and adjusted to 0.5 absorbance which corresponds to 1.5 x 10-4 colony

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forming units (CFU). Ten μl of each venom dose containing 500 and 250 µg, and RP-HPLC collected

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fractions were added and incubated at 37°C during 24 hours. Physiological saline solution was

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used as negative control and chloramphenicol as a reference control. Each test was performed in

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duplicate.

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All venoms were evaluated against MCF-7 cells (adenocarcinoma mammary cells). Only the RP-

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HPLC fractions from those venoms enhancing cytotoxic activity were evaluated against the same

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cell line. In this case we selected peaks with the best intensity and resolution. Each test was

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performed in triplicate.

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2.9.1. Cell cultures.

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MCF-7 cells were cultivated using DMEM supplemented with 5% of SBF, 100 µg/ml of penicillin

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and 100 µg/ml of streptomycin. All were incubated at 37ºC using a 5% atmosphere of CO2.

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2.9.2. Cell viability. 11

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Twenty-four-well plates were used to seed 5 × 104 MCF-7 cells and kept under normal conditions

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of culture incubated for 24 h to guaranty the adhesion. After this initial incubation period, the cell

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cultures were treated using complete venoms and their respective fractions in triplicate.

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Treatment of each compound was carried out for 24 h, and after this period of exposure, cells

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were detached using a solution of tripsine-EDTA (0.1%). A solution of trypane-blue (0.4%) was

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used to quantify the cell viability using a hemocytometer. The percentage of live cells was

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determined dividing the number of live cells by the total number of cells. Each test was performed

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in triplicate.

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2.9.3. Cytotoxic assay.

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Dose-dependent cytotoxicity was examined using the MTT assay. Ninety six-well plates were used

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to seed 2 × 104 MCF-7 cells, and incubated for 24 h to guaranty the adhesion. After this initial

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incubation period, cell cultures were treated using complete venoms and their respective fractions

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in triplicate. Treatment of each compound was carried out for 24 h, and after this period of

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exposure, 10μl de MTT (5 mg/ml) were added into each well with a final concentration of 0.5

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mg/ml and incubated for 4 h under darkness conditions. Finally, to dissolve all formazan crystals

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formed, 100 µl of isopropanol/HCl 0.04M was added and the absorbance was measured at 570 nm

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using a 96-well plat lector. Viability was expressed as a percentage compared to the negative

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control. All assays with complete venoms and selected RP-HPLC fractions were performed in

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triplicate. Concentration of the RP-HPLC fractions were determined using a BSA kit.

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2.10. Statistical analysis. 12

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Results were expressed as mean ± standard error media (S.E.M.) and statistical comparisons were

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done using an ANOVA with a Bonferroni post-test assuming a significance of p<0.05. All data

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analysis was done using GraphPad PRISM 5 (GraphPad Software, Inc; La Jolla, California, USA).

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278 3. Results.

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280 Electrophoretic profile.

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Each venom shows a specific profile, most of the compounds present on the venoms have

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molecular masses above 21 kDa and below 97 kDa (red bracket on Fig 1). A few compounds over

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97 kDa are observed in all venoms (see Fig 1). Venoms from G. grandidieri (Gg), Pandinus

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imperator (Pi) and Hottentotta gentili shows a similar electrophoretic profile with common bands

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between 21 kDa and 31 kDa, close to 31 kDa and close to 45 kDa.

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Fig 1: Scorpion venoms electrophoretic profiles. 13

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Crude venom (reduced) SDS-PAGE profile of all scorpion species in a 12% gel followed by

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Coomassie blue staining. Venoms were loaded at a concentration of 1.5 mg/ml and a final volume

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of 20 µl. MW: Molecular Weight, Gg: Grosphus grandidieri, Aa: Androctonus amoreuxi, Pi:

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Pandinus imperator, Bj: Babycurus jacksoni, Tf: Tityus fuhermanni, Hg: Hottentotta gentili.

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293 Chromatographic profile.

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The chromatographic profile showed clear differences between venoms from each species. We

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obtained well-defined fractions that allowed a posterior collection for the respective MS/MS

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analysis identification and evaluation of cytotoxic activity over MCF-7 cells (see Fig 2). P. imperator

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and T. fuhermanni venoms showed a homogenous distribution of hydrophobic and hydrophilic

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compounds eluting from 0% to 70% of ACN. T. fuhermanni showed a higher concentration of

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compounds eluting between 30 % and 35 %. In the other venoms, most of the compounds elute

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between 20% and 40% of ACN indicating a moderate hydrophilic nature of each venom content. 3.

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After minute 70, we did not found any more fraction eluting on G. grandidieri.

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Fig 2: Scorpion venoms chromatographic profiles.

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RP-HPLC chromatographic profiles of the crude venom of all scorpions using a C18 column (250

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mm - 4.6 mm). Elution gradient used: 0-70% of acetonitrile (99% in TFA 0.1%). The run was

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monitored at 215 nm. Roman numbers in chromatograms corresponds to the fractions evaluated

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against MCF-7 cells. Arrows indicate fraction subjected to MS/MS analysis.

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310 Enzymatic activity.

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No LAAO activity was detected in the evaluated venoms. Only G. grandidieri and P. imperator

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venoms were evaluated due to the yellow color of the venom after the lyophilization process and

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the molecular masses present in the electrophoretic profile matching other LAAO enzymes.

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Proteolytic activity using azocasein was detected in G. grandidieri and P. imperator using 5 mg of

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venom with a p<0.05 when compared with the blank. Using 2.5 mg, activity was only detected in

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G. grandidieri (p<0.05). Proteolytic activity using the substrate N-alpha-benzoyl-DL-arginine-p-

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nitroanilide (BapNA) was detected in all venoms. G. grandidieri and H. gentili are the most active

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venoms, with a p<0.0001 when compared with the blank. Phospholipase A2 activity using 4-nitro-

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3-octanoyloxy-benzoic acid (NOBA) substrate, was detected in the venoms of P. imperator, G.

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grandidieri, H. gentili and A. amoreuxi. P. imperator is the most active venom with a p<0.0001

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when compared with the blank. Fig 3 shows the result of the assays.

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Fig 3: Scorpion venoms enzymatic activity.

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Enzymatic activity of each venom; Pi: Pandinus imperator, Gg: Grosphus grandidieri, Tf: Tityus

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fuhrmanni, Aa: Androctonus amoreuxi, Hg: Hottentotta gentili, Bj: Babycurus jacksoni, Control:

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negative control or blank. A: Proteolytic activity using azocasein as substrate (absorbance

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monitored at 450 nm). B: Proteolytic activity using BapNA as substrate (absorbance monitored at

329

405 nm). C: Phospholiase activity using NOBA as substrate (absorbance monitored at 405 nm). All

330

significant differences are relative to the negative control or blank. **** p<0,0001 / *** p<0,001 /

331

** p<0,01 / * p<0,05.

AC C

EP

324

332 17

ACCEPTED MANUSCRIPT

MS/MS and PTM analysis.

334

After the preliminary MS/MS analysis, fragments showed sequence similarity with proteins from

335

different scorpion species, some of these proteins were reported as neurotoxic, antimicrobial or to

336

display metalloproteinase activity, among others. Table 1 summarizes all preliminary sequences

337

found in the selected fractions. It is important to consider that there are no previous reports of

338

proteomic characterization of T. fuhrmanni, G. grandidieri and B. jacksoni venom, while P.

339

imperator and A. amoreuxi has been widely studied and characterized. In all venoms, except B.

340

jacksoni,

341

/KGMANSGGICNSRWKF/KESIGCANNYTDKC) matching the 25 kDa hypothetical secreted protein

342

from Hottentotta judaicus. In the first fragment the 6th residue can vary to be I (isoleucine), A

343

(alanine), N (asparagine) or V (valine). In all venoms, we found a total of 17 fragments with PTM

344

and the most common PTM were (K) acetylations and (M) oxidations.

found

a

total

of

3

different

fragments

(KISSV[IANVC]NKDKI

M AN U

we

SC

RI PT

333

TE D

345

Given names in P. imperator are just for documentation purposes in this paper since the

347

corresponding proteins already exist and are reported. The fraction from P. imperator with

348

cytotoxic activity over MCF-7 cells showed fragments with 100% sequence similarity with three

349

different toxins affecting potassium channels (see table 1A). PiP1 fragments matched a potassium

350

channel toxin KTx 7.1 corresponding to the signal peptide (PiP1b and PiP1c) and the mature toxin

351

(Pip1a) (see Fig 4). PiP2 fragments, shows amino acid sequence similarity with the potassium

352

channel toxin KTx 6.1, a four disulfide bridge toxin. PiP2a is part of PiP2b leaving one complete

353

fragment KCRGTSDCGRPCQQQTGCPNSKC corresponding to 65.7% of the complete related toxin. In

354

addition to these fragments, other sequences were detected matching with two different peptides

AC C

EP

346

18

ACCEPTED MANUSCRIPT

355

affecting potassium channels reported in the scorpion Heterometrus laoticus, and one putative

356

metalloprotease fragment from Hottentotta judaicus.

RI PT

357 The fraction from G. grandidieri showed amino acid sequence similarity with proteins like a

359

hypothetical secreted protein from Hottentotta judaicus (like the same found on P. imperator), a

360

NADH dehydrogenase subunit from Tityus discrepans and a two fragments showed sequence

361

similarity with a defensine-like peptide (see table 1B).

M AN U

362

SC

358

In the active fraction against MCF-7 cells of T. fuhrmanni, we found fragments matching four

364

different metalloprotease-like proteins from different scorpion species, some of which were

365

reported as zinc dependents (see table 1C). In the same fraction, one fragment showed 100%

366

amino acid sequence similarity with different segments of an antimicrobial peptide from the

367

scorpion Urodacus yaschenkoi. The same hypothetical secreted protein from Hottentotta judaicus

368

detected on P. imperator and G. grandidieri were found in this venom with an additional fragment

369

(K-ESIGCANNYTDK-C). The same fraction of the NADH dehydrogenase fragment detected on G.

370

grandidieri was found in this venom. Additionally, one fragment of an antimicrobial peptide was

371

found.

EP

AC C

372

TE D

363

373

The fraction from A. amoreuxi showed sequence similarity with toxins from other Buthidae genera

374

like Buthus, Mesobuthus, Leiurus, and Isometrus, some of them distributed in North Africa (see

375

table 1D). Additionally, A. amoreuxi showed sequence similarity specifically with other

376

Androctonus species like A. australis and A. crassicauda (see table 1D). Sequences found in A.

19

ACCEPTED MANUSCRIPT

amoreuxi correspond to alpha and beta toxins, insect toxins and toxins affecting sodium channels.

378

AaP1 fragments are similar with the mature peptide of the beta-insect excitatory toxin from

379

Androctonus australis and the beta-toxin Isom1 from Isometrus vittatus. AsP1d fragment is part of

380

the protein N-terminal (see Fig 5). All sequences that we reported matching beta toxins are

381

identical to the respective toxin. The AsP3a fragment is part of the C-terminal portion of the

382

matched alpha-toxin from Buthus occitanus tunetanus. Two fragments showed sequence similarity

383

with toxins affecting sodium and potassium channels, and one more has sequence similarity with a

384

neuropeptide from Mesobuthus eupeus. Interestingly, the same hypothetical secreted protein

385

found in P. imperator, G. grandidieri and T. fuhrmanni, was found in A. amoreuxi but only the

386

Isoleucine (I) variant.

M AN U

SC

RI PT

377

387

The analyzed fraction from B. jacksoni gave different sequences, all reported for first time in this

389

species. Only two of the five fractions showed a score above 100. Sequences showed sequence

390

similarity with alpha toxins, neurotoxins, and other proteins from different scorpions. Although

391

scores are very low in some cases, is important to consider that is the first report of this venom.

392

We also found possible isomers of the protein kinase reported in Mesobuthus gibbosus with amino

393

acids varying on position 7th of the internal peptide found (RMMDPK[MNAIY]SKR). The alpha-like

394

toxin fragment corresponds to the C-terminal portion of the complete protein.

EP

AC C

395

TE D

388

396

Table 1: Venoms preliminary MS/MS analysis and their respective similar peptide or protein.

397

Summary of the identified fragments in A: Pandinus imperator. B: Grosphus grandidieri. C: Tityus

398

fuhrmanni. D: Androctonus amoreuxi. E: Babycurus jacksoni. All MS/MS derived sequences

399

correspond to internal peptides. All database numbers are from UniProtKB, only PiP5a is from 20

ACCEPTED MANUSCRIPT

GenBank. Z: charge. Amino acid within brackets [ ] on the hypothetical secreted protein and the B.

401

jacksoni protein kinase like sequence indicates that those amino acids con vary on that position.

402

Superscript £ indicates molecular weight calculated with alanine residue and without any PTM.

403

Superscript ¥ indicates molecular weight calculated with methionine residue and 2 methionine

404

oxidations. Asterisk (*) indicates fragments with (K) acetylations and pilcrow (¶) indicates

405

fragments with (M) oxidations.

RI PT

400

PiP2a

PiP2b PiP3a PiP4a

PiP5a PiP6a

K-ETGYPNAK-C K-TISCTNPK-Q KDDDDKTISCTNPK -Q RGTSDCGRPCQQQ TGCPNSK-C KCRGTSDCGRPCQ QQTGCPNSK-C

362 362

K-ETGYPNAK-C

240

*KISSV[IANVC]NKD K-I ¶ KGMANSGGICNSR WK-F

P55927

362

282

Q10726

282

KKTGCPNAKC

Expected peptide m/z

Z

Calculated peptide mass

440.209 454.227

+2 +2

878.413 906.448

499.207

+3

1494.651

525.478

+4

2097.88

Pandinus imperator

601.262

+4

2401.024

Pandinus imperator

878.413

Pandinus imperator

M AN U

PiP1c

Matching peptide acc. number

Score

Matched peptide family

Potassium channel toxin.

TE D

PiP1a PiP1b

MS/MS peptide sequence

Potassium channel toxin.

Matched organism

Pandinus imperator

P55928

Potassium channel toxin.

440.209

+2

F1CJ08

Hypothetical secreted prot.

£ 502.296

+2 1002.571

20

GB HQ288187

Metalloproteinas e

770.849

+2

1539.692

Hottentotta judaicus

18

C0HJN0

α-toxina KTx6

431.71

+2

861.437

Hottentotta laoticus

EP

Fragment given name

A: Pandinus imperator.

26

AC C

407

SC

406

£ Hottentotta judaicus

408 409

B: Grosphus grandidieri.

21

ACCEPTED MANUSCRIPT

Fragment given name

MS/MS peptide sequence

Score

Matching peptide acc. number

Matched peptide family

Expected peptide m/z

GgP1a

* KISSV[IANVC]NKD K-I

46

F1CJ08

Hypothetical secreted prot.

£ 502.296

+2 1002.571

GgP2a

R-GILTVIPR-I

28

C9X4I7

NADH dehydrogenase

434.766

+2

867.554

GgP3a

* R-KTPTPEK-R

17

Defensine-like

421.759

+2

841.454

Defensine-like

435.756

+2

869.460

GgP3b

17

* K-TPTPEKR-E

TfP1a TfP1b

MS/MS peptide sequence

Score

* KISSV[IANVC]NKDKI K-ESIGCANNYTDKC

+2

1260.711

Tityus pachyurus.

631.364

+2

1260.711

Tityus sp.

412.751

+2

823.491

574.958

+2

1721.816

687.333

+2

1372.619

690.369

+2

1378.764

15

531.271

+3

1590.844

17 17

421.758 435.755

+2 +2

841.454 869.460

593.613

+3

1777816

979,4

+2

979.406

R-TIPPGAIR-A ¶ KYMEVYIFNLMNAA K-N * KINGMKFSPCCK-R ¶ KMLFNTLDLRIK-V RVQEVYLANLMNAV K-M * R- KTPTPEK-R * K-TPTPEKR-E KFKGACLSSSNCASIC R-T

18

V9Z548

Venom metalloproteinase Antarease-like Znmetalloprotease

V9Z553

F1CIY1

Putative M12B metalloprotease.

EP

18 15

AC C

* K-EGYCHGTK-C

Matched organism

631.364

TfP4a

TfP7a

Calculated peptide mass

1399.593

35

TfP6c

Z

+2

¶ K-SADIIMLLITR-T

TfP6a TfP6b

Expected peptide m/z

Tityus discrepans Aegilops tauschii Aegilops tauschii

700.805

44

TfP3a

TfP5c

F1CJ08

Hypothetical secreted protein.

£ Hottentotta judaicus

+2 1002.571

35

TfP5b

Matched peptide family

Matched organism

£ 502.296

¶ K-SADIIMLLITR-K

TfP5a

Matching peptide acc. number

44

TfP2a

TfP4b

M AN U

Fragment given name

C: Tityus fuhrmanni.

TE D

411

SC

410

Calculated peptide mass

RI PT

R7W9E7

Z

15

E4VNZ7

R7W9E7

17 18

L0GCW2

Venom metalloprotease.

Defensin-like protein. Antimicrobial peptide

£ Hottentotta judaicus.

Hottentotta judaicus.

Mesobuthus eupeus.

Aegilops tauschii. U. yaschenkoi 22

ACCEPTED MANUSCRIPT

TfP8a

R-GILTVIPR-I

28

C9X4I7

NADH dehydrogenase

434.766

Matched peptide family

Expected peptide m/z

Z

Calculated peptide mass

530,800 530,800

+2 +2

1059,592 1059,592

499,229

+2

996,451

601,288

+2

1200.562

499,229

+2

996,451

+2

867.554

Tityus discrepans

412

AaP1c AaP1d AaP2a

Score

K- KVLEISDTR-K K-VLEISDTRK-S K- NGYAVDSSGKA R-KSYCDTTIIN K-NGYAVDSSGKV

Matching peptide acc. number

1068 1068

P01497

Beta-insect toxin.

1068 1068 198

O61668

Beta-insect toxin

LKDGYIVDDR-N

113

P01486

AaP4a

K- ALPDNVPIK-D

25

M1JBC0

23

A0A088D9U2

AaP6a

AaP7a

KLTSMSEYACPVIE K-W RNFDEIDNVGFND FGPASRPGSGR-S * KISSV[IANVC]NKD K-I

Fragment given name

BjP1a BjP2a

Mesobuthus martensii Buthus occitanus tunetanus Androctonus crassicauda

597,308

+2

1192,608

Sodium channel alpha-toxin

483,782

+2

965,554

Potassium channel blocker

807,888

+2

1613,768

Mesobuthus eupeus

E4VP42

Venom neuropeptide

823,377

+3

2467,115

Mesobuthus eupeus

F1CJ08

Hypothetical secreted protein.

£ 502.296

+2

1002.571

£

Hottentotta judaicus.

E: Babycurus jacksoni.

AC C

415

44

Androctonus australis.

EP

414

20

Matched organism

Alpha-toxin

TE D

AaP3a

AaP5a

RI PT

AaP1a AaP1b

MS/MS peptide sequence

SC

Fragment given name

D: Androctonus amoreuxi.

M AN U

413

Score

Matching peptide acc. number

Matched peptide family

Expected peptide m/z

Z

Calculated peptide mass

207

P58779

Alpha-like toxin

504,736

+2

1007,492

K-EGYPVDWGNCK118 Y

E7CLN7

Alpha-neurotoxin

656,272

+2

1310,560

433,213

+2

864,448

MS/MS peptide sequence

* KDGYPVDSK-G

BjP3a

R-ICSARSGK-F

32

H6UDU1

Defensin-like protein

BjP3a

*¶ RMMDPK[MNAIY]S K-R

19

Q7Z1X7

Protein kinase

500.002

¥

+2

998,423

Matched organism

Centruroides sculpturatus Rhopalurus junceus Wolffia australiana

¥ Mesobuthus gibbosus 23

ACCEPTED MANUSCRIPT

416

RI PT

417

418

Fig 4: Alignment of PiP1 fragments of P. imperator with potassium channel toxin alpha-KTx 7.1

420

(P55927).

421

Alignment of PiP1 fragments matching the potassium channel toxin alpha-KTx 7.1 (UniProt code:

422

P55927) toxin from P. imperator.

AC C

424

EP

TE D

423

M AN U

SC

419

425

Fig 5: Alignment of A. amoreuxi MS/MS AaP1 fractions.

426

Alignment of fragments of A. amoreuxi matching with a Beta-insect excitatory toxin (UniProt code:

427

P01497) from Androctonus australis.

428 429

Biological activity.

24

ACCEPTED MANUSCRIPT

No antibiotic activity was detected at the doses evaluated. In our measurements of cytotoxic

431

effects using MCF-7 cells after 24 hours of exposure to each venom, no morphological changes or

432

inhibitory growth activity was observed with A. amoreuxi, B. jacksoni and H. gentili venoms when

433

compared with the control (see Fig 6 A, B, C). Venom of G. grandidieri and T. fuhrmanni induced an

434

aggregation and cells adopted a rounded shape (see Fig 6 D, E). With P. imperator venom, the

435

same rounded shape can be observed in the cells, as well as a decrease in their number and size

436

(see Fig 6 F). The measurement of cytotoxic activity using MTT did not result in any detectable

437

activity. Figure 7 shows viability of cells after using trypane blue. All three venoms from P.

438

imperator, G. grandidieri and T. fuhrmanni were effective to reduce viability of cells, with the

439

venom of P. imperator being the more potent. These three venoms were subjected to a new RP-

440

HPLC process and collected fractions were used to measure cytotoxic effects using MCF-7 cells.

441

AC C

EP

TE D

M AN U

SC

RI PT

430

442

Fig 6: Cytotoxic activity of each venom over MCF-7 adenocarcinoma cells.

443

Micrographs obtained using an optical microscope (40x) of MCF-7 cell cultures after 24 hours of

444

exposure to each venom. A: Control, B: Androctonus amoreuxi, C: Babychurus jacksoni, D: 25

ACCEPTED MANUSCRIPT

445

Hottentotta gentili, E: Grosphus grandidieri, F: Tityus fuhrmanni, G: Pandinus imperator. Arrows

446

indicate cellular morphological changes for each venom.

TE D

M AN U

SC

RI PT

447

EP

448

Fig 7: Dose-response relationship of venoms enhancing cytotoxic activity over MCF-7

450

adenocarcinoma cells.

451

Cytotoxic activity of complete venoms and the respective dose-response relationship from

452

Pandinus imperator: Pi, Grosphus grandidieri: Gg and Tityus fuhrmanni: Tf.

AC C

449

453 454

The RP-HPLC collected fractions from those venoms displaying cytotoxic activity over MCF-7 cells

455

were evaluated against the same cell line using trypane blue. Only fraction III of P. imperator and 26

ACCEPTED MANUSCRIPT

fraction I of T. fuhrmanni (see Fig 8) showed cytotoxic activity after 24 h of treatment of MCF-7

457

cells (See Fig 2). Percentage of cell viability were 77% and 68.79% respectively when compared

458

with the control cell viability (p<0.05). No representative activity was observed using fractions

459

from Grosphus grandidieri. After treatment with each fraction, cells lose cohesion and viability

460

(see Fig 8). Fractions suspected to display cytotoxic activity were subjected to MS/MS preliminary

461

analysis. Fractions concentrations for Pi: 4.06 mg/lt and Tf: 11.36 mg/lt.

M AN U

SC

RI PT

456

462

Fig 8: Cytotoxic activity of Tityus fuhrmanni RP-HPLC fraction number I over MCF-7

464

adenocarcinoma cells.

465

Micrographs obtained using an optical microscope (40x) of MCF-7 cell cultures after 24 hours of

466

exposure to fraction number I collected from RP-HPLC of Tityus fuhrmanni (according Fig 2). A:

467

Control. B: RP-HPLC collected fraction Tityus fuhrmanni.

469

EP

AC C

468

TE D

463

4. Discussion.

470 471

Pharmaceutical research has focused efforts on venoms as a potential source of bio-active

472

compounds. The high diversity of molecules found in venoms is one of the main reasons to 27

ACCEPTED MANUSCRIPT

473

propose these venoms as potential sources for drug candidates. Scorpion venoms in particular are

474

a rich source of molecules with a diverse range of biological activities like antibacterial, antifungal,

475

antiviral, antimalarial and anticancer activities (3-5).

RI PT

476

The electrophoretic and chromatographic profile obtained, showed a specific outline to each

478

species. We report the biochemical analysis of six different venom including in the cases of B.

479

jacksoni, G. grandidieri and T. fuhrmanni the first MS/MS and TPM analysis of the venom. We

480

found significant, dose-dependent cytotoxic activity on MCF-7 mammary carcinoma cell lines in

481

the venoms of P. imperator, G. grandidieri and T. fuhrmanni, indicating the possible anticancer

482

activity of these three venoms. This activity may be associated with the presence of different

483

proteins and peptides found in these venoms, acting synergistically as discussed below.

M AN U

SC

477

TE D

484

Scorpion venoms are widely known for the rich content of toxins affecting sodium channels (31,

486

32). The biochemical profiles of the scorpion venoms evaluated indicate that the venoms of these

487

scorpions contains proteins with enzymatic activity. Only P. imperator, G. grandidieri and T.

488

fuhrmanni venoms may contain proteins displaying metalloproteinase activity. Electrophoretic

489

analysis corroborates these findings, due to the different conserved bands with molecular masses

490

close to 31 and 45 kDa observed in all three venoms. This is a common molecular mass of

491

metalloproteinases (33). In addition, we found the sequences of at least three different internal

492

peptides, in T. fuhrmanni and P. imperator, matching metalloproteinases previously reported in

493

other scorpions like Tityus, Mesobuthus and Hottentotta. For instance, BumaMPs1 was isolated,

494

identified and cloned from the scorpion Mesobuthus martensi; Metalloproteinases VP-1 (GenBank

495

EF442045) and VP-2 (GenBank EF442046) were isolated from Mesobuthus eupus; Antarease-like

AC C

EP

485

28

ACCEPTED MANUSCRIPT

proteins were identified from Tityus pachyurus and one other Tityus species. (34-36) (37, 38). The

497

reported antarease-like proteins are ubiquitous in a broad range of scorpion species, and their

498

catalytic activity is related to the cleavage of vesicle associated membrane proteins (VAMP) of the

499

pancreas, via the cleavage of the soluble N-ethylmaleimide-sensitive factor attachment protein

500

receptors (SNAREs) involved in pancreatic secretion (37). Venoms from Androctonus amoreuxi,

501

Babycurus jacksoni, Grosphus grandidieri, Hottentotta gentili, Pandinus imperator and Tityus

502

fuhrmanni showed the presence of serine-proteinase activity with the higher activity expressed by

503

Hottentotta gentili venom. These kind of enzymes have been previously reported in the scorpion

504

venoms of Tityus serrulatus and Titytus bahiensis (39). Although the physio-pathological effects of

505

serine-proteinases are not clear in scorpion venoms, these proteins have been reported to affect

506

blood coagulation by triggering proteolysis of some coagulation factors as performed by

507

thrombine-like enzymes (40). Pandinus imperator showed the highest level of phospholipase A2

508

(PLA2) activity. This high activity corresponds to the two different Cav dependent phospholipases

509

A2 previously reported; phospholipin (P0DKU2) and imperatoxin-1 (P59888). All other five venoms

510

showed a low to moderate activity, with a difference statistically significant when compared with

511

the blank, indicating the presence of these compounds in all venoms. These kind proteins have

512

been previously reported in other scorpions from Colombia like Opisthacanthus elatus (41). These

513

PLA2 are esterases that cleave glycerophospholipids at the sn-2 ester of phospholipids and has

514

been deeply studied specially in snakes (42).

SC

M AN U

TE D

EP

AC C

515

RI PT

496

516

We found a common sequence in the venoms of A. amoreuxi, G. grandidieri, T. fuhrmanni and P.

517

imperator. The preliminary MS/MS analysis showed 3 different sequences corresponding to a 25

518

kDa secreted protein from H. judaicus with different isoforms varying the amino acids at position

519

138 between I (isoleucine), A (alanine), N (asparagine) and V (valine) (43). We found this similar 29

ACCEPTED MANUSCRIPT

fragments in 5 of the 6 analyzed venoms and showing PTM corresponding to lysine (K)

521

acetylations. The distribution of this protein in the Buthidae family is common in the clade of

522

scorpions from America and Madagascar named “new world” and one clade of the “old world” by

523

Fet et al (44). This protein may correspond to an ancestral toxin shared by the families

524

Scorpionidae and Buthidae. The last phylogenomic study positioned both families in distant clades

525

and different superfamilies indicating that this protein is basal in the parvorder Buthida (45).

526

Further studies must be performed using different individuals from other superfamilies of the

527

same parvororder. The pharmacological and biological activity of this protein still unknown.

SC

RI PT

520

M AN U

528

Venom content from P. imperator showed similarity with toxins affecting potassium channels

530

already reported in this species (46-50). Other, hitherto unreported fragments were found,

531

corresponding to metalloproteinases. This finding suggests an active component related to the

532

proteolytic activity of the venom over the azocasein substrate. After these findings, we can state

533

for first time that the venom of P. imperator contains metalloproteinases. We analyzed for first

534

time the venom from G. grandidieri and the fractions showed sequence similarity with the

535

mentioned hypothetical protein from H. judaicus, and other proteins like NADH dehydrogenase

536

and defensin-like proteins from other scorpions. Although the scores are very low, these fractions

537

have 100% amino acid sequence similarity with the respective similar protein. Moreover, these

538

sequences are considered new in this species and are reported for first time for G. grandidieri.

539

Fragments found in T. fuhrmanni corresponding to metalloproteinases has been never reported

540

before in this species. The enzymatic activity, together with the MS findings allows us to confirm

541

the presence of these proteins in the T. fuhrmanni venom. Although we did not find any

542

antimicrobial activity in this venom, we found a sequence similar to an antimicrobial peptide.

543

However, this peptide may not be present in sufficient concentration to elicit the antimicrobial

AC C

EP

TE D

529

30

ACCEPTED MANUSCRIPT

activity. Androctonus amoreuxi venom poses alpha and beta toxins commonly found in closely

545

related Buthidae species distributed in Africa, the Arabian Peninsula and India. Alpha toxins are

546

commonly found in A. amoreuxi venom. Chen et al previously reported 3 alpha toxins named

547

AamH1, AamH2 and AamH3, in the same scorpion (51). The A. amoreuxi sequence reported here,

548

although showed similarity with AamH1, AamH2 and AamH3, it is not 100% identical, indicating

549

that it may be a fragment of a new alpha toxin of A. amoreuxi. Beta toxins have been previously

550

reported in other Androctonus species like A. australis and A. mauritanicus. All sequences that we

551

reported matching beta toxins are identical with the respective similar toxin, indicating that these

552

toxins may be widely distributed among the species of the genus Androctonus. We found a

553

fragment of peptide corresponding to a sodium channel alpha-toxin eluting at 40% of ACN. Other

554

toxins affecting sodium channels isolated and identified in different Buthidae scorpions, commonly

555

elute between 30% and 50% of ACN (52-54). There have thus far not been any MS analyses

556

published for B. jacksoni. The preliminary MS/MS analysis of B. jacksoni indicates that this venom

557

has alpha toxins and other interesting peptides. In this venom, we also found a possible isomers of

558

the protein kinase reported in Mesobuthus gibbosus.

SC

M AN U

TE D

EP

559

RI PT

544

Cytotoxic activity found in the venom of T. fuhrmanni is related with the venom content reported

561

in the preliminary MS/MS analysis. In this venom, micrographs showed a loss of cohesion of MCF-7

562

cells when exposed to the venom fraction containing metalloproteinase-like fragments. This loss

563

of cohesion, together with the presence of the antimicrobial peptides, may cause direct damage to

564

the MCF-7 cells and loss of viability related with a membranolytic mechanism. These proteins play

565

an important role affecting internal cellular processes such as cell signaling, cell adhesion, invasion

566

and motility (43, 55). Although there is not enough evidence for scorpion metalloproteinases

567

enhancing any cytolytic activity in cancer cells, one snake Zn-metalloproteinase from Porthidium

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nasutum was reported to induce specific apoptosis activity in JurKat (T-cell acute lymphocytic

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leukemia) and K562 (Chronic myeloid leukemia) cells (56).

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metalloproteinases may be the main factor to propose these proteins in the venom of T.

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fuhrmanni as promising anticancer proteins. Although the score of the fragment showing

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sequence similarity with a DBP antimicrobial peptide from the scorpion Urodacus yaschenkoi,

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found in the fraction with cytotoxic activity is very low, the presence of this kind of compounds in

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T. fuhrmanni venom cannot be discarded, since we found a 100% similarity with the antimicrobial

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peptide segment. Its presence could result in the synergistic activity proposed above. Further

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analyses remain to be performed, such as the isolation, characterization and cytotoxic evaluation

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of the fraction with metalloproteinase activity.

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The catalytic activity of

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In the case of P. imperator venom, the presence of phospholipases and metalloproteinases may be

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essentials for the anticancer activity explained as a synergistic activity and evidenced by the

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complete loss of cohesion and cell viability. These PLA2 proteins may be the clue of the anticancer

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activity. These esterases have been proposed as potential anticancer candidates mainly due to

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their function in cell death (42). The anticancer activity of these proteins has been previously

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reported in leukemia cells lines. The main mechanism by which these proteins mediate cell death

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is the release of arachidonic acid (AA) (42, 57). AA can disrupt membranes by acting as a

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detergent, or its metabolic products can stimulate caspase activation (42, 57). Although no PLA2

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sequences were observed in the proteomic analysis of the fraction with cytotoxic activity, the

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biochemical profile indicates the presence of these esterases in this venom. As exposed above,

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metalloproteinases may be essential to induce a loss of cohesion of cells allowing the interaction

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of phospholipases derivate with MCF-7 cells. The higher cytolytic activity of P. imperator venom,

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compared with the isolated RP-HPLC fraction, explains the synergic mechanism proposed of the

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phospholipases A2 and metalloproteinases. This synergic effect of the whole venom of P.

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imperator showed a decrease in cell size suggesting an interaction of venom components with the

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cell membrane surface. Chloride and potassium channels are overexpressed in some cancer cells,

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like glioma cells, and become targets to anticancer peptides (58-61) and peptides affecting

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potassium channels could play an important role in the cytotoxic activity as well. Some DBP,

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isolated from the deathstalker scorpion Leiurus quinquestriatus (Buthidae), and is one the most

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studied anticancer peptides affecting gliomas, among the deadliest forms of cancer (62-65).

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5. Conclusions

The biochemical profiles of all venoms indicate the presence of phospholipases A2,

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metalloproteinases and serine-proteinases. All venoms showed a specific electrophoretic and

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chromatographic profile. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a

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dose-response cytotoxic activity over MCF-7 mammary carcinogenic cells, proposing these venoms

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as a potential source of anticancer molecules. No cytotoxic activity was seen with the venoms

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from A. amoreuxi, B. jacksoni, and H. gentili. The activity may be mediated by a synergic

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mechanism involving proteins and peptides like metalloproteinases, phospholipases antimicrobial

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peptides and DBP peptides that targeting ionic channels. Metalloproteinases may be essential to

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cause a loss of cohesion of cells allowing the interaction with the mentioned proteins and

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peptides. In the case of T. fuhrmanni, the loss of cohesion and the presence of the antimicrobial

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peptides, may cause direct damage to MCF-7 cells and a loss of viability related to a

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membranolytic mechanism. We reported the first MS/MS and PTM of the venoms of B. jacksoni,

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G. grandidieri and T. fuhrmanni. These venoms contain alpha toxins, neurotoxins, antimicrobial

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peptides and proteins with enzymatic activity, in some cases never reported before in these

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venoms.

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617 Acknowledgments

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The authors are grateful with the Sostenibilidad program of the Universidad de Antioquia (UdeA),

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and Comité para el Desarrollo de la Investigación CONADI, Universidad Cooperativa de Colombia.

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This research was financed with the Project CIQF-211 through the Comité para el Desarrollo de la

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Investigación (CODI-UdeA). AvdM is supported by a grant by Fundação para a Ciência e Tecnologia

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(FCT, Portugal) under the Programa Operacional Potencial Humano – Quadro de Referência

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Estratégico Nacional funds from the European Social Fund and Portuguese Ministério da Educação

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e Ciência (SFRH/BPD/101057/2014). AvdM is also funded by FEDER funds through the Operational

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Program for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation

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for Science and Technology under the project FCT-PTDC/BIA-EVF/2687/2012 and FCOMP-01-0124-

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FEDER-028340.

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Author Contributions

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Sebastián Estrada-Gómez contributed with the conceptualization, writing – review & editing,

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funding acquisition and methodology; Lyz Gomez-Rave contributed investigation and formal

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analysis; Leidy Johana Vargas Muñoz contributed with funding acquisition, writing – review &

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editing, investigation and formal analysis ; Arie van der Meijden contributed with scorpion

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collection, venom extraction, funding acquisition, and writing – review & editing.

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637 References

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Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family.

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Venoms showed the presence of proteolytic and phospholipase activity. All venoms analyzed contained defensin-like proteins, alpha toxins, metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity, among others. Venoms from P. imperator, G. grandidieri and T. fuhrmani showed a dose-dependent cytotoxic activity over MCF-7 cells. Two isolated RP-HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF-7.

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