Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin

Accepted Manuscript Title: Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin Author: Edinara Targino de Melo Andr´eia Bergamo Estrela Elizabeth Cristina Gomes Santos Paula Renata Lima Machado Kleber Juvenal Silva Farias Taffarel Melo Torres En´eas de Carvalho Jo˜ao Paulo Matos Santos Lima Arn´obio Antonio Silva-J´unior Euz´ebio Guimar˜aes Barbosa Matheus de Freitas Fernandes-Pedrosa PII: DOI: Reference:

S0196-9781(15)00061-3 http://dx.doi.org/doi:10.1016/j.peptides.2015.03.003 PEP 69420

To appear in:

Peptides

Received date: Revised date: Accepted date:

15-12-2014 2-3-2015 5-3-2015

Please cite this article as: Melo ET, Estrela AB, Santos ECG, Machado PRL, Farias KJS, Torres TM, de Carvalho E, Lima JPMS, Silva-J´unior AA, Barbosa EG, FernandesPedrosa MF, Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin, Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.003 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.

Structural characterization of a novel peptide with antimicrobial activity from the

2

venom gland of the scorpion Tityus stigmurus: Stigmurin

3 4 5 6

Edinara Targino de Melo a¥, Andréia Bergamo Estrela a¥, Elizabeth Cristina Gomes Santos a, Paula Renata Lima Machado b, Kleber Juvenal Silva Farias b, Taffarel Melo Torres c, Enéas de Carvalho d, João Paulo Matos Santos Lima e, Arnóbio Antonio SilvaJúnior a, Euzébio Guimarães Barbosa f, Matheus de Freitas Fernandes-Pedrosa a, e, *.

7 8

a. Laboratório de Tecnologia e Biotecnologia Farmacêutica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.

9 10

b. Laboratório de Imunologia Clínica, Universidade Federal do Rio Grande do Norte, RN, Brazil

11 12

c. Departamento de Ciências Animais, Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brazil.

13

d. Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.

14 15

e. Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.

16 17

f. Laboratório de Química Farmacêutica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.

M

an

us

cr

ip t

1

18

¥

19 20

* Author to whom correspondence should be addressed: e-mail: [email protected]; Tel.: +55-84-3342-9820; Fax: +55-84-3342-9833.

21

Abstract

d

te

Ac ce p

22

Both authors contributed equally to this work

A new antimicrobial peptide, herein named Stigmurin, was selected based on a

23

transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland,

24

an underexplored source for toxic peptides with possible biotechnological applications.

25

Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in

26

vitro. The CD spectra suggested that this peptide interacts with membranes, changing its

27

conformation in the presence of an amphipathic environment, with predominance of

28

random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal

29

activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5 μM. It was

30

also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results

31

suggest that Stigmurin can be considered a potential anti-infective drug.

32 33

Page 1 of 29

34

Keywords:

35

Scorpion venom; Tityus stigmurus; Antimicrobial peptide (AMP); Non-disulfide-

36

bridged

(NDBP);

Amphipathic

peptide;

Circular

Dichroism

(CD).

Ac ce p

te

d

M

an

us

cr

ip t

peptide

Page 2 of 29

37

Introduction Antimicrobial peptides (AMPs) are important components of the innate immune

39

system, showing a broad spectrum of action against microbes [33,40]. It is believed that

40

the mechanism of action of these peptides is not dependent on interaction with a

41

specific receptor, making them interesting therapeutic alternatives when compared to

42

available antibiotics; the latter act mainly by inhibiting biosynthetic pathways and are

43

more prone to induce microbial resistance [32,54,85]. AMPs are distributed across a

44

wide variety of species, including unicellular microorganisms, invertebrates and

45

vertebrates, which strongly suggests the importance of these molecules in the process of

46

survival [48,66].

us

cr

ip t

38

The presence of AMPs in the venom of scorpions is frequently reported in the

48

literature as an early evolved primitive component of the immune system [1,56]. Some

49

examples of antimicrobial peptides found in scorpion venom include Hadrurin from

50

Hadrurus

51

Parabutoporin from Parabuthus schlechteri [76], IsCTs from Opisthacanthus

52

madagascariensis [18,19], Pandinins from Pandinus imperator [15], Mucroporin from

53

Lychas mucronatus [17], Imcroporin from Isometrus maculatus [88], VmCt1, VmCt2

54

and Vejovine from Vaejovis mexicanus [36,63], BmKn2 and BmKb1 from Mesobuthus

55

martensii [87], Ctriporin from Chaerilus tricostatus [27], Meucin 13 and Meucin 18

56

from Mesobuthus eupeus [29], StCT2 from Scorpiops tibetanus [10], TsAP-1 and

57

TsAP-2 both from Tityus serrulatus [31]. A recent comprehensive review on the

58

different classes of AMP found in the venom of several scorpion species can be found

59

elsewhere [35]. Taken together, these examples indicate that scorpion venoms are rich

60

resources for the study of antimicrobial peptides.

Opistoporins

from

Opistophtalmus

carinatus

[50],

te

d

M

[75],

Ac ce p

61

aztecus

an

47

The members of Buthidae scorpions are dangerous to humans; to date,

62

approximately 500 species have been identified, of which 25 can cause lethal accidents

63

[60]. These animals have a wide distribution around the globe: species of the genera

64

Androctonus, Leiurus and Buthus are found in North Africa, Middle East and India,

65

genus Parabuthus is found in South Africa and genus Tityus in South America,

66

especially Brazil [73]. In Brazil, studies on the chemical properties of scorpion venoms

67

have been performed more frequently in Tityus serrulatus, T. bahiensis and T.

68

fasciolatus [5,39]. In contrast, the venom of T. stigmurus was not yet explored as an

Page 3 of 29

arsenal of toxins with potential biotechnological applications. Our group has previously

70

reported the identification of a transcript encoding an antimicrobial peptide (AMP) from

71

T. stigmurus, which was the most abundant transcript in the venom gland [3]. In the

72

present study, this peptide (herein named Stigmurin) had its structure evaluated in silico

73

and by circular dichroism, and its biological activities investigated against different

74

microorganisms and in different mammal cell lines in vitro.

75

Materials and methods

cr

76

ip t

69

Bioinformatic and Phylogenetic analyses

78

The complete nucleotide sequence TSTI0001C selected from the full length

79

cDNA library [3] was analyzed by ORF-Finder [70]. Multiple alignments were

80

performed with CLUSTAL X [44]. Putative signal peptide, propeptide and mature

81

peptide were predicted using SignalP v4.0 [57] and ProP 1.0 [24]. The amino acid

82

sequence was subjected to a similarity search in the database of the National Center for

83

Biotechnology Information (NCBI) using BLASTp [4].

M

an

us

77

The phylogenetic analysis was perfomed using AMP sequences with known

85

three-dimensional structure, obtained from the Antimicrobial Peptide Database (APD;

86

http://aps.unmc.edu/AP/main.php) [78]. Selected sequences were aligned using the

87

programs MAFFT [41] and MUSCLE [25]. A best-fit adjustment of the amino acids

88

substitution model was performed using the ProtTest tool [20]. The dendrogram was

89

calculated based on a Bayesian analysis using the BEAST tool [23]. Inferences were

90

conducted in two independent runs with four simultaneous chains each, and fixed WAG

91

model, allowing gamma distributed rates among sites. Each Markov Chain was initiated

92

with a random tree, ran for 106 generations, and sampled every 100 generations.

93

Consensus tree was estimated following a discarded burn-in of 1,000,000 trees. The

94

convergence of the simultaneous runs was assessed using the Tracer tool v.1.5 [22], to

95

evaluate the statistic support and robustness of the Bayesian analysis. Generated trees

96

were edited in the software FigTree (http://tree.bio.ed.ac.uk/software/figtree/) [62].

Ac ce p

te

d

84

97

Peptide modeling

98

Stigmurin modeling was performed using I-TASSER [68], and 5 models were

99

generated. The structures were validated by checking Ramachandran plots, Cβ

100

derivation, clash scores, rotamers, backbone bonds and backbone angles [12]. The

101

models were analyzed and sorted by score values, and pdb files were visualized using

Page 4 of 29

102

USFC Chimera software [83]. The top three models were submitted to a molecular

103

dynamics analysis. Models refinement

105

Molecular dynamics simulations were performed to optimize the obtained

106

models. All simulations were performed in explicit water (TIP3P) or 2,2,2-

107

trifluoroethanol (TFE) 70%, using GROMACS [71]. Simulation package and AMBER

108

99SB-ILDN force field were used [45]. The protonation state of the protein residues at

109

pH 7.0 was determined by PROPKA web server [53]. The simulation time step was 0.5

110

microseconds (µs); the minimum distance between any atom of the protein and the box

111

wall was 1.2 nm. Coulomb and van der Waals interactions within a shorter-range cutoff

112

of 1.0 nm were computed every time step. Particle Mesh Ewald was employed to

113

minimize the effects of truncating the electrostatic interactions beyond the 1.2 nm long-

114

range cutoff. Covalent bonds in the protein were constrained using the LINCS algorithm

115

[37].

an

us

cr

ip t

104

Circular Dichroism (CD)

117

Far-UV spectra of the peptide were recorded at 25 °C in water, TFE 20%, 50%

118

or 70% (v/v) or sodium dodecyl sulfate (SDS), using 0.1 cm pathlength quartz cuvette

119

in a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) with Peltier-type temperature

120

controller. Each spectrum corresponds to an average of five scans taken from 182 to 260

121

nm, at a scan rate of 50 nm/min [77,80]. Secondary structure of Stigmurin was

122

determined by deconvoluting CD data using CONTIN/LL [72], CDSSTR [14] and K2D

123

[6] algorithms. We have also used CAPITO web-server [81] to verify the results.

125

d

te

Ac ce p

124

M

116

Peptide synthesis

Mature peptide (without both, signal peptide and propeptide), herein named

126

Stigmurin, was synthesized in its amidated form by Invitrogen Life Technologies, USA,

127

using FMOC solid-phase technology [11,49]. C-terminal amidation was selected based

128

on the presence, in the complete translated sequence, of a highly conserved post-

129

translational processing signal (Gly-Arg-Arg-Lys), of which the proteolytic cleavage

130

typically precedes the amidation of the adjacent residue in the mature peptide, as

131

described previously for several similar scorpion peptides [17,31,42,86,88]. The

132

synthetic peptide was purified by reverse-phase HPLC (>90% purity) and its molecular

133

weight

134

(Supplementary figure 1).

135

was

confirmed

by

mass

spectrometry

performed

by the

supplier

Microorganism strains and in vitro antimicrobial assay

Page 5 of 29

136

For antimicrobial assays, the following strains were used: Staphylococcus

137

aureus (ATCC 29213), Escherichia coli (ATCC 25922), Methicillin-resistant

138

Staphylococcus aureus MRSA (ATCC 33591), Candida albicans (ATCC 90028),

139

Candida krusei (ATCC 6258) and Candida glabrata (ATCC 90030). Minimal inhibitory concentration (MIC) was determined by broth microdilution

141

method in Mueller Hinton Broth (MHB), following the guidelines of the Clinical and

142

Laboratory Standards Institute CLSI [13], with small variations as indicated below.

143

Inocules were prepared at 1x105 colony forming units per milliliter (CFU/mL) for

144

bacteria and to 1x104 CFU/mL for yeasts. One hundred microliters of the bacterial

145

suspension and 100 μl of serially diluted Stigmurin were added to each well in

146

microtitre plates, to a final peptide concentration of 1.1 to 139.5 μM. The plates were

147

incubated at 35 °C for 24 h (bacteria) or at 30 °C for 48 h (yeasts) with continuous

148

shaking in a humidified atmosphere. Positive growth controls were inoculated as

149

describe above, without addition of peptide. Sterile MHB was used as negative control.

150

The MIC was defined as the lowest concentration able to prevent microbial growth,

151

based on optical density (absorbance at 550 nm) [82].

M

an

us

cr

ip t

140

Hemolysis assay

153

Hemolytic activity was assessed by incubating a suspension of healthy human

154

donor erythrocytes with increasing concentrations of synthetic peptide. Cells were first

155

washed three times by centrifugation at 8,000 g for 5 min in 1X PBS, pH 7.4 [52,58],

156

then incubated with Stigmurin (1.1– 139.5 μM) at 37 °C for 180 min. Optical density

157

(absorbance at 540 nm) of supernatants was recorded using a plate reader (Epoch-

158

Biotek, Winooski, VT, USA).

160

te

Ac ce p

159

d

152

Cytotoxicity assay

The cytotoxicity of the synthetic peptide was evaluated in SiHa cells (human

161

cervical cancer cell line) and Vero E6 cells (African green monkey kidney epithelial cell

162

line). Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

163

tetrazolium bromide (MTT) assay (Sigma–Aldrich, Germany). Confluent cell-

164

monolayers contained in 96-well plates were incubated with serially diluted Stigmurin

165

(2.17– 279 μM) in RPMI-1640 for SiHa cells, or Leibovitz (L-15) for Vero E6 cells, at

166

37 °C for 24 h. Then, 50 μL of MTT (1 mg/mL) was added to each well and further

167

incubated for 4 h at 37 °C. Supernatants were removed and replaced by 100 μL of

168

dimethyl sulfoxide (DMSO) to solubilize formazan crystals [51], and absorbance was

Page 6 of 29

169

measured at 540 nm. Peptide concentration necessary to reduce cell viability in 50%

170

(IC50) was calculated by comparison with untreated cells. Statistical analysis

172

For the interpretation of bioassays, multiple-group comparisons were evaluated

173

by analysis of variance (ANOVA) using Graph Pad Prism software (Version 5.00,

174

Graph Pad, San Diego, CA, USA). Differences were considered significant when p <

175

0.05.

ip t

171

177

cr

176

Results Bioinformatic and Phylogenetic analyses

179

From the cDNA library obtained from T. stigmurus venom glands [3], the clone

180

TSTI0001C was isolated and identified, coding for a mature protein with 73 amino acid

181

residues [GenBank: JK483709], named Stigmurin. The protein sequence shows a 22

182

amino acids-long signal peptide, and propeptide starting with the highly conserved post-

183

translational processing signal: Gly-Arg-Arg-Lys-Arg (GRRKR), and 29 amino acids

184

preceding the mature protein of 17 amino acids (Figure 1).

M

an

us

178

Multiple sequence alignment with peptides retrieved from GenBank (Figure 2)

186

showed high similarity between Stigmurin and PAMs of different scorpion species, all

187

exhibiting experimentally proved in vitro antimicrobial activity [2,8,17,21,31,47,88].

188

Several amino acid residues are observed in conserved positions of these peptides,

189

especially in mature toxin region. Probably all aligned sequences undergo post-

190

translational modification, given the presence of the signal peptide, propeptide and

191

cleavage site [31]. Interestingly, the phylogenetic analysis (Figure 3) demonstrated that

192

Stigmurin, along with the scorpion peptides Mucroporin and Bmkb1, exhibited

193

significant sequence similarity (as well as an evolutive proximity) to phylloseptins, a

194

family of antimicrobial peptides from South/Central American Phyllomedusinae frog

195

skin [43,61].

te

Ac ce p

196

d

185

According to structural modeling, it was observed that Stigmurin shows alpha-

197

helical conformation (Figure 4A). The stability of the secondary structure depends on

198

the environment by which it is surrounded; this is supported by two simulation

199

experiments where the peptide changed from alpha-helix structure to a beta-sheet

200

conformation when in water, whereas it assumed a random coil structure when the

201

simulation was performed in TFE 70% (Figure 4 B and C, and Supplementary video

202

1 and 2).

Page 7 of 29

Secondary structure evaluated by CD spectroscopy

204

The CD analysis of synthetic Stigmurin in aqueous solution showed a typical

205

spectrum of disordered structure. When 20% TFE was added to the system, the dichroic

206

spectrum changed; in 50% TFE, the deviation of the spectra from zero-value of

207

ellipticity became more prominent, and the results were similar in 70% TFE, as well as

208

in SDS above critical micelle concentration (CMC), a membrane-mimetic environment

209

(Figure 5A). The spectra were analyzed by deconvolution using four different

210

approaches [6,14,72,81]. Figure 5B presents the proportion of the sequence assuming

211

alpha-helical, beta-sheet or random coil secondary structure in the different

212

environments, calculated as the mean of the values found by the four methods. The

213

results show a general predominance of random coil and beta-sheet conformation.

us

cr

ip t

203

Antimicrobial activity

215

Stigmurin was active against Gram-positive bacterial strains Staphylococcus

216

aureus and Methicillin-resistant S. aureus MRSA, and the yeasts Candida albicans, C.

217

krusei, and C. glabrata, but it was ineffective against Gram-negative Escherichia coli

218

(Table 1). Interestingly, Stigmurin was found to possess low hemolytic activity (22 %)

219

at the highest concentration tested (139.5 μM), as shown in Figure 6.

M

an

214

In vitro cytotoxicity

221

Stigmurin showed toxicity against SiHa cells, leading to 36%, 69% and 92% of

222

non-viable cells after exposition to the peptide at concentrations of 69.8 μM, 139.5 μM

223

and 279 μM, respectively (Figure 7A). Cytotoxic effect it was also observed in Vero E6

224

cells, where 57% and 89% of the cells were non-viable after incubation with Stigmurin

225

at concentrations of 139.5 μM and 279 μM, respectively (Figure 7B). The IC50

226

parameter verifies that viability reduction was more prominent on human carcinoma

227

lineage SiHa (IC50 118 μM), than on monkey epithelial cells Vero E6 (IC50 150 μM).

229

te

Ac ce p

228

d

220

Discussion

230

The identification and analysis of Stigmurin demonstrated that this peptide has

231

high sequence similarity to other non-disulfide-bridged antimicrobial peptides

232

[2,8,17,21,31,47,88]. Through multiple sequence alignment and phylogenetic analysis,

233

it could be noticed a significant conservation of key amino acid residues that are

234

characteristic of antimicrobial peptides [73]. This observation is consistent with the

235

hypothesis that many AMPs are evolutionarily related within the Scorpionidae taxon,

Page 8 of 29

236

having arisen from multiple duplications of an ancestral gene [73,86]. The

237

diversification of AMPs could then be part of an evolutionary strategy driven by the

238

rapid evolution of pathogens [67]. Furthermore, bioinformatic analysis showed evidence

239

that Stigmurin is a new AMP. Structural analysis of Stigmurin by molecular dynamics and CD showed that its

241

structure differs when in the presence of water, SDS or TFE. These results suggest that

242

it may assume different conformations depending on the environment, which is a

243

common behavior in AMPs [74]. It is essential to highlight that, according to CD

244

analysis, Stigmurin exhibited a more regular secondary structure when in contact with

245

TFE or SDS. TFE diminishes the peptide interaction with the solvent and favors

246

intramolecular interactions, thus increasing the chance of secondary structure

247

conformation (in this work, mainly β-sheet) [65]. Stigmurin presented a different

248

structure in the presence of water, mostly random coil. Furthermore, the contact with a

249

micellar solution led to a conformational shift in Stigmurin's structure, indicating that it

250

may interact with micelles, which mimic membranes [74]. This suggests that the

251

mechanism of action of Stigmurin is through interaction with membranes, which is very

252

common among other AMPs already characterized [33,40,65,74,85].

d

M

an

us

cr

ip t

240

It is noticed a discrepancy between the homology models (all alpha-helical

254

structure, Figure 4A) with the findings from dynamics simulation (Figure 4B and 4C)

255

and experimental CD results (Figure 5A and 5B). Altogether, the present study revealed

256

that Stigmurin alters its secondary structure according to the medium, which shows that

257

the residues can be structured or flexible in specific conditions, possibly having a

258

functional significance [33,34,74]. Another point to be discussed is that the molecular

259

dynamics simulation agrees with deconvoluted CD results regarding the type of

260

predominant structures, but not the tendency of variation in different media. This could

261

derive from the deconvolution algorithms, which are optimized for larger proteins rather

262

than for small peptides [6,14,72], or from issues to accurately represent the dielectric

263

properties with the force field employed in the simulations [38]. Nevertheless, it can be

264

concluded that Stigmurin has an environment-dependent conformation.

Ac ce p

te

253

265

In this work we have demonstrated that Stigmurin, a novel antimicrobial peptide,

266

possesses antimicrobial activity against Gram-positive bacteria, including antibiotic-

267

resistant pathogens (MRSA), and fungi. Peptide TsAP-1 from Tityus serrulatus venom,

Page 9 of 29

a highly similar scorpion AMP (96% identity) from a closely related species, presented

269

poor antimicrobial activity (MIC > 120 µM for S. aureus, E. coli and C. albicans) when

270

compared to Stigmurin [31]. Both peptides have the same net charge (+2 considering

271

the C-terminal amidation), and there are only two residues which differ in the mature

272

peptide sequences, exchanging Phe24 and Leu34 in Stigmurin for a Leu and a Ser,

273

respectively, in TsAP-1 [31]. It is possible that the different activity is a result of a

274

decreased hydrophobicity conferred by Ser to TsAP-1. Peptide hydrophobicity, together

275

with a positive net charge in an amphipatic arrangement, is considered a major

276

requirement for their antimicrobial activity [59]. In contrast, despite a lower sequence

277

similarity to Stigmurin (78%), peptide TsAP-2 from T. serrulatus has identical

278

proportion of hydrophobic residues (10 out of 17) and presented a more similar activity,

279

causing deleterious effects in Gram-positive bacterium S. aureus development, and in

280

C. albicans, but being less effective against Gram-negative E. coli [31].

an

us

cr

ip t

268

In fact, activity against Gram-positive bacteria with a weaker activity against

282

Gram-negative bacteria was also described for other non-dissulfide-bridged peptides

283

from scorpions such as Pandinin1 and -2, IsCT and IsCT2 [15,18,19]. Among these

284

peptides, IsCT, IsCT2 and TsAP-2 present a conserved consensus sequence (X(0-

285

5)IX3IX3IX4)

286

This consensus is partially present in Stigmurin and Pandinin2, where the second Ile

287

residue is substituted by other hydrophobic residues (Val and Phe, respectively). Here

288

again, hydrophobicity seems to be a key aspect in the antimicrobial activity of cationic

289

peptides. The initial electrostatic interaction with negatively charged surface molecules

290

(e.g. teichoic acids in the Gram-positive cell wall), and subsequent membrane insertion

291

are important steps in the process of killing; however, the mechanisms of action and the

292

specific cellular targets can vary among peptides and among organisms for a given

293

peptide [28]. Furthermore, it is important to note that the differences in the cell surface,

294

or even differences in the absorption of the agent by each type of microorganism are

295

other factors that can influence the MIC values [69].

d

M

281

Ac ce p

te

where the three Ile residues seem to play an important functional role [86].

296

Infections by multi-drug resistant pathogens such as MRSA are reaching

297

alarming numbers worldwide, frequently causing the death of hospital patients [55]. It is

298

appreciated that the mechanism of action of AMPs, in contrast to most conventional

299

antibiotics, is less prone to induce microbial resistance, and peptide antibiotics have

300

been considered valuable alternatives in the treatment of such infections [55]. Our

Page 10 of 29

results suggest that Stigmurin can be a candidate molecule for use in antimicrobial

302

therapy. Furthermore, at the concentration which Stigmurin has prominent antimicrobial

303

activity, its hemolytic activity is only mild, supporting the potential use of this molecule

304

for therapeutic purposes [88]. Interestingly, Stigmurin combines a good antimicrobial

305

activity with a fairly low hemolytic activity, in contrast to the closely related peptides

306

from T. serrulatus which are either poorly antimicrobial (TsAP-1) or highly hemolytic

307

(TsAP-2) [31].

ip t

301

There are fewer studies reporting AMPs activity against Candida species, but it

309

is believed that the mechanism of action is similar to that observed in bacteria [40]. The

310

cell wall of yeasts is an anionic surface characterized by its rigidity, being glucans (1,3-

311

β and 1,6-β) the most abundant polysaccharides (around 60%), followed by mannans.

312

Chitin, the less abundant component, corresponds to approximately 1% of all

313

saccharides present in the cell wall [46]. Some scorpions AMPs tested against Candida

314

albicans yielded similar results to those we found for Stigmurin, including: TSAP-2

315

(MIC 10 μM) [31], Meucin-18 (MIC 24.1 μM) [29], and Ctriporin (MIC 20 μg/mL)

316

[27].This data strengthens our results suggesting that Stigmurin could be considered for

317

the treatment of candidiasis.

d

M

an

us

cr

308

The results on the cytotoxic activity of Stigmurin indicate a possible differential

319

toxicity towards tumor cells. This is in accordance with observations by other

320

researchers reporting variations in sensitivity of different cell cultures to antitumor

321

AMPs [7,79]. For instance, peptides present in the venom of the scorpion Tityus

322

discrepans were active against SKBR3 breast cancer cell line, having negligible effect

323

upon monkey kidney cells (MA104) [16]. The exact mechanism of toxicity which

324

differs in different cell types is not yet fully elucidated, but may be related to the

325

negative charge of cancer cell membranes, due to increased expression of molecules

326

with anionic character, such as glycosylated mucins, sialylated gangliosides, and

327

heparin sulfate [30]. Furthermore, it has been reported that cell intrinsic factors

328

influence their sensitivity to toxic substances, including chemical biotransformation and

329

binding forms, characteristics of membrane permeability and surface determinants,

330

intracellular synthesis and mechanisms of adaptation and recovery [9,64,84].

Ac ce p

te

318

331

From T. serrulatus, peptide TsAP-2 showed cytotoxicity against five different

332

human cancer cell lines, with IC50 ranging from 4 to 15 µM, whereas TsAP-1 had a less

Page 11 of 29

effective antitumoral action [31]. This was in line with their antimicrobial activity; it is

334

thus possible that, like the effect upon microorganisms, Stigmurin's antitumoral

335

potential is more similar to TsAP-2, despite a lower sequence similarity, as discussed

336

above. The IC50 values obtained in the high µM range for Stigmurin upon the two cell

337

lines tested in the present study are not per se indicators of an antitumoral activity;

338

besides potency, other parameters such as maximum effect and the slope of the dose-

339

response curve, should also be taken into account when analyzing putative novel

340

antitumoral drugs [26]. Further studies on a wider panel of tumor cell lineages will be

341

required to suggest a possible anticancer potential for Stigmurin. Nevertheless, this is to

342

our knowledge the first scientific evidence demonstrating the effect of a component of

343

T. stigmurus venom on cell viability of a human cancer cell line.

us

cr

ip t

333

Acknowledgements

345

We would like to thank CAPES (Toxinology 063/2010) and FAPERN

346

(PRONEM 2011) for the financial support. The authors are also grateful to Kobashi,

347

L.S. from the Butantan Institute who helped in the discussion of circular dichroism

348

experiments. A.B.E. acknowledges a research fellowship from Conselho Nacional de

349

Desenvolvimento Científico e Tecnológico (CNPq). M.F.F.P. is a researcher from

350

CNPq.

te

Ac ce p

351

d

M

an

344

352

References

353 354 355

[1]

Almaaytah A, Albalas Q. Scorpion venom peptides with no disulfide bridges: A review. Peptides 2014;51:35–45. doi:http://dx.doi.org/10.1016/j.peptides.2013.10.021.

356 357 358 359 360

[2]

Almaaytah A, Zhou M, Wang L, Chen T, Walker B, Shaw C. Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: biochemical and functional characterization of natural peptides and a single site-substituted analog. Peptides 2012;35:291–9. doi:10.1016/j.peptides.2012.03.016.

361 362 363 364

[3]

Almeida DD, Scortecci KC, Kobashi LS, Agnez-Lima LF, Medeiros SRB, SilvaJunior AA, et al. Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey. BMC Genomics 2012;13:362. doi:10.1186/1471-2164-13-362.

365 366 367

[4]

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402. doi:10.1093/nar/25.17.3389.

Page 12 of 29

[5]

Alvarenga ER. Transcriptome analysis of the Tityus serrulatus scorpion venom gland. Open J Genet 2012;02:210–20. doi:10.4236/ojgen.2012.24027.

370 371 372

[6]

Andrade MA, Chacón P, Merelo JJ, Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 1993;6:383–90.

373 374 375

[7]

Arnusch CJ, Pieters RJ, Breukink E. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides. PLoS One 2012;7:e39768. doi:10.1371/journal.pone.0039768.

376 377 378 379

[8]

Baradaran M, Jalali A, Jolodar A, Ghasemian S. A new caerin-like antibacterial peptide from the venom gland of the Iranian scorpion Mesobuthus eupeus: cDNA amplification and sequence analysis. African J Biotechnol 2012;11:10176–81. doi:10.5897/AJB11.3373.

380 381

[9]

Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005;3:238–50. doi:10.1038/nrmicro1098.

382 383 384

[10]

Cao L, Li Z, Zhang R, Wu Y, Li W, Cao Z. StCT2, a new antibacterial peptide characterized from the venom of the scorpion Scorpiops tibetanus. Peptides 2012;36:213–20. doi:10.1016/j.peptides.2012.04.010.

385 386 387

[11]

Carpino LA, Han GY. 9-Fluorenylmethoxycarbonyl function, a new basesensitive amino-protecting group. J Am Chem Soc 1970;92:5748–9. doi:10.1021/ja00722a043.

388 389 390 391

[12]

Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr Sect D Biol Crystallogr 2010;66:12–21. doi:10.1107/S0907444909042073.

392 393 394 395 396

[13]

Cockerill FR, Wikler MA, Alder J, Dudley MN, Eliopoulos GM, Ferraro MJ, et al. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically ; Approved Standard — Ninth Edition. Methods Dilution Antimicrob Susceptibility Tests Bact That Grow Aerob Approv Standar- Ninth Ed 2012;32.

397 398 399

[14]

Compton LA, Johnson WC. Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 1986;155:155–67. doi:10.1016/0003-2697(86)90241-1.

400 401 402 403

[15]

Corzo G, Escoubas P, Villegas E, Barnham KJ, He W, Norton RS, et al. Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator. Biochem J 2001;359:35–45. doi:10.1042/02646021:3590035.

404 405 406

[16]

D’Suze G, Rosales A, Salazar V, Sevcik C. Apoptogenic peptides from Tityus discrepans scorpion venom acting against the SKBR3 breast cancer cell line. Toxicon 2010;56:1497–505. doi:10.1016/j.toxicon.2010.09.008.

407 408 409

[17]

Dai C, Ma Y, Zhao Z, Zhao R, Wang Q, Wu Y, et al. Mucroporin, the first cationic host defense peptide from the venom of Lychas mucronatus. Antimicrob Agents Chemother 2008;52:3967–72. doi:10.1128/AAC.00542-08.

410 411 412

[18]

Dai L, Yasuda A, Naoki H, Corzo G, Andriantsiferana M, Nakajima T. IsCT, a novel cytotoxic linear peptide from scorpion Opisthacanthus madagascariensis. Biochem Biophys Res Commun 2001;286:820–5. doi:10.1006/bbrc.2001.5472.

Ac ce p

te

d

M

an

us

cr

ip t

368 369

Page 13 of 29

[19]

Dai L, Corzo G, Naoki H, Andriantsiferana M, Nakajimaa T. Purification, structure-function analysis, and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis. Biochem Biophys Res Commun 2002;293:1514–22. doi:10.1016/S0006-291X(02)00423-0.

417 418 419

[20]

Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 2011;27:1164–5. doi:10.1093/bioinformatics/btr088.

420 421 422

[21]

Diego-García E, Batista CVF, García-Gómez BI, Lucas S, Candido DM, GómezLagunas F, et al. The Brazilian scorpion Tityus costatus Karsch: genes, peptides and function. Toxicon 2005;45:273–83. doi:10.1016/j.toxicon.2004.10.014.

423 424

[22]

Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 2007;7:214. doi:10.1186/1471-2148-7-214.

425 426 427

[23]

Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 2012;29:1969–73. doi:10.1093/molbev/mss075.

428 429

[24]

Duckert P, Brunak S, Blom N. Prediction of proprotein convertase cleavage sites. Protein Eng Des Sel 2004;17:107–12. doi:10.1093/protein/gzh013.

430 431

[25]

Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004;32:1792–7. doi:10.1093/nar/gkh340.

432 433 434

[26]

Fallahi-Sichani M, Honarnejad S, Heiser LM, Gray JW, Sorger PK. Metrics other than potency reveal systematic variation in responses to cancer drugs. Nat Chem Biol 2013;9:708–14. doi:10.1038/nchembio.1337.

435 436 437 438

[27]

Fan Z, Cao L, He Y, Hu J, Di Z, Wu Y, et al. Ctriporin, a new anti-methicillinresistant Staphylococcus aureus peptide from the venom of the scorpion Chaerilus tricostatus. Antimicrob Agents Chemother 2011;55:5220–9. doi:10.1128/AAC.00369-11.

439 440 441

[28]

Friedrich CL, Moyles D, Beveridge TJ, Hancock REW. Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria. Antimicrob Agents Chemother 2000;44:2086–92. doi:10.1128/AAC.44.8.2086-2092.2000.

442 443 444 445

[29]

Gao B, Sherman P, Luo L, Bowie J, Zhu S. Structural and functional characterization of two genetically related meucin peptides highlights evolutionary divergence and convergence in antimicrobial peptides. FASEB J 2009;23:1230–45. doi:10.1096/fj.08-122317.

446 447

[30]

Gaspar D, Veiga AS, Castanho MARB. From antimicrobial to anticancer peptides. A review. Front Microbiol 2013;4:294. doi:10.3389/fmicb.2013.00294.

448 449 450 451

[31]

Guo X, Ma C, Du Q, Wei R, Wang L, Zhou M, et al. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities. Biochimie 2013;95:1784–94. doi:10.1016/j.biochi.2013.06.003.

452 453 454

[32]

Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 2000;8:402–10. doi:10.1016/S0966842X(00)01823-0.

455 456 457

[33]

Hancock REW, Sahl H-G. Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies. Nat Biotechnol 2006;24:1551–7. doi:10.1038/nbt1267.

Ac ce p

te

d

M

an

us

cr

ip t

413 414 415 416

Page 14 of 29

[34]

Hansson T, Oostenbrink C, van Gunsteren W. Molecular dynamics simulations. Curr Opin Struct Biol 2002;12:190–6. doi:10.1016/S0959-440X(02)00308-1.

460 461 462

[35]

Harrison PL, Abdel-Rahman M a, Miller K, Strong PN. Antimicrobial peptides from scorpion venoms. Toxicon 2014;88:115–37. doi:10.1016/j.toxicon.2014.06.006.

463 464 465 466

[36]

Hernandez-Aponte CA, Silva-Sanchez J, Quintero-Hernandez V, RodriguezRomero A, Balderas C, Possani LD, et al. Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus. Toxicon 2011;57:84–92. doi:10.1016/j.toxicon.2010.10.008.

467 468 469

[37]

Hess B, Bekker H, Berendsen HJC, Fraaije JGEM. LINCS: A linear constraint solver for molecular simulations. J Comput Chem 1997;18:1463–72. doi:10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H.

470 471 472

[38]

Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 2006;65:712–25. doi:10.1002/prot.21123.

473 474 475 476 477

[39]

Horta CCR, de Magalhães BF, Oliveira-Mendes BBR, Carmo AO do, Duarte CG, Felicori LF, et al. Molecular, Immunological, and Biological Characterization of Tityus serrulatus Venom Hyaluronidase: New Insights into Its Role in Envenomation. PLoS Negl Trop Dis 2014;8. doi:10.1371/journal.pntd.0002693.

478 479

[40]

Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev 2006;19:491–511. doi:10.1128/CMR.00056-05.

480 481

[41]

Katoh K, Toh H. Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 2010;26:1899–900. doi:10.1093/bioinformatics/btq224.

482 483 484 485

[42]

Van Kesteren RE, Smit AB, Dirks RW, de With ND, Geraerts WP, Joosse J. Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis. Proc Natl Acad Sci U S A 1992;89:4593–7.

486 487 488

[43]

König E, Bininda-Emonds ORP. Evidence for convergent evolution in the antimicrobial peptide system in anuran amphibians. Peptides 2011;32:20–5. doi:10.1016/j.peptides.2010.10.009.

489 490

[44]

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8.

491 492 493

[45]

Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins Struct Funct Bioinforma 2010;78:1950–8. doi:10.1002/prot.22711.

494 495

[46]

Lipke PN, Ovalle R. Cell wall architecture in yeast : New structure and new challenges. Jourunal Bacteriol 1998;180:3735–40.

496 497 498 499

[47]

Luo F, Zeng XC, Hahin R, Cao ZJ, Liu H, Li WX. Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: Gaining insight into evolutionary mechanism. Peptides 2005;26:2427– 33. doi:10.1016/j.peptides.2005.06.008.

500 501 502

[48]

Melo MN, Ferre R, Castanho MARB. Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat Rev Microbiol 2009;7:245–50. doi:10.1038/nrmicro2095.

Ac ce p

te

d

M

an

us

cr

ip t

458 459

Page 15 of 29

[49]

Merrifield RB. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J Am Chem Soc 1963;85:2149–54. doi:10.1021/ja00897a025.

505 506 507 508

[50]

Moerman L, Bosteels S, Noppe W, Willems J, Clynen E, Schoofs L, et al. Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa. Eur J Biochem 2002;269:4799–810. doi:10.1046/j.1432-1033.2002.03177.x.

509 510 511

[51]

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. doi:10.1016/0022-1759(83)90303-4.

512 513 514

[52]

Oguiura N, Boni-Mitake M, Affonso R, Zhang G. In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus. J Antibiot 2011;64:327–31. doi:10.1038/ja.2011.10.

515 516 517

[53]

Olsson MHM, SØndergaard CR, Rostkowski M, Jensen JH. PROPKA3: Consistent treatment of internal and surface residues in empirical p K a predictions. J Chem Theory Comput 2011;7:525–37. doi:10.1021/ct100578z.

518 519 520

[54]

Oyston PCF, Fox MA, Richards SJ, Clark GC. Novel peptide therapeutics for treatment of infections. J Med Microbiol 2009;58:977–87. doi:10.1099/jmm.0.011122-0.

521 522 523

[55]

Park SC, Park Y, Hahm KS. The role of antimicrobial peptides in preventing multidrug-resistant bacterial infections and biofilm formation. Int J Mol Sci 2011;12:5971–92.

524 525 526

[56]

Pasupuleti M, Schmidtchen A, Malmsten M. Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 2012;32:143–71. doi:10.3109/07388551.2011.594423.

527 528 529

[57]

Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8:785–6. doi:10.1038/nmeth.1701.

530 531 532 533

[58]

Polyansky AA, Vassilevski AA, Volynsky PE, Vorontsova O V., Samsonova O V., Egorova NS, et al. N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins. FEBS Lett 2009;583:2425–8. doi:10.1016/j.febslet.2009.06.044.

534 535 536

[59]

Powers J-PS, Hancock REW. The relationship between peptide structure and antibacterial activity. Peptides 2003;24:1681–91. doi:10.1016/j.peptides.2003.08.023.

537 538 539

[60]

Prendini L, Wheeler WC. Scorpion higher phylogeny and classification, taxonomic anarchy, and standards for peer review in online publishing. Cladistics 2005;21:446–94. doi:10.1111/j.1096-0031.2005.00073.x.

540 541 542 543 544

[61]

Raja Z, André S, Piesse C, Sereno D, Nicolas P, Foulon T, et al. Correction: Structure, Antimicrobial Activities and Mode of Interaction with Membranes of Bovel Phylloseptins from the Painted-Belly Leaf Frog, Phyllomedusa sauvagii. PLoS One 2013;8:10.1371/annotation/dbb3e614–dc4c – 40dd – b9e0–37787ae. doi:10.1371/annotation/dbb3e614-dc4c-40dd-b9e0-37787ae6b150.

545 546

[62]

Rambaut A. FigTree, a graphical viewer of phylogenetic trees. Inst Evol Biol Univ Edinburgh 2009.

Ac ce p

te

d

M

an

us

cr

ip t

503 504

Page 16 of 29

[63]

Ramírez-Carreto S, Quintero-Hernández V, Jiménez-Vargas JM, Corzo G, Possani LD, Becerril B, et al. Gene cloning and functional characterization of four novel antimicrobial-like peptides from scorpions of the family Vaejovidae. Peptides 2012;34:290–5. doi:10.1016/j.peptides.2012.02.002.

551 552 553 554

[64]

Ran S, He J, Huang X, Soares M, Scothorn D, Thorpe PE. Antitumor effects of a monoclonal antibody that binds anionic phospholipids on the surface of tumor blood vessels in mice. Clin Cancer Res 2005;11:1551–62. doi:10.1158/10780432.CCR-04-1645.

555 556 557 558

[65]

Roccatano D, Colombo G, Fioroni M, Mark AE. Mechanism by which 2,2,2trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study. Proc Natl Acad Sci U S A 2002;99:12179–84. doi:10.1073/pnas.182199699.

559 560

[66]

Rodríguez de la Vega RC, Schwartz EF, Possani LD. Mining on scorpion venom biodiversity. Toxicon 2010;56:1155–61. doi:10.1016/j.toxicon.2009.11.010.

561 562 563

[67]

Roelants K, Fry BG, Ye L, Stijlemans B, Brys L, Kok P, et al. Origin and Functional Diversification of an Amphibian Defense Peptide Arsenal. PLoS Genet 2013;9:e100. doi:10.1371/journal.pgen.1003662.

564 565 566

[68]

Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 2010;5:725–38. doi:10.1038/nprot.2010.5.

567 568

[69]

Russell AD. Similarities and differences in the responses of microorganisms to biocides. J Antimicrob Chemother 2003;52:750–63. doi:10.1093/jac/dkg422.

569 570 571

[70]

Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2010;38:5–16. doi:10.1093/nar/gkh073.

572 573 574

[71]

Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC. GROMACS: Fast, flexible, and free. J Comput Chem 2005;26:1701–18. doi:10.1002/jcc.20291.

575 576 577 578

[72]

Van Stokkum IH, Spoelder HJ, Bloemendal M, van Grondelle R, Groen FC. Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal Biochem 1990;191:110–8. doi:10.1016/00032697(90)90396-Q.

579 580 581 582

[73]

Sunagar K, Undheim EAB, Chan AHC, Koludarov I, Muñoz-Gómez SA, Antunes A, et al. Evolution stings: The origin and diversification of scorpion toxin peptide scaffolds. Toxins (Basel) 2013;5:2456–87. doi:10.3390/toxins5122456.

583 584 585

[74]

Takahashi T, Nomura F, Yokoyama Y, Tanaka-Takiguchi Y, Homma M, Takiguchi K. Multiple membrane interactions and versatile vesicle deformations elicited by melittin. Toxins (Basel) 2013;5:637–64. doi:10.3390/toxins5040637.

586 587 588

[75]

Torres-Larios A, Gurrola GB, Zamudio FZ, Possani LD. Hadrurin, A new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus. Eur J Biochem 2000;267:5023–31. doi:10.1046/j.1432-1327.2000.01556.x.

589 590 591

[76]

Verdonck F, Bosteels S, Desmet J, Moerman L, Noppe W, Willems J, et al. A novel class of pore-forming peptides in the venom of Parabuthus schlechteri Purcell (scorpions: buthidae). Cimbebasia 2000;16:247–60.

Ac ce p

te

d

M

an

us

cr

ip t

547 548 549 550

Page 17 of 29

[77]

Wallace BA, Lees JG, Orry AJW, Lobley A, Janes RW. Analyses of circular dichroism spectra of membrane proteins. Protein Sci 2003;12:875–84. doi:10.1110/ps.0229603.

595 596 597

[78]

Wang G, Li X, Wang Z. APD2: The updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 2009;37. doi:10.1093/nar/gkn823.

598 599 600

[79]

Wang WX, Ji YH. Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro. J Neurooncol 2005;73:1–7. doi:10.1007/s11060-004-4205-6.

601 602 603

[80]

Whitmore L, Wallace BA. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 2008;89:392–400. doi:10.1002/bip.20853.

604 605 606

[81]

Wiedemann C, Bellstedt P, Görlach M. CAPITO--a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics 2013;29:1750–7. doi:10.1093/bioinformatics/btt278.

607 608 609

[82]

Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008;3:163–75. doi:10.1038/nprot.2007.521.

610 611 612

[83]

Yang Z, Lasker K, Schneidman-Duhovny D, Webb B, Huang CC, Pettersen EF, et al. UCSF Chimera, MODELLER, and IMP: An integrated modeling system. J Struct Biol 2012;179:269–78. doi:10.1016/j.jsb.2011.09.006.

613 614

[84]

Yount NY, Yeaman MR. Peptide antimicrobials: Cell wall as a bacterial target. Ann N Y Acad Sci 2013;1277:127–38. doi:10.1111/nyas.12005.

615 616

[85]

Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389–95. doi:10.1038/415389a.

617 618

[86]

Zeng X-C, Corzo G, Hahin R. Scorpion venom peptides without disulfide bridges. IUBMB Life 2005;57:13–21. doi:10.1080/15216540500058899.

619 620 621 622

[87]

Zeng X-C, Wang S-X, Zhu Y, Zhu S-Y, Li W-X. Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch. Peptides 2004;25:143–50. doi:10.1016/j.peptides.2003.12.003.

623 624 625

[88]

Zhao Z, Ma Y, Dai C, Zhao R, Li S, Wu Y, et al. Imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion Isometrus maculates. Antimicrob Agents Chemother 2009;53:3472–7. doi:10.1128/AAC.01436-08.

627

cr

us

an

M

d

te

Ac ce p

626

ip t

592 593 594

628 629 630 631 632 633

Page 18 of 29

Figure 1 - Nucleotide and translated amino acid sequences of the cDNA clone encoding

635

Stigmurin precursor, as analyzed by ORF-Finder. Predicted amino acid sequence is

636

shown below corresponding codons. Putative signal peptide (Bold), propeptide (Italics)

637

and mature peptide (dark gray box) were predicted using SignalP v4.0 and ProP 1.0.

638

The conserved propetide convertase cleavage site is indicated in light gray box.

ip t

634

639

Figure 2 - Alignment of Stigmurin (GenBank: JK483709) with that of known

641

antimicrobial peptides from scorpions by CLUSTAL X, indicating different peptide

642

regions. Gray scale indicates the residues’ degree of conservation. Dashed line: signal

643

peptide; Stars: probable active peptide; Asterisks: C-terminal amidated lysine and

644

adjacent amide donor glycine residues; Crosses: cleavage site for proprotein convertase;

645

Rectangle: propeptide. GenBank accession numbers are: S6CWV8.1: TsAP-1 from

646

Tityus serrulatus; Q5G8B3.1: antimicrobial peptide clone 6 from T. costatus;

647

Q5G8B4.1: antimicrobial peptide clone 5 from T. costatus; S6D3A7.1: TsAP-2 from T.

648

serrulatus; AGC92780.1: caerin-like peptide from Mesobuthus eupeus; AAW23032.1:

649

toxin peptide from M. martensii; G8YYA5.1: AamAP1 from Androctonus amoreuxi;

650

B9UIY3.1: Mucroporin, from Lychas mucronatus; C7B247.1: Imcroporin from

651

Isometrus maculatus.

652 653

Figure 3 - Phylogenetic analysis of Stigmurin and antimicrobial peptides with known

654

secondary structure, obtained from multiple alignment using MAFFT program.

655

Bayesian analysis was performed in BEAST tool using fixed WAG model. Posterior

656

predictive p-values are shown in the nodes. Color code represent: Red = scorpion

657

AMPs, Blue = Stigmurin; Green = Phylloseptins, Black = Peptides from hylids.

658

Figure 4 - Modeled patterns of Stigmurin’s secondary structure A) Stigmurin modeled

659

ab initio by I-TASSER, visualized using USFC Chimera software. Hydrophobic

660

residues and hydrophilic residues are shown in red and blue, respectively. Also,

661

representative snapshots from the dynamics simulations are shown in B) in explicit

662

water and C) in 70% (v/v) TFE solution. Simulations were performed in GROMACS

663

with 0.5-μs time steps, starting from full α-helices and using AMBER 99SB-ILDN

664

force field.

Ac ce p

te

d

M

an

us

cr

640

665

Page 19 of 29

Figure 5 - Stigmurin evaluation by Far-UV circular dichroism. (A) CD spectra in water

667

(light gray), TFE 20 % (dotted), 50 % (dashed), and 70 % (solid black), 20 mM SDS

668

(dark gray). (B) Predominance of secondary structures according to deconvolution data

669

of CD spectra using different algorithms (CONTIN/LL, SELCON and K2D) and

670

CAPITO server (CD Analysis and Plotting Tool). Values presented are mean ± SD of

671

the percentages obtained by the four different methods, except in water, which was

672

performed exclusively using CAPITO.

673

Table 1 - Minimum inhibitory concentrations (MIC) of synthetic Stigmurin peptide

674

against standard bacterial strains. Values represent mean ± SD (n = 3). ** p < 0.01 and

675

*** p < 0.001 compared to positive control.

676

Figure 6 - Hemolytic activity of Stigmurin. Human red blood-cells were incubated with

677

serially diluted Stigmurin (1.1– 139.5 μM), at 37 °C for 180 min. The activity was

678

determined by measuring the absorbance at 540 nm, using PBS and distilled water as

679

negative and positive lysis control, respectively. Percentage hemolysis was calculated

680

relative to the positive control (100 %). Values represent mean ± SD (n = 3). ** p <

681

0.01 compared to positive control.

cr

us

an

M

d

682

ip t

666

Figure 7 - Citotoxicity of Stigmurin. Dose-response curves of cell viability as measured

684

by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in cell

685

lines SiHa (A) and Vero E6 (B). Cells were cultured in 96-well microtitre plates using

686

RPMI-1640 or L-15 media in the presence of serially diluted Stigmurin (2.17– 279 μM)

687

for 24 h. The activity was determined by measuring the absorbance at 540 nm after

688

addition of MTT for 4 h and subsequent solubilization with DMSO. Values represent

689

mean ± SD (n = 3). Statistical significance was analyzed using a paired t-test . * p <

690

0.05, ** p < 0.01 and *** p < 0.001 compared to positive control.

Ac ce p

691

te

683

692

Page 20 of 29

692 693 694

Highlights  This work describes a novel peptide (Stigmurin) from Tityus stigmurus scorpion

699

 Stigmurin´s structure was evaluated in silico and by circular dichroism

700 701

 The peptide assumes different conformations depending on the environment

702

 Stigmurin exhibited antibacterial and antifungal activities

703

 Stigmurin was toxic against SiHa and Vero E6 cell lines

us

cr

ip t

695 696 697 698

Ac ce p

te

d

M

an

704

Page 21 of 29

Strains Gram-positive Staphylococcus aureus (ATCC 29213) Methicillin-resistant S. aureus MRSA (ATCC 33591)

MIC (µM ± SD)

> 139

Yeasts Candida albicans (ATCC 90028) C. krusei (ATCC 6258) C. glabrata (ATCC 90030)

Ac ce p

te

d

M

an

us

34.75 ± 1.4 *** 69.5 ± 4.02 ** 69.5 ± 2.83 ***

cr

Gram-negative Escherichia coli (ATCC 25922)

ip t

8.68 ± 1.1 *** 17.37 ± 1.2 ***

Page 22 of 29

Ac

ce

pt

ed

M

an

us

cr

i

Figure 1

Page 23 of 29

Ac

ce

pt

ed

M

an

us

cr

i

Figure 2

Page 24 of 29

cr us

0,04

0,2 0,1 0,12 0,14

0,29

M an ed

1

0,27

ce pt

0,14 0,16

0,24

Ac

0,69

Mucroporin (Lychas mucronatus -AP01327) Bmkb1 (Buthus martensii Karsch -AP01977) Stigmurin, Tityus stigmurus (JK483709.1) Phylloseptin-H5 (Phylllomedusa hypochondrialis - AP00762) 0,04 Phylloseptin-H1 (Phylllomedusa hypochondrialis- AP00546) 0,02 0,06 Phylloseptin-O2 ( Phyllomedusa hypochondrialis-AP00760) Phylloseptin-H11 (Phyllomedusa hypochondrialis-AP00972) Phylloseptin-H3 (Phyllomedusa hypochondrialis- AP00758) 0,02 Phylloseptin-H2 (Phyllomedusa hypochondrialis- AP00757) Phylloseptin-1( Phyllomedusa sauvagei -AP01581) Phylloseptin 12 (Phyllomedusa hypochondrialis- AP01350) Temporin A (Rana temporaria - AP00094 0,04 Temporin B ( Rana temporaria- AP00095) 0,02 0,06 Bombinin H4 (Bombina variegata -AP00056) Temporin L (Rana temporaria -AP00101) Bombinin-like peptide 3 (Bombina orientalis- AP00052) 0,02 Bombinin-like peptide 2 (Bombina orientalis- AP00051) 0,04 0,06 Bombinin-like peptide 4 (Bombina orientalis- AP00053) 0,08 Maximin 4 (Bombina maxima - AP00061) Ranatuerin-2CSa (Rana cascadae- AP00592) Caerin 1.5 (Litoria caerula- AP00244) 0,04 Caerin 1.4 (Litoria caerula- AP00243) 0,02 0,08 Caerin 1.3 (Litoria caerula- AP00242) Caerin 1.2 (Litoria caerula- AP00241) 0,02 Caerin 1.1 (Litoria splendida -AP00240) Caerin 1.8 (Litoria chloris- AP00247) 0,04 Caerin 1.9 (Litoria chloris- AP00248) 0,02 Caerin 1.6 (Litoria xanthomera- AP00245) Maculatin 1.1 (Litoria eucnemis- AP00260) Sarcotoxin IA (Drosophila melanogaster- AP00230) 0,02 Cecropin 2 (Musca domestica -AP00135) 0,04 Cecropin A (Hyalophora cecropia- AP00139) Clavanin E (Styela clava- AP00280) Clavanin B (Styela clava- AP00277) 0,08 0,02 Clavanin A (Styela clava- AP00276) 0,06 Clavanin D (Styela clava- AP00279) 0,02 Clavanin C (Styela clava- AP00278) Chrysophsin-3 (Chrysophrys major- AP00341) 0,04 Chrysophsin-2 (Chrysophrys major- AP00340) 0,02 0,08 Chrysophsin-1 (Chrysophrys major- AP00339) Cathepsin G1-5 (Homo sapiens -AP00334) 0,02 Piscidin 1 (Morone saxatilis- AP00473) Aurein 2.4 (Litoria aurea- AP00017) 0,04 Aurein 2.3 (Litoria aurea- AP00016) 0,02 Aurein 2.2 (Litoria aurea- AP00015) Citropin 1.1(Litoria citropa- AP00351) 0,04 Aurein 1.2 (Litoria aurea - AP00013) 0,02 Aurein 1.1 (Litoria aurea - AP00012) Uperin 3.6 (Uperoleia inundata- AP00325) Page 25 of 29 Ponericin G6 (Pachycondyla goeldii- AP00381) 0,02

ip t

Figure 3

0,18

0,41

0,1 0,12 0,14

Ac

ce

pt

ed

M

an

us

cr

i

Figure 4

Page 26 of 29

Ac ce p

te

d

M

an

us

cr

ip t

Figure 5

Page 27 of 29

Ac

ce

pt

ed

M

an

us

cr

i

Figure 6

Page 28 of 29

Ac

ce

pt

ed

M

an

us

cr

i

Figure 7

Page 29 of 29