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Biochimie xxx (2012) 1

Contents lists available at SciVerse ScienceDirect

Biochimie
journal homepage: www.elsevier.com/locate/biochi

Graphical Abstract
1
2
3 Biochimie 2012, -, ---
Functional characterization of a synthetic hydrophilic antifungal peptide derived
4 from the marine snail Cenchritis muricatus
5
6 Carlos López-Abarrategui a, Annia Alba-Menéndez b, Osmar N. Silva d, Osvaldo Reyes-Acosta c,
7 Ilka M. Vasconcelos f, Jose T.A. Oliveira f, Ludovico Migliolo d, Maysa P. Costa g,
8 Carolina R. Costa g, Maria R.R. Silva g, Hilda E. Garay c, Simoni C. Dias d, Octávio L. Franco d, e,
9 Anselmo J. Otero-González a, *
10
a
11 Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Calle 25 entre J e I, Vedado,
Municipio Plaza, La Habana 10400, Cuba
12
13
Cm-p1, a novel antifungal peptide analog from the Caribbean Sea mollusk Cenchritis
14
muricatus.
15
16
17
18
19
20
21
22
23
24 Highlights
25
26 < Identification of a novel analog peptide from sea mollusks. < Reduced activity of Cm-P1 against mammalian cells. < 3D model showing the
27 probable regions involved in antifungal activity.
28
29
30
31
32
33

0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.12.016

Please cite this article in press as: C. López-Abarrategui, et al., Functional characterization of a synthetic hydrophilic antifungal peptide derived
from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016
 BIOCHI3772_proof ■ 28 December 2011 ■ 1/7

Biochimie xxx (2012) 1e7

Contents lists available at SciVerse ScienceDirect

Biochimie
journal homepage: www.elsevier.com/locate/biochi

Research paper
1 56
2 57
3
Functional characterization of a synthetic hydrophilic antifungal 58
4 peptide derived from the marine snail Cenchritis muricatus 59
5 60
6
Q3 Carlos López-Abarrategui a, Annia Alba-Menéndez b, Osmar N. Silva d, Osvaldo Reyes-Acosta c, 61
7 62
8
Ilka M. Vasconcelos f, Jose T.A. Oliveira f, Ludovico Migliolo d, Maysa P. Costa g, Carolina R. Costa g, 63
9 Maria R.R. Silva g, Hilda E. Garay c, Simoni C. Dias d, Octávio L. Franco d, e, Anselmo J. Otero-González a, * 64
10 a
Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Calle 25 entre J e I, Vedado, Municipio Plaza, La Habana 10400, Cuba 65
11 b
Subdirección de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”, La Habana, Cuba 66
c
12 Sección de Síntesis Química, Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba 67
d
Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB. Brasília, DF, Brazil
13 e 68
Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil
14 f
Universidade Federal do Ceará, Fortaleza, CE, Brazil 69
15 g
Instituto de Patologia Tropical e Saúde Pública, GO, Brazil 70
16 71
17 72
18 a r t i c l e i n f o a b s t r a c t 73
19 74
20 Article history: Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract 75
21 Received 2 August 2011 of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple 76
Accepted 17 December 2011
22 deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and 77
Available online xxx
23 functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and 78
24 a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally 79
Keywords:
25 characterized. This peptide demonstrated the capacity to prevent the development of yeasts and 80
Cenchritis muricatus
filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular
26 Antifungal peptide 81
Molecular modeling
modeling analyses showed that this peptide possible forms a single hydrophilic a-helix and the probable
27 82
Synthetic peptide cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate
28 the importance of sea animals peptide discovery for biotechnological tools development that could be
83
29 useful in solving human health and agribusiness problems. 84
30 Ó 2011 Elsevier Masson SAS. All rights reserved. 85
31 86
32 87
33 88
34 89
35 1. Introduction challenge for antifungal therapy. The development of novel thera- 90
36 peutic agents may overcome this problem [6]. Additionally, because 91
37 In the last three decades, several agents of new infectious the search for original and useful antifungals has been limited, 92
38 diseases have been identified, some of which are responsible for especially for drug-resistant pathogenic fungi, screening for novel 93
39 entirely novel and life-threatening disorders [1,2]. A lack of new antifungal peptides in multiple biomes could help to reduce 94
40 antibiotics for treatment of illnesses associated with the appear- infections in plants and animals. 95
41 ance of multi-drug-resistant strains has demanded the urgent Antimicrobial peptides (AMPs) can exhibit a broad spectrum of 96
42 development of innovative strategies for the control of microor- activities against a wide range of microorganisms, including Gram- 97
43 ganisms [3]. Certain human fungal infections, such as those caused positive and Gram-negative bacteria [7], yeasts [8], fungi [9], 98
44 by Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma viruses [10], protozoa [11] and parasites, such as nematodes [12]. 99
45 capsulatum and Candida albicans, are gaining importance due to the Several mechanisms of action have been proposed for these 100
46 increasing number of immunocompromised patients [4]. Thus far, molecules [13], which indicate that many of them have more than 101
47 existing treatments for these infections are limited to only a small one antimicrobial target at the cellular level [14]. Initially, peptides 102
48 number of antifungal drugs, such as azoles, echinocandins, and can interact with the cell membrane, causing an increase in 103
49 polyenes [5]. Indeed, pathogenic fungi possess many complicated permeability concomitant with a loss of membrane function. 104
50 mechanisms for resisting these drugs, constituting a critical Moreover, peptides can affect intracellular targets, such as the 105
51 nucleus and its DNA, leading to apoptosis [15]. 106
52 * Corresponding author. There are multiple sources of antimicrobial peptides, including 107
53 E-mail address: aoterog@infomed.sld.cu (A.J. Otero-González). plants, mammals and invertebrates. Invertebrates base their 108
54 109
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
55 110
doi:10.1016/j.biochi.2011.12.016

Please cite this article in press as: C. López-Abarrategui, et al., Functional characterization of a synthetic hydrophilic antifungal peptide derived
from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016
 BIOCHI3772_proof ■ 28 December 2011 ■ 2/7

2 C. López-Abarrategui et al. / Biochimie xxx (2012) 1e7

111 humoral defense against infectious agents on a wide and effective (Bruker Daltonics). A 1 mL sample was mixed with 3 mL of matrix 176
112 repertoire of AMPs that may interact directly with microbes or with solution (1% [w/v] a-cyano-4-hydroxycinnamic acid, 3% [v/v] tri- 177
113 their toxic molecules [16]. Due to this fact, many researchers have fluoroacetic acid and 50% [v/v] acetonitrile) and applied onto 178
114 used mollusks and other marine sea animals as sources for the a MALDI target plate (1 mL in duplicate). After crystallization at 179
115 development of novel antimicrobials [16]. Mollusks are an abun- room temperature, samples were analyzed using a spectrometer 180
116 dant and significant group in the trophic chain of the animal operated in reflector mode for MS acquisitions and LIFT mode for 181
117 kingdom. Among the mollusks, gastropods, including snails and tandem MS (MS/MS). Protein identification was achieved by 182
118 slugs, represent the most abundant class. Snails in particular are peptide mass fingerprinting (PMF) and manual de novo sequencing. 183
119 successful animals from an evolutionary point of view, due to their The sequenced peptide was directly aligned at the non-redundant 184
120 capacity to adapt to different environments and to reach dry land protein database (NCBI) and APD data bank similarity searches 185
121 [17]. Although antimicrobial peptides have been purified from with bioactive peptides. 186
122 several marine invertebrates, only a few have been reported in 187
123 mollusks [16]. Most research has focused on species such as Mytilus 2.3. Amino acid sequence analysis by automated Edman 188
124 edulis Linnaeus, 1758 and Mytilus galloprovincialis Lamarck, 1819. degradation 189
125 These species are capable of synthesizing defensins, mytilins and 190
126 the antifungal peptide mitomycin [16]. The amino acid sequences of the peptides were analyzed by 191
127 To contribute to this research field, a preliminary screening of automated Edman degradation. Microsequencing was performed 192
128 antimicrobial activities from marine invertebrates from the Carib- using a HewlettePackard 1000A protein sequencer equipped with 193
129 bean Sea was performed and identified a remarkable antimicrobial a HPLC system. 194
130 activity in the littoral snail Cenchritis muricatus (Beaded Peri- 195
131 winkle). Here, we report the isolation and biochemical character- 2.4. Peptide synthesis 196
132 ization of different forms of the antimicrobial peptide fragments 197
133 from C. muricatus. To expand our functional analyses, a lower- Cm-p1 was synthesized by the solid-phase method using 9- 198
134 molecular-weight peptide, Cm-p1, was synthesized and further fluorenyl-methoxycarbonyl chemistry [19], purified by reverse- 199
135 evaluated regarding its antimicrobial actions toward multiple fungi phase high-performance liquid chromatography to >98% purity 200
136 and mammalian cells. An in silico evaluation was also conducted, on an acetonitrile/H2O-TFA gradient and confirmed by ion-spray 201
137 which showed that the molecular surface and the presence of mass spectrometry (Micromass, Manchester, United Kingdom). 202
138 several residues could be essential for the peptide’s antifungal 203
139 activity. 2.5. Determination of protein concentration 204
140 205
141 2. Materials and methods Protein concentrations were estimated using Coomassie Blue 206
142 staining [20]. Bovine serum albumin (BSA, 0.1 mg ml1) was used as 207
143 2.1. Extraction and isolation of proteinaceous compounds the standard protein. All determinations were performed in 208
144 triplicate. 209
145 C. muricatus Linnaeus, 1758 (Mollusca: Gastropoda) snails were 210
146 hand-collected on the northern coast of Havana, Cuba. C. muricatus 2.6. Bioassays against bacteria 211
147 snails were homogenized in a solution containing 0.6 M NaCl and 212
148 0.1% HCl (2:1 [w/v]) in a blender. The homogenate was centrifuged Pathogenic bacteria were cultured in 2.0 mL of LB (Luria-Ber- 213
149 at 10,000
g for 30 min at 4  C. Soluble proteins were precipitated tani) broth (10 g L1 NaCl, 5 g L1 yeast extract and 45 g L1 bacto 214
150 with (NH4)2SO4 at 100% saturation with constant stirring for 1 h at peptone) for 18e24 h at 37  C. Protein-rich fractions, the purified 215
151 4  C. After centrifuging again at 10,000
g for 30 min at 4  C, the peptides and Cm-p1 were resuspended in distilled water and 216
152 precipitate was resuspended in 10 mM TriseHCl buffer, pH 8.0 and filtered through 0.22-mm nylon membranes. Dilution series for all 217
153 was further desalted using PD MidiTrap G-10 columns (GE samples were prepared for an initial concentration of 200 mg ml1. 218
154 Healthcare, USA). The resulting desalted, protein-rich fraction was Samples were incubated for 6 h at 37  C with 5
106 CFU mL1 of 219
155 separated into low- (<10 kDa) and high-molecular-weight fractions each bacterium tested. The assayed bacteria were Escherichia coli, 220
156 (>10 kDa) using Amicon Ultra-15 (10 K) centrifugal filter devices Staphylococcus aureus, Klebsiella sp., and Shigella sp. These micro- 221
157 (Millipore, USA). The low-molecular-weight fraction was lyophi- organisms were obtained from the microbiology collection of the 222
158 lized, and 3.0 mg of the dry fraction was dissolved in 0.1% tri- Universidade Católica de Brasília. Sterile distilled water and chlor- 223
159 fluoroacetic acid (TFA) and then applied onto a reversed-phase amphenicol (40 mg mL1) were used as the negative and positive 224
160 HPLC Vydac C18-TP analytical column (Hesperia, CA, USA) equili- controls, respectively. Bacterial growth was determined spectro- 225
161 brated with 0.1% TFA. Retained proteins were eluted with a linear photometrically at 595 nm every hour during the incubation period 226
162 methanol gradient (0e100%) at a flow rate of 1.0 ml min1. Protein according to the Clinical and Laboratory Standards Institute (CLSI) 227
163 detection was performed at 280 nm. guidelines [21]. 228
164 229
165 2.2. Protein identification by mass spectrometry 2.7. Bioassays against fungi 230
166 231
167 After elution, a peptide digestion was performed using Bioassays against fungi were performed using the broth 232
168 sequencing grade modified trypsin (Promega, USA) according to microdilution method [22]. Fusarium oxysporum, Botrytis cinerea, 233
169 [18] with minor modifications. Briefly, 600 ng of buffered trypsin and Aspergillus niger were obtained from the Universidade Católica 234
170 was added to the peptide and incubated on ice for 30 min. Then Collection and were grown on solid medium potato dextrose broth 235
171 40 mL of 50 mM NH4HCO3 was added, and the solution was incu- (Difco, USA). Liquid potato dextrose broth was used for the inhi- 236
172 bated at 37  C for 22 h. The digestion supernatant was collected and bition assays against these fungi. Amphotericin-B (30 mg ml1) was 237
173 stored at 20  C. The peptides derived from the tryptic digestion used as the positive control, and liquid potato dextrose broth was 238
174 were analyzed using an UltraFlex III MALDI-TOF/TOF (Bruker Dal- used as the negative control. The bioassays against human patho- 239
175 tonics) precisely calibrated with peptide calibration standard II genic yeast (Candida parapsilosis [ATCC 22019] and clinically 240

Please cite this article in press as: C. López-Abarrategui, et al., Functional characterization of a synthetic hydrophilic antifungal peptide derived
from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016
 BIOCHI3772_proof ■ 28 December 2011 ■ 3/7

C. López-Abarrategui et al. / Biochimie xxx (2012) 1e7 3

241 isolated strains of C. albicans [01U, 38U], C. neoformans [L26, L30], alignments and iterative structural assembly simulations [35,55]. 306
242 Trichophyton mentagrophytes [28d, 28e] and Trichophyton rubrum The RMSD among model construct by MODELLER, I-Tasser and 307
243 [329]) were performed in RPMI-1640 medium (Gibco BRL, USA). HHPred server were calculated from the overlap of Ca traces and 308
244 Itraconazole and fluconazole (0.5e4 mg ml1) were used as the backbones onto the template structure using the program 3DSS. 309
245 positive controls. Sterile distilled water was used as the negative Electrostatic surfaces were calculated with the ABPS tool [36]. The 310
246 control. The minimum inhibitory concentrations (MICs) were ob- protein structure was visualized and analyzed with PyMOL (Delano 311
247 tained according to the CLSI guidelines [23]. All tests were con- Scientific) [37]. 312
248 ducted in triplicate. 313
249 3. Results 314
250 2.8. Hemolytic assay 315
251 3.1. Identification of antimicrobial peptides 316
252 Cm-p1 hemolytic activity was evaluated by determining the 317
253 release of hemoglobin from an 8% suspension of fresh human First, the low-molecular-weight fraction (<10 kDa) of the 100% 318
254 erythrocytes at 414 nm with an ELISA plate reader [24]. The assay ammonium sulfate precipitation pellet was applied onto 319
255 was performed with different concentrations of Cm-p1 a reversed-phase HPLC C-18 column. Proteins and peptides were 320
256 (0e400 mg ml1). The percentage of hemolysis was calculated eluted using a linear methanol gradient (0e100%), which yielded 14 321
257 using the following equation: % hemolysis ¼ [(Abs414 nm in the fractions (Fig. 1A). The antimicrobial activities of all peaks were 322
258 peptide solution  Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X- evaluated against diverse pathogens. The second fraction was 323
259 100  Abs414 nm in PBS)]
100. PBS was used as the negative eluted with 49.4% methanol and showed the highest antimicrobial 324
260 control, and Triton X-100 was used as the positive control. All activity (data not shown). 325
261 assays were performed in triplicate. 326
262 3.2. Amino acid sequencing and bioinformatics 327
263 2.9. Cytotoxicity assay 328
264 To identify the fraction responsible for the higher activity, 329
265 The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium fraction 2 was analyzed by MALDI-TOF/TOF. Fraction 2 consisted of 330
266 bromide; SigmaeAldrich, USA) assay was performed according to a single peptide that exhibited an m/z value of 1485 (Fig. 1B). It was 331
267 [25]. RAW 264.7 murine macrophage-like cells (Rio de Janeiro Cell not possible to determine the sequence of this peptide by MS/MS; 332
268 Bank, Brazil) were plated at a concentration of 1
105 cells per well in therefore, the peptide was digested with trypsin. The digestion 333
269 supplemented DMEM medium (4 mM glutamine, 10% FCS and yielded various peptide fragments that exhibited a more intense 334
270 100 units/ml penicillin/streptomycin) and incubated with different ion at an m/z value of 1224 (data not shown). This ion was then 335
271 concentrations of peptides (0e400 mg ml1). After overnight incu- sequenced by MS/MS and yielded the 10-amino acid peptide 336
272 bation, 10 ml of the MTT solution (5 mg mL1 in PBS) was added to 337
273 each well. Plates were incubated for 4 h in 5% CO2 at 37  C. The 338
274 generated blue formazan product was dissolved by the addition of 339
275 100 ml of 100% DMSO (Mallinckrodt Chemical, USA) per well. The 340
276 absorbance was monitored at 575 nm in an ELISA plate reader (Bio- 341
277 Tek, USA). 342
278 343
279 2.10. In silico analyses and molecular modeling 344
280 345
281 Initially, the physiochemical parameters were calculated using 346
282 the software ProtParam from the ExPASy server [26]. ClustalW was 347
283 used for aligning multiple sequences [27]. Helical wheel projection 348
284 was performed using the tool wheel from Gromacs software [28]. 349
285 PSI-BLAST was used to find the best templates for homology 350
286 modeling, but no results were returned. Alternatively, data mining 351
287 in a non-redundant database (APD2) highlighted reliable templates 352
288 [29]. The Protein Data Bank (PDB) structure 2RMI showed 22% of 353
289 identical residues and was thus used as the template. This template, 354
290 a peptide named astressin, binds to corticotrophin-releasing factor 355
291 (CRF)-R1 and CRF-R2 receptors with low nanomolar affinity. These 356
292 receptors have been implicated in stress-related disorders, such as 357
293 anxiety, depression, eating disorders, gastrointestinal maladies, 358
294 irritable bowel syndrome, and postoperative stress [30].Fifty 359
295 theoretical three-dimensional peptide models were constructed 360
296 with Modeller v.9.8 [31] using the selected template. The final 361
297 models were evaluated according to geometry, stereochemistry, 362
298 and energy distributions. PROSA II was utilized to analyze packing 363
299 and solvent exposure characteristics, and PROCHECK was used 364
300 for additional stereochemical quality checks [32,33]. Additionally, 365
301 the root mean square deviation (RMSD) was calculated from 366
302 the overlap of Ca traces and backbones onto the template Fig. 1. A: HPLC reversed-phase chromatogram profile (Vydac C18-TP) of the low- 367
molecular-weight fraction from Cenchritis muricatus snails. The diagonal line indi-
303 structure using the program 3DSS [34]. In order to confirm model 368
cates a linear methanol gradient. B: Mass spectrum of Cm-p1 isolated by HPLC in
304 reliability, I-Tasser and HHPred were also utilized for additional a MALDI-ToF/ToF mass spectrometer operating in reflector mode and using a matrix of 369
305 tridimensional model construction according multiple-threading a-cyano-4-hydroxycinnamic acid. Q 4 370

Please cite this article in press as: C. López-Abarrategui, et al., Functional characterization of a synthetic hydrophilic antifungal peptide derived
from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016
 BIOCHI3772_proof ■ 28 December 2011 ■ 4/7

4 C. López-Abarrategui et al. / Biochimie xxx (2012) 1e7

371 SRSELIVHQR. This peptide fragment was named Cm-p1 and was Table 1 436
372 compared to the AMP database [29]. The comparison of the primary Antifungal activity of Cm-p1 against different yeasts and mycelial fungi. The 437
minimum inhibitory concentration (MIC) [13] of the peptide is represented for each
373 structure of Cm-p1 showed clear identity with other antimicrobial Q2 438
fungus. The asterisk indicates that the MIC is lower than the displayed value.
374 peptides. Cm-p1 shared 30% and 40% identity with ure- 439
375 chistachykinin I and hyposin-H5, respectively. Moreover, we also Microorganism Minimum Inhibitory 440
Concentration (mM)
376 observed a sequence relationship between Cm-p1 and different 441
Botrytis cinerea 20
377 members of the hyposin family (Fig. 2). Additionally, similarity was 442
Fusaryum oxysporum 20
378 observed with Vesp-VB1 from the invertebrate V. bicolor, which Aspergillus niger 41 443
379 exhibits both antifungal and bactericidal activities (data not shown) Candida albicans 01U 13 444
380 [38]. In all cases, we noted a conserved hydrophobic core (italicized Candida albicans 38U 7 445
381 residues, Fig. 2). Furthermore, cationic residue number 10 (bold Candida parapsilosis 105 446
Cryptococcus neoformans L26 209
382 type, Fig. 2), which ended the hydrophobic region, was also 447
Cryptococcus neoformans L30 209
383 conserved in all the antimicrobial peptides we analyzed. In parallel, Trichophyton mentagrophytes e 448
384 a similar purification procedure was performed using the same Trichophyton rubrum 3 449
385 crude extract, and a peptide with an m/z value of 5282 was also 450
386 isolated (data not shown). This peptide, named Cm-p2, was 451
387 partially sequenced by automated Edman degradation and yielded 452
amphipathic molecule, as was demonstrated by helix-wheel
388 the sequence SESILIVHQQQSSRSSGS. Cm-p1 and Cm-p2 share 70% 453
projection using the tool wheel of Gromacs software (data not
389 identity between them, suggesting that they could be two 454
shown). To improve peptide structure analyses, a three-
390 members of a single family of antimicrobial peptides or could be 455
dimensional model of the hydrophilic Cm-p1 peptide was con-
391 significantly correlated. 456
structed (Fig. 3). The model exhibited a a-helix conformation when
392 457
generated by three different methodologies as previously described
393 3.3. Antimicrobial activity 458
before in session 2.11. The model constructed present cationic
394 459
arginines (Arg2 and Arg10) and histidine (His8) residues and also an
395 460
To demonstrate the antimicrobial activity of Cm-p1, the peptide exposed hydrophobic region (Leu5eVal7) that favors a mem-
396 was synthesized in solid-phase. Despite the high bactericidal 461
braneepeptide interaction (Figs. 2 and 3). The model shared 22%
397 activity of the C. muricatus crude extract, synthesized Cm-p1 was 462
identity with the neuropeptide PDB structure 2RMI [30]. A valida-
398 incapable of inhibiting either the growth of Gram-negative bacteria 463
tion of the three-dimensional model of Cm-p1 by Ramachandran
399 (E. coli and Klebsiella pneumoniae) or the development of Gram- plot showed that 100% of the modeled amino acid residues were in 464
400 positive bacteria (S. aureus) (data not shown). Nevertheless, 465
physically acceptable regions for secondary structure formation in
401 a broad spectrum of antifungal activity was observed for the relation to the torsion angles phi and psi. The z-score value given in 466
402 synthetic peptide (Table 1). Cm-p1 was active against filamentous 467
PROSA II was used to verify whether the input structure was within
403 fungi and yeasts of both medical and agricultural interest. As the range of scores typically found for native proteins of similar 468
404 469
Table 1 shows, a lower MIC was obtained against T. rubrum size. The z-score value was 1.36 and is comparable to NMR
405 (4 mg ml1), whereas a higher MIC was obtained against different structures of antimicrobial peptides of similar length, which have z- 470
406 clinical isolates of C. neoformans (256 mg ml1). 471
scores of 1 to 1.5 [39e41]. The RMSD value obtained for Cm-p1
407 model acquired by using Modeller strategy was 1.7 Å. Moreover 472
408 473
3.4. Toxicity against mammalian cells when this helical model was compared with models constructed by
409 I-Tasser and HHPred servers, 0.8 Å RMSD values were obtained. 474
410 475
To evaluate the toxicity of Cm-p1 against mammalian cells, These values clear demonstrate the similar structural conformation
411 among all models proposed and strongly suggests a helical 476
412 different doses of the peptide were incubated in the presence of 477
human erythrocytes. No concentrations of Cm-p1 were capable of conformation.
413 478
414 provoking hemoglobin release (data not shown). Additionally, the 479
415 viability and proliferation of cultured RAW 264.7 cells incubated 4. Discussion 480
416 with different doses of Cm-p1 were evaluated using the MTT 481
417 reagent. The viability and proliferation of RAW 264.7 cells were Functional studies have determined the residues or domains 482
418 unaffected by Cm-p1 (Fig. 3). that could be responsible for antimicrobial properties. In many 483
419 cases, the N-terminal region has been hypothesized to act as an 484
420 3.5. Theoretical structural analysis initial membrane anchor [42]. Although structural and functional 485
421 differences can be observed among the different antimicrobial 486
422 Initial analyses show that Cm-p1 is a hydrophilic molecule peptides, some properties could be very common, such as hydro- 487
423 scoring a grand average of hydropathicity (GRAVY) of 0.830 and phobicity and amphipathicity [16]. In this regard, Cm-p1 is not 488
424 exhibits a small central hydrophobic region flanked by basic amino a classical antimicrobial peptide. In fact, it is a small, hydrophilic 489
425Q1 acids at the extremes (Fig. 4). Furthermore, Cm-p1 is not an peptide; there is no clear evidence of the amphipathicity properties 490
426 491
427 492
428 493
429 494
430 495
431 496
432 497
433 498
434 Fig. 2. Primary structure alignment of Cm-p1 and Cm-p2 against other antimicrobial peptides from different animal sources. Italicized residues correspond to the conserved 499
435 hydrophobic region, and bold residues correspond to the conserved cationic residues. 500

Please cite this article in press as: C. López-Abarrategui, et al., Functional characterization of a synthetic hydrophilic antifungal peptide derived
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 BIOCHI3772_proof ■ 28 December 2011 ■ 5/7

C. López-Abarrategui et al. / Biochimie xxx (2012) 1e7 5

501 30% identity with Cm-p1 [43]. This peptide exhibits a broad spec- 566
502 trum of antibacterial activity against the Gram-positive bacteria 567
503 Streptococcus mutans, S. aureus and Enterococcus faecium and the 568
504 Gram-negative bacteria E. coli O-157, Pseudomonas aeruginosa and 569
505 Vibrio vulnificus with MIC values in the 10.6e42.5 mM range [43]. 570
506 Despite the similarities between these peptides, Cm-p1 is not an 571
507 antibacterial peptide, possibly due to its lower molecular charge 572
508 (þ1) in comparison to urechistachykinin I (þ2). Moreover, hyposin- 573
509 H5, an antimicrobial peptide identified from the skin secretions of 574
510 the frog Phyllomedusa hypochondrialis azurea [44], shares 40% 575
511 identity with Cm-p1. Despite the clear identity shared with hypo- 576
512 sins (Fig. 2), Cm-p1 exhibits different physiochemical properties, 577
513 such as hydrophobicity (GRAVY: 0.007) and net charge (þ4). 578
514 Another antimicrobial peptide that shares sequence similarity with 579
515 Cm-p1 is VESP-VB1. This peptide was isolated from the venom of 580
516 Fig. 3. Effects of Cm-p1 on RAW 264.7 murine macrophage-like cells viability. the wasp Vespa bicolor Fabricius. Interestingly, despite the low 581
517 Different concentrations of peptide were added to 1000 cells per well and incubated cationicity of VESP-VB1 (þ1), this peptide exhibits antimicrobial 582
518 for 72 h at 37  C in 5% CO2. Triton X-100 was used as a positive control for toxicity, and activity against different isolates of E. coli and S. aureus, possibly 583
PBS was used as a negative control. Cm-p1 were assayed at 200 mg ml1 (Cm-p1 200)
519 and at 400 mg ml1 (Cm-p1 400).
due to the molecule’s hydrophobicity (GRAVY: 1.131) [38]. 584
520 To confirm that the Cm-p1 sequence was derived from C. mur- 585
521 icatus, we identified a parallel sequence (Cm-p2) that was part of 586
522 that are commonly found in other antimicrobial peptides. Indeed, a larger peptide (5282 Da). Cm-p1 shared 70% identity with Cm-p2. 587
523 of the 1755 peptides in the AMP database, only three have features The divergence between these sequences could be attributed to the 588
524 similar to Cm-p1. One of these, urechistachykinin I, is a neuropep- existence of isoforms, which is relatively probable for AMPs, or 589
525 tide derived from the invertebrate Urechis unicinctus that shares simply the fragmentation of similar peptides. Even with this 590
526 divergence, Cm-p1 could be interpreted as a closely related protein 591
527 sequence from C. muricatus. Taking into account that Cm-p1 is more 592
528 positively charged and less hydrophilic than Cm-p2, we synthe- 593
529 sized Cm-p1 to explore its antimicrobial potential. 594
530 Although Cm-p1 was not capable of inhibiting bacterial 595
531 growth, even at high doses, it exhibited a broad spectrum anti- 596
532 microbial activity against fungi. The MIC values of Cm-p1 for yeast 597
533 and filamentous fungi were in the 4e256 mg ml1 range. The 598
534 peptide was less effective against the yeasts C. neoformans and 599
535 Candida parapsilosis. Interestingly, some echinocandins/pneumo- 600
536 candins are also less effective against these fungi [45]. Further- 601
537 more, it has been demonstrated that C. parapsilosis has developed 602
538 a major resistance to amphotericin-B compared to other Candida 603
539 species [46]. Taking into account the other evaluated yeast and 604
540 filamentous fungi, the range of MIC values was 4e50 mg ml1. This 605
541 range of effectiveness is similar to the antifungal activities re- 606
542 ported for other peptides [47,48]. Antimicrobial peptides that 607
543 primarily or exclusively have antifungal properties are less ubiq- 608
544 uitous than those with broad antimicrobial actions. Nevertheless, 609
545 both natural and synthetic peptides do exist that exhibit primarily 610
546 antifungal activity [48]. The plant defensins Hs-AFP1, Dm-AMP1 611
547 and Rs-ARF2 and the insect defensins heliomycin and drosomycin 612
548 belong to this group of peptides [49,50]. Furthermore, ure- 613
549 chistachykinin I is also capable of inhibiting fungal strains at 614
550 concentrations of 21.3e42.5 mM [43]. 615
551 In addition to functional studies, theoretical modeling shows 616
552 that Cm-p1 could assume an a-helix conformation with a distribu- 617
553 tion of net charge (þ2) caused by exposed cationic arginine (Arg2 618
554 and Arg10) and histidine (His8) residues, which probably favor 619
555 a membraneepeptide interaction. Additionally, despite the hydro- 620
556 philicity of Cm-p1, the hydrophobic conserved region Leu5eVal7 621
557 seems to play a critical role in the peptide’s antifungal activity, as 622
558 has been previously demonstrated [51]. Moreover, the presence of 623
559 the amino acid residue Val7 might also be important in fungal 624
560 interaction. Several studies have demonstrated that an amidated 625
561 valine residue at the C-terminus had lethal effects against fungi and 626
562 Fig. 4. Three-dimensional structural modeling of Cm-p1. Blue and red regions corre- a broad spectrum of pathogenic microorganisms [52]. 627
spond to the cationic and anionic areas, respectively. Labeled residues may be related
563 One of the current antifungal therapeutic problems is the 628
to the peptide’s possible mechanism of action. The structure was visualized using
564 PyMOL. (For interpretation of the references to colour in this figure legend, the reader toxicity of many approved drugs, such as amphotericin-B, echino- 629
565 is referred to the web version of this article.) candins and pneumocandins. For this reason, it is essential to 630

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from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016
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6 C. López-Abarrategui et al. / Biochimie xxx (2012) 1e7

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from the marine snail Cenchritis muricatus, Biochimie (2012), doi:10.1016/j.biochi.2011.12.016