Journal Identification = PEP Article Identification = 68374 Date: July 8, 2011 Time: 3:14 pm

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Peptides 32 (2011) 1477–1483

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa ge: www.elsev ier .com/ locate /pept ides

ntimicrobial proline-rich peptides from the hemolymph of marine snail Rapanaenosa

avlina Dolashkaa,∗, Vesela Moshtanskaa, Valika Borisovab, Aleksander Dolashkia,tefan Stevanovicc, Tzvetan Dimanovb, Wolfgang Voelterd

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, G. Bonchev 9, Sofia 1113, BulgariaSME – SYCO-PHARMA, OOD, Ltd. 47 Bregalnitza Str., 1303 Sofia, BulgariaInstitute for Cell Biology, University of Tuebingen, GermanyInterfacultary Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany

r t i c l e i n f o

rticle history:eceived 17 February 2011eceived in revised form 3 May 2011ccepted 3 May 2011vailable online 22 June 2011

eywords:ntimicrobial proline-rich peptides

a b s t r a c t

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically activecomponents such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobialalternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first timewe have explored the isolation, identification and characterisation of 11 novel antimicrobial peptidesproduced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafil-tration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weightsbetween 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the

emolymph of Rapana venosaram-positive (Staphylococcus aureus) and

Gram-negative (Klebsiella pneumoniae)acteria

peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicat-ing novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristicsof protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimi-crobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.

. Introduction

The recent appearance of a growing number of bacteria resistanto conventional antibiotics has become a serious medical problem.o overcome this resistance, the development of antibiotics, withovel mechanisms of action is a persisting issue [20,27,43]. Theew generation of native peptides seems to fit to this urgent issue.s a consequence, these native peptides have been termed “natu-al antibiotics”, because they are active against a large spectrumf microorganisms including bacteria, filamentous fungi, proto-oan and metazoan parasites [21,32,33]. Antimicrobial peptidesAMPs) are important components of the non-specific host defenser innate immune system in a variety of organisms ranging fromlants and insects to animals including molluscs and arthropods,mphibians and mammals [21,26]. Therefore, in the last years theres a great interest in studying new antimicrobial peptides.

AMPs are classified into several groups based on amino acidequences, secondary structures, and functional similarities e.g.orming �-helices, �-sheet, containing thioether rings, overrep-

∗ Corresponding author. Tel.: +359 29606163; fax: +359 8700225.E-mail address: pda54@abv.bg (P. Dolashka).

196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2011.05.001

© 2011 Elsevier Inc. All rights reserved.

resentation of one or two amino acids (e.g. Pro, His or Trp),attached to lipids and macrocyclic cystine-knot peptides [21].Many proline-rich peptides were described by Otvos [30]. Severalcysteine-rich peptides were purified from scorpion Centruroideslimpidus limpidus [8], while a new type of short antimicrobialpeptides, designated temporin-SHf, were identified in the skinof the frog Pelophylax saharica [1]. It was also found that thehemolymph of molluscs and arthropods is rich in peptides andaromatic polypeptides [16,17,24,35,37,46].

Despite their variations in structure and size, AMPs are usuallycharacterized by their cationic and hydrophobic nature, as human�-defensin 28 (hBD28) which is a strongly cationic AMP againstEscherichia coli K12 [25,44]. The nature of the peptides was con-sidered to be crucial for the initial interaction between the peptideand the bacterial membrane [3,41].

Most of the peptides exhibit a broad spectrum of microbialactivity against Gram-positive and Gram-negative bacteria andyeasts. In the hemolymph of the blue crab Callinestes sapidus, Scyllaserrata, Thalamita crenata, houseflies (Musca domestica), Galleria

mellonella, female thick Amblyomma hebraeum AMPs which arestrong inhibitors against gram-negative bacteria were identified[3,10,34–36]. Several novel AMPs with antimicrobial activity werealso isolated from the body wall of the sea hare Dolabella auric-

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laria [22]. Besides peptides, the presence of glycoproteins withntimicrobial activity was reported from the mucus of the giantnail Achatina fulica and from the egg mass and purple fluid of theea hare Aplysia kurodai [45].

It was found that beside antimicrobial activity against Micro-occus luteus and E. coli, the cysteine-rich peptides from Mytilusdulis exhibit also antifungal activity against Neurospora crassa [5].tudies on the antibacterial and antifungal peptides in marine mol-uscs are mainly focused on the mussels of Mytilus galloprovincialisnd M. edulis [5,28]. Beside the wide spectrum of AMPs’ antimi-robial activities their potential benefit for the treatment of cancernd viral or parasitic infections is suggested [6,7,11]. There is also

significant interest in the development of therapeutic antibioticsased on AMPs; however, the poor understanding of the fundamen-al mechanism of action of these peptides has largely hampereduch efforts.

Therefore, identification and isolation of the active substancesnd determination of their primary structures or DNA sequencesre of enormous importance to understand non-specific immuneesponse mechanism of mollusks and arthropods against pathogennvasion and for the development of new biopharmaceutical con-ept and finally products.

In this study, we report for the first time about the structure androperties of several new antimicrobial peptides, isolated from theemolymph of marine snail Rapana venosa.

. Materials and methods

.1. Animals and collection of hemolymph

The marine snails R. venosa were collected from the Blackea and provided by “Delta Industry” AD, Sozopol, Bulgaria. Theemolymph was isolated from the foot of animals as described byolashka-Angelova et al. [14,19]. For preliminary purification theemolymph was centrifuged in two steps: first for 30 min at 4 ◦Cnd 5000 × g to remove the cellular contents, and second then theentrifugation was elongated for 4 h at 4 ◦C at 30,000 × g to precip-tate the hemocyanin.

.2. Purification of peptides

The supernatant was concentrated and separated into sev-ral fractions using Millipore filters with different size (3, 10 and0 kDa). A fraction with mass between 3 and 10 kDa was concen-rated and applied on a HPLC system, using a Nucleosil 7 C18olumn (250 mm × 10 mm; Macherey-Nagel, Düren, Germany),quilibrated with buffer A (H2O, containing 0.1% TFA). Elution waserformed stepwise in three steps with 20, 50 and 80% of solu-ion B (80% acetonitrile, containing 0.08% TFA) at a flow rate of.5 ml min−1. Ultraviolet absorption was monitored at 280 and14 nm and the eluted fractions were collected and dried by Speed-ac. The isolated fractions were reconstituted in MilliQ water with.10% TFA (v/v) before being applied again on a Nucleosil 7 C18olumn for rechromatography. For elution, a linear gradient from% solvent A (0.1% TFA in water) to 100% solvent B (0.085% TFA inCN) within 50 min at a flow rate of 1 ml min−1 was used. Again,

he HPLC fractions were detected at a wavelength of 214 nm andollected.

.3. Determination of N-terminal amino acid sequences and masspectrometric analysis

Isolated HPLC fractions were dried and after dissolving in 40%ethanol/1% formic acid their N-terminal amino acid sequencesere determined by automated Edman N-terminal sequencing on a

2 (2011) 1477–1483

Pulsed Liquid Protein Sequencer (Applied Biosystems GmbH, FosterCity, CA).

The molecular masses of isolated peptides were measuredby AutoflexTM III, High-Performance MALDI-TOF & TOF/TOF Sys-tems (Bruker Daltonics). For mass spectrometric analysis about50 pmol of the HPLC fractions were dissolved in 0.1% (v/v) TFA andapplied to the target. Analysis was carried out using �-cyano-4-hydroxycinnamic acid as a matrix. The mass spectrometer uses a200 Hz frequency-tripled Nd–YAG laser operating at a wavelengthof 355 nm. 3500 shots were acquired in the MS mode and collisionenergy of 4200 was applied. A solution of peptide standard wasused to calibrate the mass scale. The mass values assigned to theamino acid residues are the average masses.

2.4. Carbohydrate analysis

Glycopeptides were analyzed by orcinol/sulphonic method.2–4 �l of the purified peptide solutions was applied to the thin-layer plate and air-dried, taking care to restrict the size of the spotto 2–3 mm in diameter. The plate was sprayed with orcinol/H2SO4and heated for 20 min at 100 ◦C.

2.5. Spectroscopic studies

Spectroscopic properties of peptides were analyzed by UV spec-troscopy, circular dichroism and fluorescence spectroscopy. UVspectra were measured on a Shimadzu spectrophotometer. Thespectra of the peptide solutions with A280 = 0.2 in 50 mM Tris/HClbuffer, pH 8.0, were measured by circular dichroism (CD) in theUV region from 195 to 250 nm using a Jasco J-720 dichrograph,equipped with a personal computer IBM PC-AT, PS/2, and a cuvetof 0.2 mm. A software DOS version was used for calculation of theCD data [18].

Peptide solutions in 50 mM Tris/HCl buffer, pH 8.0, withabsorbance at the excitation wavelength <0.05 to minimizethe inner filter or absorption effects, were analyzed by aspectrofluorimeter (Perkin-Elmer Model LS5), equipped with athermostatically controlled sample holder and a Model 3600 datastation. Fluorescence emission spectra were recorded in the regionfrom 290 to 520 nm. Excitation at 295 nm was used for predom-inant measuring the fluorescence of tryptophyl residues. Spectrawere corrected for background due to the solvent.

2.6. Antibacterial assays of the peptides

2.6.1. Liquid growth inhibition assayTwo different species of bacteria, a Gram-positive (Staphylococ-

cus aureus) and a Gram-negative (Klebsiella pneumoniae) one, wereused as reference to analyze the antimicrobial activity of the pep-tides. Both bacteria are isolates from patients of the Medical CenterPolyMed®, Sofia, Bulgaria. The concentrations of the peptideswere determined spectrophotometrically as: Peptide 2–242 �g/ml,Peptide 3–158 �g/ml, Peptide 4–113 �g/ml, Peptide 5–189 �g/ml,Peptide 6–171 �g/ml, Peptide 7–598 �g/ml, Peptide 8–108 �g/ml.Three different concentrations (2, 15 and 50 �l of the solutions)were used for testing their antimicrobial activity. The eluted pep-tides were qualitatively checked according to the liquid growthinhibition assay. Briefly, 10 �l of the samples was mixed with 6 mlof a mid-logarithmic phase culture of bacteria in poor broth nutri-ent medium (1% dextrose) with definite OD. Microbial growth wasassessed by an increase in the McF value after incubation (24 h,35 ◦C). The nutrient medium with the bacterial culture, but without

peptides and incubated for 24 h at 35 ◦C was used as a control. 1 unitMcF corresponds to 3 × 108 cells/ml. The turbidity was measuredwith DENSIMAT (BioMerieux, France) instrument.

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tides 32 (2011) 1477–1483 1479

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Fig. 1. RP-HPLC purification of the peptides. The hemolymph fraction between 3and 10 kDa was subjected to Nucleosil C18 RP-HPLC column (250 mm × 10 mm;Macherey-Nagel, Düren, Germany), equilibrated with 0.1% (v/v) trifluoroaceticacid/water. Elution (1.5 ml min−1) was performed according to the proceduredescribed in Section 2. UV-absorbing peaks at 214 nm were collected. The fractionswith antimicrobial activities are marked by arrows. (Insert) Orcinol/H2SO4 test ofthe peptides eluted in Fig. 3. 1 �l of peptides: (1A) buffer; (2A) Peptide 1; (3A) Pep-tide 2; (4A) Peptide3; (5A) Peptide 4; (6A) Peptide 5; (1B) Peptide 6; (2B) Peptide7; (3B) Peptide 8; (4B) Peptide 9; (5B) Peptide 10; (6B) Peptide 11 were applied toa thin layer silica gel plate and air-dried. The plate was sprayed with orcinol/H2SO4

and heated for 20 min at 100 ◦C.

Fig. 2. RP-HPLC rechromatography of Peptide 8 with antimicrobial activity. Peptide8 was subjected to Nucleosil C18 RP-HPLC column (250 mm × 10 mm; Macherey-Nagel, Düren, Germany), equilibrated with 0.1% (v/v) trifluoroacetic acid/water.Elution was performed with linear gradient. UV-absorbing peaks at 214 nm werecollected. (Insert) Radial diffusion assay performed according to the procedure

P. Dolashka et al. / Pep

.6.2. Radial diffusion assayBacteria were grown overnight at 37 ◦C in TSB, sub-cultured and

rown to an optical density (OD) of 0.6 at 620 nm. The cells wereentrifuged for 10 min at 900 × g. The supernatant was discardednd the cells were washed once with 10 ml cold PBS buffer by cen-rifugation. Finally, the cells were diluted to an OD of 0.6 in PBSuffer. A gel solution containing 1% (w/v) of powdered TSB medium,%, w/v agarose, and 0.02% (v/v) Tween 20 made up in PBS bufferas prepared and autoclaved. 10 ml of the media was aliquoted

nd added to 1 ml of the diluted bacterial culture and dispersed for0 s using a laboratory vortex. Once the bacteria were adequatelyispersed, the gel was poured into a circular culture dish on a levellatform. The gel was then allowed to set for 1 h, before wells wereade using a 5 mm punch. After adding 10 �l of sample material

o each well, the plates were incubated for 3 h at 37 ◦C and thenurned over and incubated for a further 14 h at 37 ◦C. The areas ofhe clear zones surrounding the wells were calculated.

. Results

This work, presented here provides information on the defenseystem of mollusk and is the first peptidomic study on new AMPsriginated from the hemolymph of molluscan Rapana snail.

.1. Purification of the peptides

Three fractions with molecular masses between 3 and up to0 kDa were separated from the extracted hemolymph of marinenails R. venosa after centrifugation and concentration by Milliporelters. The obtained fractions: Fraction 1 (3–10 kDa), Fraction 210–30 kDa), and Fraction 3 (up to 30 kDa) were tested for antimi-robial activity. Only one of them (Fraction 1 with mass rangeetween 3 and 10 kDa) showed a positive result. Therefore, thisraction was further studied, the containing compounds were puri-ed and their structures and properties analyzed.

Two steps were applied, linear gradients and stepwise in threeteps with 20, 50 and 80% of solution B (80% acetonitrile, containing.08% TFA) for purification of the peptides from the Nucleosil C18olumn, equilibrated with 0.1% (v/v) TFA/water. Four fractions wereluted from the column by stepwise gradients with 20% ACN (Peaks–4, Fig. 1) and six fractions with 50% ACN (Peaks 5–11, Fig. 1).n some of these fractions a higher content of sugar was identi-ed (Fig. 1, inset). For further purification the pre-purified fractionsere again subjected to a Nucleosyl C18 column for rechromatog-

aphy, mainly one peak was observed in the chromatograms forractions no. 5, 6 and 7, while seven peaks were eluted by a linearradient of ACN (0–80% ACN, 0.8% TFA) after rechromatographyf Fractions no. 8 (Fig. 2), 9, 10 and 11. Totally eleven pure pep-ides, numbering Peptides 1–11, were obtained from the Fractions–11 and further studied. The preliminary analyses for antibacterialctivity of the isolated fractions showed that four (Fractions 8–11)f them, eluted with 50% ACN, revealed an antimicrobial activi-ies against Gram-negative (K. pneumoniae) and Gram-positive (S.ureus) bacterial strains (Fig. 2, inset).

.2. Identification of the peptides

To identify and analyze the isolated eleven peptides, sev-ral methods and techniques were applied. The peptides weredentified by their molecular masses, carbohydrate content and-terminal sequences.

The molecular masses of the peptides in the hemolymph of

apana snails were measured by MALDI MS and were determinedo be in the region from 3000 to 9500 Da. Some of them, like Pep-ides 1–4 have short chains with masses between 3 and 4 kDa, whilehe masses of four of AMPs (Peptides 8–11) were found to be in the

described in Section 2 of the separated fractions (shown on Fig. 1) against Grampositive S. aureus.

range from 7500 to 9500 Da. As shown in the spectrum on Fig. 3,the measured mass of the Peptide 8 is 9044.304 Da.

To identify the glycopeptides the isolated fractions were ana-lyzed by orcinol/sulphonic acid test. As shown on Fig. 1 (inset) thefractions on positions A2, 3, 4, 6 and B4 and 5 contain glycopeptidesand the intensity of spots A4 and A5 (Fig. 1, inset) is stronger com-pared to the other spots what indicates a higher content for sugarin Peptides 3 and 4.

N-terminal amino acid sequences of the peptides were alsodetermined by Edman degradation is shown in Fig. 4. The alignmentof the obtained sequence of the peptides with the known short part

of the gene sequence of the R. venosa showed a high identity onlybetween Peptide no. 3 (ELVRKNVDHLSTPDVLELV) and the aminoacid sequence of the hemocyanin molecule. The region – VRKNVD

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1480 P. Dolashka et al. / Peptides 32 (2011) 1477–1483

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Fig. 5. (A) The UV absorbance spectrum of the peptide 8 in 50 mM Tris/HCl buffer,pH 8.0, exhibited antimicrobial activity in the region of 200–600 nm; (Insert) fluo-rescence spectrum of peptide 8 in 50 mM Tris/HCl buffer, pH 8.0 at �ex 295 nm andthe fluorescence emission spectra were recorded in the region from 290 to 520 nm;(B) CD spectra in the UV region from 195 to 250 nm, a cuvet of 0.2 mm of: Peptide 8

FP

ig. 3. MALDI-MS, spectrum of the Peptide no. 8 isolated as shown in Fig. 3. Standardeptide solution was used to calibrate the mass scale of the AutoflexTM III, High-erformance MALDI-TOF & TOF/TOF Systems (Bruker Daltonics).

is a conserved part in the N-terminal sequences of functionalnits of molluscan hemocyanins, which indicates that Peptide 3

s probably released from the hemocyanin. Most of the purifiedeptides from the hemolymph of Rapana snails are highly cationicith a proline-rich N-terminal region (Fig. 4). Two Pro residues are

ncluded in the N-terminal region of Peptides 2, 4 and 5, while threef them are found for Pro residues in Peptides 6, 7, 8 and 9. In thisegion they also show high homology with the proline-rich pep-ides isolated from the plasma of the shrimp Penaeus vannamei andenaeus stylirostris [12].

.3. Spectroscopic studies of the peptides

Three spectroscopic methods, UV absorption, fluorescence spec-roscopy and circular dichroism, were applied for further analysesf the structure of new AMPs. The UV-absorption spectrum of Pep-ide 8 showed an intense band at 210–220 nm and a smaller oneetween 250 and 285 nm (Fig. 5A). The absorption at 210–220 nm

s mainly due to the peptide bond while the absorption at50–285 nm is attributed to the aromatic side chains of amino acids

ike phenylalanine, including the phenolic groups of tyrosine, thendole rings of tryptophan, the imidasole ring of histidines, and the

isulphide of cystine.

The fluorescence properties (quantum yield and �max) of Pep-ide 8 were analyzed upon excitation at 295 nm by the fluorescencemission spectrum, recorded in the region from 290 to 520 nm. The

1 5 10

Peptide 3 E L V R K N V D H L S

Peptide 2 S P P N Q P S

Peptide 4 S L P P T L E E

Peptide 5 S P P S E Q L G K

Peptide 6 S P P P G E S K V

Peptide 7 A P P P G L S A G

Peptide 8 A P P P G Y A M EPeptide 9 F P P P G E S A V

Pen-1 R P P P I G R P - P

Pen-2 R P P P I G R P - P

Pen-3a R P P P F V R P L P

Pen-3b R P P P F V R P L P

ig. 4. Sequence comparisons of the isolated peptides from the hemolymph of Rapana ve. stylirostris.

in 50 mM Tris/HCl buffer, pH 8.0 ( ) and peptide nigrocin 2 in the presence of TFE(—–).

properties are expected to be similar to those of hemocyanins, astyrosine and tryptophan residues are found in the native molecule.The emission spectrum of Peptide 8 is characterized by an emis-sion band with a maximum at 350 nm upon excitation at 295 nm,where the tryptophan residues are exclusively and fully excited(Fig. 5A, inset). The shift of the emission maximum toward shorterwavelengths is diagnostic for tryptophyl side chains in a non-polarenvironment, since �max for Trp in water is 355–360 nm [15].

Circular dichroism spectroscopy was also applied to analyze the

secondary structure of the new AMPs. CD spectra of the peptides,isolated from hemolymph of Rapana, were measured in the regionof 195–260 nm and not Cotton effect was observed (Fig. 5B) as well

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P. Dolashka et al. / Peptides 3

Table 1Results from the liquid growth antimicrobial assays.

Peptides K. pneumoniae After 24 hincubation

S. aureus After24 h incubation

1 0.9 >7.5 1.7 3.72 0.9 >7.5 1.1 2.43 0.9 6.5 1.5 3.14 0.9 4.9 1.1 3.05 0.9 5.3 1.5 2.56 0.9 5.2 1.3 3.17 0.9 4.3 1.6 2.68 1.0 5.2 1.4 1.89 0.9 5.9 1.4 1.710 1.1 >7.5 1.5 1.711 1.0 5.8 2.0 1.9Control 1.1 4.7 1.3 4.5

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cF – McFarland standards are devised to replace the counting of individual cellsnd are designed to correspond to approximate cell densities as required by theethod of antibacterial testing. 1 unit McF corresponds to 3 × 108 cells/ml.

s for Pro-rich peptides, isolated from the skin of a Korean frogana rugosa [31]. The CD spactra of two peptides, Peptide 8 fromhe hemolymph of Rapana and the known peptide nigrocin 2 fromana nigromaculata [38], are shown on Fig. 5B. Peptide nigrocin 2as no regular secondary structure in aqueous solution, however,

n the presence of Trifluoroethanol (TFE), CD spectra showed twoinima at 208 and 222 nm, which is characteristic of the presence

f a helical conformation.

.4. Antimicrobial activity of the peptides

Three different concentrations in the solutions of the peptides2, 10 and 50 �l), isolated from the hemolymph of Rapana were ana-yzed for antibacterial activity against Gram-positive (S. aureus) andne Gram-negative (K. pneumoniae) bacteria. Seven from eleveneptides exhibited antimicrobial activity against these two bac-erial strains. In the liquid growth inhibition assay (described inection 2) several fractions showed antimicrobial activity against. aureus. The growth inhibition of Fractions 8, 9, 10 and 11 wasver 90%. The strongest growth inhibition showed Fractions 10nd 11, while Fractions 2, 5 and 7 showed only mild growth inhi-ition (about 50%), and Fractions 1, 3, 4 and 6 had no significantffect (Table 1). However, the growth inhibition effect of all theleven peptides on K. pneumoniae was lower compared to S. aureusTable 1). Fractions 4 and 7 showed about 50% inhibition, the restf the samples had no antimicrobial activity on this bacterium.

. Discussion

Since the discovery of the microbial peptides cecropin fromhe insect Hyaophora cecropia [6], reports on the occurrence andharacterisation of low molecular mass AMPs from a wide vari-ty of organisms has accumulated rapidly [20,27,40]. Most of thextracts are complex mixtures of biochemically and pharmacolog-cally active components such as peptides and proteins. It was alsoound that the hemolymph of mollusk and arthropods containsarge amounts of biologically active proteins and peptides withifferent molecular masses and properties. One of these proteins,emocyanin R. venosa, was isolated from the hemolymph of marinenails and its antitumor and antiviral activities were established15,16,19,45].

These findings raise the question whether the compounds in the

emolymph of Rapana snail possess also an antibacterial activity.o answer to this question three fractions with molecular massesetween 3 and up to 30 kDa were separated from the extractedemolymph of marine snails R. venosa and were tested for antibac-

2 (2011) 1477–1483 1481

terial activity. Only one of the tested fractions (Fraction 1) withmass range between 3 and 10 kDa, gives a positive result. Moreover,eleven pure peptides, numbering Peptides 1–11, were isolated fromthis fraction and their molecular masses, carbohydrate content andN-terminal sequences were determined.

Some of the peptides, like Peptides 1–4 have short chainswith masses between 3 and 4 kDa, measured by MALDI MS. Sev-eral authors reported on the low molecular weight AMPs isolatedfrom different sources. One of them is a peptide with mass of4322.94 Da, isolated from G. mellonella [10] and the smallest nat-ural linear antimicrobial peptide, temporin-SHf, composed of onlyeight residues, which was found in the skin of the frog P. saharica[1].

Beside these short chain peptides, several longer-chain AMPswere identified in the hemolymph of the Rapana snails (Peptides8–11). The measured mass of the Peptide 8 (9044.304 Da) is closeto the mass of glycine-rich AMP (9 kDa), isolated from the insectPhormia terranovae [13]. The masses of four other peptides from thehemolymph of Rapana snails were found to be in the range from7500 to 9500 Da which is usual for AMP’s of molluscs and arthro-pods and correlates also to the masses of antimicrobial peptidesisolated from different sources. Most of the AMPs isolated fromhemocytes have masses in the region of 3–12 kDa [28] as e.g. thetwo AMPs from the hemocytes of Carcinus maenas with masses of6.5 kDa and 11.5 kDa [36,39], as well as AMPs with masses from 5.48to 6.62 kDa, isolated in the active form from the mollusk penaeidshrimp [11,12].

Some of peptides as Peptides 1–5, Peptides 6, 9 and 10 areglycopeptides which is of importance for their function in thehemolymph of Rapana. Antibacterial glycopeptides with O-linkedsugars were also isolated from insects [12] and with N-linked sugarsfrom the hemolymph of mollusc Biomphalaria glabrata [29].

The analyzed peptides from the hemolymph of mollusks andarthropods have different structure. Some of them are obtainedfrom N- or C-terminal peptides from the hemocyanins. It wasreported that three peptides with molecular masses of 2.7, 7.9, and8.3 kDa, isolated from the plasma of the shrimp P. vannamei andP. stylirostris, display 95–100% sequence identity with a C-terminalsequence of hemocyanin, and probably, they are cleaved fragmentsof the respiratory proteins [13].

To identify the new antibacterial peptides from the hemolymphof Rapana the separated fractions were analyzed by Edmandegradation and compared to the hemocyanin, dissolved in thehemolymph. However, the full amino acid sequence of Rapanahemocyanins is still unknown. That’s why the alignment of theobtained N-terminal amino acid sequences of the peptides showeda high identity only between Peptide no. 3, ELVRKNVDHLST-PDVLELV and the amino acid sequence of the known short partof the gene sequence of R. venosa hemocyanin. However, their N-terminal amino acid sequences show that most of peptides arehighly cationic with two (Peptides 2, 4 and 5) or three (Peptides6, 7, 8 and 9) proline residues. In prolin-rich N-terminal regionthey reveal a high homology with the proline-rich peptides iso-lated from the plasma of the shrimp P. vannamei and P. stylirostris[12]. Penaeidins are also highly cationic AMPs and are composed ofan N-terminal proline-rich region followed by a C-terminal domainstabilized by three intramolecular disulfide cross-links [12]. Theyshow a high degree of similarity with the sequence of arthropodandefensins in the cysteine-rich cationic region. The positions of thecysteines in arthropodan defensins are highly conserved, and thisarray is also identical to that of defensins A and B from M. edulis [5].

Beside AMPs with an N-terminal proline-rich region and

cystein-rich region was also found in several arthropods [36] andmollusks [29]. In the hemocytes of arthropod C. maenas, a proline-rich AMP of 6.5 kDa [36] was found besides, a second, a cysteine-rich11.5-kDa peptide [34]. However, both AMPs differ biochemically as

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482 P. Dolashka et al. / Pep

ell as their functionality. The other cationic cysteine-rich AMPs,ytilins A and B, were isolated from mollusc M. edulis [5] as wellyticins A and B, isolated from the hemocytes and plasma of

he mussel M. galloprovincialis, comprise 40 residues with fourntramolecular disulfide bridges and a cysteine array differentrom that of previously characterized cysteine-rich antimicrobialeptides [29]. It is known that the disulphide bounds additionaltabilised the peptides.

Additional confirmation that some of isolated AMPs from theemolymph of Rapana are Pro-rich peptides was also revealed byheir CD spectra. Not Cotton effect was observed in the CD spectra ofeptide 8 from Rapana and this result correlates with the other Pro-ich peptides, isolated from the skin of a Korean frog R. rugosa [31]nd the known peptide nigrocin 2 [38]. Using this method severalther peptides were characterised in the literature, as the pres-nce of both �-sheet and �-helix conformations in the secondarytructure of Human �-defensin 28 (hBD28) [42], as well severalystein-rich peptides.

Marine molluscs are exposed to microbial pathogens in theirnvironment, which can number up to 106 bacteria/ml and09 viruses/ml of seawater [29]. Arthropods and molluscs havevolved an immune system that could distinguish different classesf pathogens [4]. The example of lebocins, longer proline-richntibacterial peptides from various sources, indicate that differ-nt insect species evolved their specific antibacterial peptides todapt the environment where they reside and the pathogens thathreaten their existence [4].

Defensins and cystein-rich peptides from marine molluscsxpress a stronger activity against Gram-positive and Gram-egative bacteria and fungi and a synthetic mytilin fragmentisplayed activity against the white spot syndrome virus [29]. At

east 18 known and putatively antimicrobial peptides from 10amilies were discovered to defend G. mellonella against invading

icrobes. Moreover, antimicrobial activity against E. coli, S. aureus,nd Candida albicans, was shown from Ixodes sinensis [46] as wells antibacterial activity in vivo and in vitro of some peptides from. mellonella against Pseudomonas aeruginosa [2].

For decades, one major area of interest for the discovery andtudy of new antibiotics was the investigation of AMPs derived fromnsect immune defense reactions [4]. Therefore, the isolated AMPsrom the hemolymph of Rapana were analyzed for antibacterialctivity against two bacterial strains, one Gram-positive (S. aureus)nd one Gram-negative (K. pneumoniae). These strains were cho-en because they are human pathogenic bacteria and commonlysed for antimicrobial tests. Despite being harmless in most indi-iduals, S. aureus is capable of causing various infections of thekin and other organs. The most common treatment for S. aureusnfection is penicillin, but in most countries, penicillin-resistances extremely common. Combination therapy with gentamicin maye used to treat serious infections like endocarditis [9], but its use

s controversial because of the high risk of damage to the kidneys23]. The duration of treatment depends on the site of infection andn severity. For this reason, the discovery of natural products withntimicrobial activity is of a great interest.

We have found that seven from eleven peptides isolated fromhe hemolymph of Rapana exhibited antimicrobial activity against. aureus. Exclusively high growth inhibition effect, over 90%,howed Fractions 8, 9, 10 and 11. However, the growth inhibitionf all the eleven peptides on K. pneumoniae was lower comparedo S. aureus (Table 1). Fractions 4 and 7 showed about 50% inhibi-ion, the rest of the samples had no antimicrobial activity on thisacterium.

Consequently, the priority for the next decades should beocused on the development of alternative drugs and/or the recov-ry of natural molecules that would allow consistent and properontrol of pathogen-caused diseases. The antibiotic peptides are

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powerful arsenal of molecules that could be the antimicrobial drugsof the new century as an innovative response to the increasingproblem of medical research. Therefore, we will undertake furthercharacterisation of the peptides from Rapana to reveal their fullstructures and to explain the antibacterial activity.

Acknowledgements

This work was supported by a research grant by the Bul-garian National Science Fund TK01-496/2009 and DFG-01/2008(Germany). We thank to Ing. Yordan Peichev, Director of SYCO-PHARMA, OOD, Sofia for his support and Ing. H. Stoyanov, Directorof “Delta Industry” AD, Sozopol, for providing the animals.

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