Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Peptides 32 (2011) 1477–1483

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Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa Pavlina Dolashka a,∗ , Vesela Moshtanska a , Valika Borisova b , Aleksander Dolashki a , Stefan Stevanovic c , Tzvetan Dimanov b , Wolfgang Voelter d a

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

a r t i c l e

i n f o

Article history: Received 17 February 2011 Received in revised form 3 May 2011 Accepted 3 May 2011 Available online 22 June 2011 Keywords: Antimicrobial proline-rich peptides Hemolymph of Rapana venosa Gram-positive (Staphylococcus aureus) and a Gram-negative (Klebsiella pneumoniae) bacteria

a b s t r a c t Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The recent appearance of a growing number of bacteria resistant to conventional antibiotics has become a serious medical problem. To overcome this resistance, the development of antibiotics, with novel mechanisms of action is a persisting issue [20,27,43]. The new generation of native peptides seems to fit to this urgent issue. As a consequence, these native peptides have been termed “natural antibiotics”, because they are active against a large spectrum of microorganisms including bacteria, filamentous fungi, protozoan and metazoan parasites [21,32,33]. Antimicrobial peptides (AMPs) are important components of the non-specific host defense or innate immune system in a variety of organisms ranging from plants and insects to animals including molluscs and arthropods, amphibians and mammals [21,26]. Therefore, in the last years there is a great interest in studying new antimicrobial peptides. AMPs are classified into several groups based on amino acid sequences, secondary structures, and functional similarities e.g. forming ␣-helices, ␤-sheet, containing thioether rings, overrep-

∗ Corresponding author. Tel.: +359 29606163; fax: +359 8700225. E-mail address: [email protected] (P. Dolashka). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.05.001

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]. Several cysteine-rich peptides were purified from scorpion Centruroides limpidus limpidus [8], while a new type of short antimicrobial peptides, designated temporin-SHf, were identified in the skin of the frog Pelophylax saharica [1]. It was also found that the hemolymph of molluscs and arthropods is rich in peptides and aromatic polypeptides [16,17,24,35,37,46]. Despite their variations in structure and size, AMPs are usually characterized by their cationic and hydrophobic nature, as human ␤-defensin 28 (hBD28) which is a strongly cationic AMP against Escherichia coli K12 [25,44]. The nature of the peptides was considered to be crucial for the initial interaction between the peptide and the bacterial membrane [3,41]. Most of the peptides exhibit a broad spectrum of microbial activity against Gram-positive and Gram-negative bacteria and yeasts. In the hemolymph of the blue crab Callinestes sapidus, Scylla serrata, Thalamita crenata, houseflies (Musca domestica), Galleria mellonella, female thick Amblyomma hebraeum AMPs which are strong inhibitors against gram-negative bacteria were identified [3,10,34–36]. Several novel AMPs with antimicrobial activity were also isolated from the body wall of the sea hare Dolabella auric-

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ularia [22]. Besides peptides, the presence of glycoproteins with antimicrobial activity was reported from the mucus of the giant snail Achatina fulica and from the egg mass and purple fluid of the sea hare Aplysia kurodai [45]. It was found that beside antimicrobial activity against Micrococcus luteus and E. coli, the cysteine-rich peptides from Mytilus edulis exhibit also antifungal activity against Neurospora crassa [5]. Studies on the antibacterial and antifungal peptides in marine molluscs are mainly focused on the mussels of Mytilus galloprovincialis and M. edulis [5,28]. Beside the wide spectrum of AMPs’ antimicrobial activities their potential benefit for the treatment of cancer and viral or parasitic infections is suggested [6,7,11]. There is also a significant interest in the development of therapeutic antibiotics based on AMPs; however, the poor understanding of the fundamental mechanism of action of these peptides has largely hampered such efforts. Therefore, identification and isolation of the active substances and determination of their primary structures or DNA sequences are of enormous importance to understand non-specific immune response mechanism of mollusks and arthropods against pathogen invasion and for the development of new biopharmaceutical concept and finally products. In this study, we report for the first time about the structure and properties of several new antimicrobial peptides, isolated from the hemolymph of marine snail Rapana venosa. 2. Materials and methods 2.1. Animals and collection of hemolymph The marine snails R. venosa were collected from the Black Sea and provided by “Delta Industry” AD, Sozopol, Bulgaria. The hemolymph was isolated from the foot of animals as described by Dolashka-Angelova et al. [14,19]. For preliminary purification the hemolymph was centrifuged in two steps: first for 30 min at 4 ◦ C and 5000 × g to remove the cellular contents, and second then the centrifugation was elongated for 4 h at 4 ◦ C at 30,000 × g to precipitate the hemocyanin. 2.2. Purification of peptides The supernatant was concentrated and separated into several fractions using Millipore filters with different size (3, 10 and 30 kDa). A fraction with mass between 3 and 10 kDa was concentrated and applied on a HPLC system, using a Nucleosil 7 C18 column (250 mm × 10 mm; Macherey-Nagel, Düren, Germany), equilibrated with buffer A (H2 O, containing 0.1% TFA). Elution was performed stepwise in three steps with 20, 50 and 80% of solution B (80% acetonitrile, containing 0.08% TFA) at a flow rate of 1.5 ml min−1 . Ultraviolet absorption was monitored at 280 and 214 nm and the eluted fractions were collected and dried by Speedvac. The isolated fractions were reconstituted in MilliQ water with 0.10% TFA (v/v) before being applied again on a Nucleosil 7 C18 column for rechromatography. For elution, a linear gradient from 5% solvent A (0.1% TFA in water) to 100% solvent B (0.085% TFA in ACN) within 50 min at a flow rate of 1 ml min−1 was used. Again, the HPLC fractions were detected at a wavelength of 214 nm and collected. 2.3. Determination of N-terminal amino acid sequences and mass spectrometric analysis Isolated HPLC fractions were dried and after dissolving in 40% methanol/1% formic acid their N-terminal amino acid sequences were determined by automated Edman N-terminal sequencing on a

Pulsed Liquid Protein Sequencer (Applied Biosystems GmbH, Foster City, CA). The molecular masses of isolated peptides were measured by AutoflexTM III, High-Performance MALDI-TOF & TOF/TOF Systems (Bruker Daltonics). For mass spectrometric analysis about 50 pmol of the HPLC fractions were dissolved in 0.1% (v/v) TFA and applied to the target. Analysis was carried out using ␣-cyano-4hydroxycinnamic acid as a matrix. The mass spectrometer uses a 200 Hz frequency-tripled Nd–YAG laser operating at a wavelength of 355 nm. 3500 shots were acquired in the MS mode and collision energy of 4200 was applied. A solution of peptide standard was used to calibrate the mass scale. The mass values assigned to the amino 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 thinlayer plate and air-dried, taking care to restrict the size of the spot to 2–3 mm in diameter. The plate was sprayed with orcinol/H2 SO4 and heated for 20 min at 100 ◦ C. 2.5. Spectroscopic studies Spectroscopic properties of peptides were analyzed by UV spectroscopy, circular dichroism and fluorescence spectroscopy. UV spectra were measured on a Shimadzu spectrophotometer. The spectra of the peptide solutions with A280 = 0.2 in 50 mM Tris/HCl buffer, pH 8.0, were measured by circular dichroism (CD) in the UV region from 195 to 250 nm using a Jasco J-720 dichrograph, equipped with a personal computer IBM PC-AT, PS/2, and a cuvet of 0.2 mm. A software DOS version was used for calculation of the CD data [18]. Peptide solutions in 50 mM Tris/HCl buffer, pH 8.0, with absorbance at the excitation wavelength <0.05 to minimize the inner filter or absorption effects, were analyzed by a spectrofluorimeter (Perkin-Elmer Model LS5), equipped with a thermostatically controlled sample holder and a Model 3600 data station. Fluorescence emission spectra were recorded in the region from 290 to 520 nm. Excitation at 295 nm was used for predominant measuring the fluorescence of tryptophyl residues. Spectra were corrected for background due to the solvent. 2.6. Antibacterial assays of the peptides 2.6.1. Liquid growth inhibition assay Two different species of bacteria, a Gram-positive (Staphylococcus aureus) and a Gram-negative (Klebsiella pneumoniae) one, were used as reference to analyze the antimicrobial activity of the peptides. Both bacteria are isolates from patients of the Medical Center PolyMed® , Sofia, Bulgaria. The concentrations of the peptides were 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 peptides were qualitatively checked according to the liquid growth inhibition assay. Briefly, 10 ␮l of the samples was mixed with 6 ml of a mid-logarithmic phase culture of bacteria in poor broth nutrient medium (1% dextrose) with definite OD. Microbial growth was assessed 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 unit McF corresponds to 3 × 108 cells/ml. The turbidity was measured with DENSIMAT (BioMerieux, France) instrument.

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2.6.2. Radial diffusion assay Bacteria were grown overnight at 37 ◦ C in TSB, sub-cultured and grown to an optical density (OD) of 0.6 at 620 nm. The cells were centrifuged for 10 min at 900 × g. The supernatant was discarded and the cells were washed once with 10 ml cold PBS buffer by centrifugation. Finally, the cells were diluted to an OD of 0.6 in PBS buffer. A gel solution containing 1% (w/v) of powdered TSB medium, 1%, w/v agarose, and 0.02% (v/v) Tween 20 made up in PBS buffer was prepared and autoclaved. 10 ml of the media was aliquoted and added to 1 ml of the diluted bacterial culture and dispersed for 10 s using a laboratory vortex. Once the bacteria were adequately dispersed, the gel was poured into a circular culture dish on a level platform. The gel was then allowed to set for 1 h, before wells were made using a 5 mm punch. After adding 10 ␮l of sample material to each well, the plates were incubated for 3 h at 37 ◦ C and then turned over and incubated for a further 14 h at 37 ◦ C. The areas of the clear zones surrounding the wells were calculated. 3. Results This work, presented here provides information on the defense system of mollusk and is the first peptidomic study on new AMPs originated from the hemolymph of molluscan Rapana snail. 3.1. Purification of the peptides Three fractions with molecular masses between 3 and up to 30 kDa were separated from the extracted hemolymph of marine snails R. venosa after centrifugation and concentration by Millipore filters. The obtained fractions: Fraction 1 (3–10 kDa), Fraction 2 (10–30 kDa), and Fraction 3 (up to 30 kDa) were tested for antimicrobial activity. Only one of them (Fraction 1 with mass range between 3 and 10 kDa) showed a positive result. Therefore, this fraction was further studied, the containing compounds were purified and their structures and properties analyzed. Two steps were applied, linear gradients and stepwise in three steps with 20, 50 and 80% of solution B (80% acetonitrile, containing 0.08% TFA) for purification of the peptides from the Nucleosil C18 column, equilibrated with 0.1% (v/v) TFA/water. Four fractions were eluted from the column by stepwise gradients with 20% ACN (Peaks 1–4, Fig. 1) and six fractions with 50% ACN (Peaks 5–11, Fig. 1). In some of these fractions a higher content of sugar was identified (Fig. 1, inset). For further purification the pre-purified fractions were again subjected to a Nucleosyl C18 column for rechromatography, mainly one peak was observed in the chromatograms for Fractions no. 5, 6 and 7, while seven peaks were eluted by a linear gradient of ACN (0–80% ACN, 0.8% TFA) after rechromatography of Fractions no. 8 (Fig. 2), 9, 10 and 11. Totally eleven pure peptides, numbering Peptides 1–11, were obtained from the Fractions 8–11 and further studied. The preliminary analyses for antibacterial activity of the isolated fractions showed that four (Fractions 8–11) of them, eluted with 50% ACN, revealed an antimicrobial activities against Gram-negative (K. pneumoniae) and Gram-positive (S. aureus) bacterial strains (Fig. 2, inset). 3.2. Identification of the peptides To identify and analyze the isolated eleven peptides, several methods and techniques were applied. The peptides were identified by their molecular masses, carbohydrate content and N-terminal sequences. The molecular masses of the peptides in the hemolymph of Rapana snails were measured by MALDI MS and were determined to be in the region from 3000 to 9500 Da. Some of them, like Peptides 1–4 have short chains with masses between 3 and 4 kDa, while the masses of four of AMPs (Peptides 8–11) were found to be in the

Fig. 1. RP-HPLC purification of the peptides. The hemolymph fraction between 3 and 10 kDa 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 (1.5 ml min−1 ) was performed according to the procedure described in Section 2. UV-absorbing peaks at 214 nm were collected. The fractions with antimicrobial activities are marked by arrows. (Insert) Orcinol/H2 SO4 test of the peptides eluted in Fig. 3. 1 ␮l of peptides: (1A) buffer; (2A) Peptide 1; (3A) Peptide 2; (4A) Peptide3; (5A) Peptide 4; (6A) Peptide 5; (1B) Peptide 6; (2B) Peptide 7; (3B) Peptide 8; (4B) Peptide 9; (5B) Peptide 10; (6B) Peptide 11 were applied to a thin layer silica gel plate and air-dried. The plate was sprayed with orcinol/H2 SO4 and heated for 20 min at 100 ◦ C.

Fig. 2. RP-HPLC rechromatography of Peptide 8 with antimicrobial activity. Peptide 8 was subjected to Nucleosil C18 RP-HPLC column (250 mm × 10 mm; MachereyNagel, Düren, Germany), equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution was performed with linear gradient. UV-absorbing peaks at 214 nm were collected. (Insert) Radial diffusion assay performed according to the procedure described in Section 2 of the separated fractions (shown on Fig. 1) against Gram positive 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 analyzed by orcinol/sulphonic acid test. As shown on Fig. 1 (inset) the fractions on positions A2, 3, 4, 6 and B4 and 5 contain glycopeptides and the intensity of spots A4 and A5 (Fig. 1, inset) is stronger compared to the other spots what indicates a higher content for sugar in Peptides 3 and 4. N-terminal amino acid sequences of the peptides were also determined by Edman degradation is shown in Fig. 4. The alignment of the obtained sequence of the peptides with the known short part of the gene sequence of the R. venosa showed a high identity only between Peptide no. 3 (ELVRKNVDHLSTPDVLELV) and the amino acid sequence of the hemocyanin molecule. The region – VRKNVD

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

– is a conserved part in the N-terminal sequences of functional units of molluscan hemocyanins, which indicates that Peptide 3 is probably released from the hemocyanin. Most of the purified peptides from the hemolymph of Rapana snails are highly cationic with a proline-rich N-terminal region (Fig. 4). Two Pro residues are included in the N-terminal region of Peptides 2, 4 and 5, while three of them are found for Pro residues in Peptides 6, 7, 8 and 9. In this region they also show high homology with the proline-rich peptides isolated from the plasma of the shrimp Penaeus vannamei and Penaeus stylirostris [12]. 3.3. Spectroscopic studies of the peptides

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) fluorescence spectrum of peptide 8 in 50 mM Tris/HCl buffer, pH 8.0 at ex 295 nm and the 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 in 50 mM Tris/HCl buffer, pH 8.0 ( ) and peptide nigrocin 2 in the presence of TFE (—–).

Three spectroscopic methods, UV absorption, fluorescence spectroscopy and circular dichroism, were applied for further analyses of the structure of new AMPs. The UV-absorption spectrum of Peptide 8 showed an intense band at 210–220 nm and a smaller one between 250 and 285 nm (Fig. 5A). The absorption at 210–220 nm is mainly due to the peptide bond while the absorption at 250–285 nm is attributed to the aromatic side chains of amino acids like phenylalanine, including the phenolic groups of tyrosine, the indole rings of tryptophan, the imidasole ring of histidines, and the disulphide of cystine. The fluorescence properties (quantum yield and max ) of Peptide 8 were analyzed upon excitation at 295 nm by the fluorescence emission spectrum, recorded in the region from 290 to 520 nm. The

properties are expected to be similar to those of hemocyanins, as tyrosine and tryptophan residues are found in the native molecule. The emission spectrum of Peptide 8 is characterized by an emission 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 shorter wavelengths is diagnostic for tryptophyl side chains in a non-polar environment, 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 region of 195–260 nm and not Cotton effect was observed (Fig. 5B) as well

1

5

10

15

20

Peptide 3

E L V RK N V D H L S T P DV L E L V

Peptide 2

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

P P N

Q

P

S I M T F D

Y AKT NK

E F N M

KKMG

S F N F

G E S K V D M S F N

Peptide 7

A P P P

G L S A G V

Peptide 8

A P P P

G Y A ME S D S F

Peptide 9

F P P P

G E S A V D M S F F

Pen-1

R P P P I G R P - P L R L V V

Pen-2

R P P P I G R P - P F R P V

Y AL S NP A Q S Y AL S NP

Pen-3a

R P P P F V R P L P G G P I G P Y NGC

Pen-3b

R P P P F V R P L P G G P I G P Y NGC P

Fig. 4. Sequence comparisons of the isolated peptides from the hemolymph of Rapana venosa snail with penaeidins isolated from the plasma of the shrimp P. vannamei and P. stylirostris.

P. Dolashka et al. / Peptides 32 (2011) 1477–1483 Table 1 Results from the liquid growth antimicrobial assays. Peptides

K. pneumoniae

1 2 3 4 5 6 7 8 9 10 11 Control

0.9 0.9 0.9 0.9 0.9 0.9 0.9 1.0 0.9 1.1 1.0 1.1

After 24 h incubation >7.5 >7.5 6.5 4.9 5.3 5.2 4.3 5.2 5.9 >7.5 5.8 4.7

S. aureus

After24 h incubation

1.7 1.1 1.5 1.1 1.5 1.3 1.6 1.4 1.4 1.5 2.0 1.3

3.7 2.4 3.1 3.0 2.5 3.1 2.6 1.8 1.7 1.7 1.9 4.5

Bold indicates peptides exhibited antibacterialen effect. McF – McFarland standards are devised to replace the counting of individual cells and are designed to correspond to approximate cell densities as required by the method of antibacterial testing. 1 unit McF corresponds to 3 × 108 cells/ml.

as for Pro-rich peptides, isolated from the skin of a Korean frog Rana rugosa [31]. The CD spactra of two peptides, Peptide 8 from the hemolymph of Rapana and the known peptide nigrocin 2 from Rana nigromaculata [38], are shown on Fig. 5B. Peptide nigrocin 2 has no regular secondary structure in aqueous solution, however, in the presence of Trifluoroethanol (TFE), CD spectra showed two minima at 208 and 222 nm, which is characteristic of the presence of a helical conformation. 3.4. Antimicrobial activity of the peptides Three different concentrations in the solutions of the peptides (2, 10 and 50 ␮l), isolated from the hemolymph of Rapana were analyzed for antibacterial activity against Gram-positive (S. aureus) and one Gram-negative (K. pneumoniae) bacteria. Seven from eleven peptides exhibited antimicrobial activity against these two bacterial strains. In the liquid growth inhibition assay (described in Section 2) several fractions showed antimicrobial activity against S. aureus. The growth inhibition of Fractions 8, 9, 10 and 11 was over 90%. The strongest growth inhibition showed Fractions 10 and 11, while Fractions 2, 5 and 7 showed only mild growth inhibition (about 50%), and Fractions 1, 3, 4 and 6 had no significant effect (Table 1). However, the growth inhibition effect of all the eleven peptides on K. pneumoniae was lower compared to S. aureus (Table 1). Fractions 4 and 7 showed about 50% inhibition, the rest of the samples had no antimicrobial activity on this bacterium. 4. Discussion Since the discovery of the microbial peptides cecropin from the insect Hyaophora cecropia [6], reports on the occurrence and characterisation of low molecular mass AMPs from a wide variety of organisms has accumulated rapidly [20,27,40]. Most of the extracts are complex mixtures of biochemically and pharmacologically active components such as peptides and proteins. It was also found that the hemolymph of mollusk and arthropods contains large amounts of biologically active proteins and peptides with different molecular masses and properties. One of these proteins, hemocyanin R. venosa, was isolated from the hemolymph of marine snails and its antitumor and antiviral activities were established [15,16,19,45]. These findings raise the question whether the compounds in the hemolymph of Rapana snail possess also an antibacterial activity. To answer to this question three fractions with molecular masses between 3 and up to 30 kDa were separated from the extracted hemolymph of marine snails R. venosa and were tested for antibac-

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terial activity. Only one of the tested fractions (Fraction 1) with mass range between 3 and 10 kDa, gives a positive result. Moreover, eleven pure peptides, numbering Peptides 1–11, were isolated from this fraction and their molecular masses, carbohydrate content and N-terminal sequences were determined. Some of the peptides, like Peptides 1–4 have short chains with masses between 3 and 4 kDa, measured by MALDI MS. Several authors reported on the low molecular weight AMPs isolated from different sources. One of them is a peptide with mass of 4322.94 Da, isolated from G. mellonella [10] and the smallest natural linear antimicrobial peptide, temporin-SHf, composed of only eight residues, which was found in the skin of the frog P. saharica [1]. Beside these short chain peptides, several longer-chain AMPs were identified in the hemolymph of the Rapana snails (Peptides 8–11). The measured mass of the Peptide 8 (9044.304 Da) is close to the mass of glycine-rich AMP (9 kDa), isolated from the insect Phormia terranovae [13]. The masses of four other peptides from the hemolymph of Rapana snails were found to be in the range from 7500 to 9500 Da which is usual for AMP’s of molluscs and arthropods and correlates also to the masses of antimicrobial peptides isolated from different sources. Most of the AMPs isolated from hemocytes have masses in the region of 3–12 kDa [28] as e.g. the two AMPs from the hemocytes of Carcinus maenas with masses of 6.5 kDa and 11.5 kDa [36,39], as well as AMPs with masses from 5.48 to 6.62 kDa, isolated in the active form from the mollusk penaeid shrimp [11,12]. Some of peptides as Peptides 1–5, Peptides 6, 9 and 10 are glycopeptides which is of importance for their function in the hemolymph of Rapana. Antibacterial glycopeptides with O-linked sugars were also isolated from insects [12] and with N-linked sugars from the hemolymph of mollusc Biomphalaria glabrata [29]. The analyzed peptides from the hemolymph of mollusks and arthropods have different structure. Some of them are obtained from N- or C-terminal peptides from the hemocyanins. It was reported that three peptides with molecular masses of 2.7, 7.9, and 8.3 kDa, isolated from the plasma of the shrimp P. vannamei and P. stylirostris, display 95–100% sequence identity with a C-terminal sequence of hemocyanin, and probably, they are cleaved fragments of the respiratory proteins [13]. To identify the new antibacterial peptides from the hemolymph of Rapana the separated fractions were analyzed by Edman degradation and compared to the hemocyanin, dissolved in the hemolymph. However, the full amino acid sequence of Rapana hemocyanins is still unknown. That’s why the alignment of the obtained N-terminal amino acid sequences of the peptides showed a high identity only between Peptide no. 3, ELVRKNVDHLSTPDVLELV and the amino acid sequence of the known short part of the gene sequence of R. venosa hemocyanin. However, their Nterminal amino acid sequences show that most of peptides are highly cationic with two (Peptides 2, 4 and 5) or three (Peptides 6, 7, 8 and 9) proline residues. In prolin-rich N-terminal region they reveal a high homology with the proline-rich peptides isolated from the plasma of the shrimp P. vannamei and P. stylirostris [12]. Penaeidins are also highly cationic AMPs and are composed of an N-terminal proline-rich region followed by a C-terminal domain stabilized by three intramolecular disulfide cross-links [12]. They show a high degree of similarity with the sequence of arthropodan defensins in the cysteine-rich cationic region. The positions of the cysteines in arthropodan defensins are highly conserved, and this array 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] and mollusks [29]. In the hemocytes of arthropod C. maenas, a prolinerich AMP of 6.5 kDa [36] was found besides, a second, a cysteine-rich 11.5-kDa peptide [34]. However, both AMPs differ biochemically as

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well as their functionality. The other cationic cysteine-rich AMPs, Mytilins A and B, were isolated from mollusc M. edulis [5] as well Myticins A and B, isolated from the hemocytes and plasma of the mussel M. galloprovincialis, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array different from that of previously characterized cysteine-rich antimicrobial peptides [29]. It is known that the disulphide bounds additional stabilised the peptides. Additional confirmation that some of isolated AMPs from the hemolymph of Rapana are Pro-rich peptides was also revealed by their CD spectra. Not Cotton effect was observed in the CD spectra of Peptide 8 from Rapana and this result correlates with the other Prorich peptides, isolated from the skin of a Korean frog R. rugosa [31] and the known peptide nigrocin 2 [38]. Using this method several other peptides were characterised in the literature, as the presence of both ␤-sheet and ␣-helix conformations in the secondary structure of Human ␤-defensin 28 (hBD28) [42], as well several cystein-rich peptides. Marine molluscs are exposed to microbial pathogens in their environment, which can number up to 106 bacteria/ml and 109 viruses/ml of seawater [29]. Arthropods and molluscs have evolved an immune system that could distinguish different classes of pathogens [4]. The example of lebocins, longer proline-rich antibacterial peptides from various sources, indicate that different insect species evolved their specific antibacterial peptides to adapt the environment where they reside and the pathogens that threaten their existence [4]. Defensins and cystein-rich peptides from marine molluscs express a stronger activity against Gram-positive and Gramnegative bacteria and fungi and a synthetic mytilin fragment displayed activity against the white spot syndrome virus [29]. At least 18 known and putatively antimicrobial peptides from 10 families were discovered to defend G. mellonella against invading microbes. Moreover, antimicrobial activity against E. coli, S. aureus, and Candida albicans, was shown from Ixodes sinensis [46] as well as antibacterial activity in vivo and in vitro of some peptides from G. mellonella against Pseudomonas aeruginosa [2]. For decades, one major area of interest for the discovery and study of new antibiotics was the investigation of AMPs derived from insect immune defense reactions [4]. Therefore, the isolated AMPs from the hemolymph of Rapana were analyzed for antibacterial activity against two bacterial strains, one Gram-positive (S. aureus) and one Gram-negative (K. pneumoniae). These strains were chosen because they are human pathogenic bacteria and commonly used for antimicrobial tests. Despite being harmless in most individuals, S. aureus is capable of causing various infections of the skin and other organs. The most common treatment for S. aureus infection is penicillin, but in most countries, penicillin-resistance is extremely common. Combination therapy with gentamicin may be used to treat serious infections like endocarditis [9], but its use is controversial because of the high risk of damage to the kidneys [23]. The duration of treatment depends on the site of infection and on severity. For this reason, the discovery of natural products with antimicrobial activity is of a great interest. We have found that seven from eleven peptides isolated from the hemolymph of Rapana exhibited antimicrobial activity against S. aureus. Exclusively high growth inhibition effect, over 90%, showed Fractions 8, 9, 10 and 11. However, the growth inhibition of all the eleven peptides on K. pneumoniae was lower compared to S. aureus (Table 1). Fractions 4 and 7 showed about 50% inhibition, the rest of the samples had no antimicrobial activity on this bacterium. Consequently, the priority for the next decades should be focused on the development of alternative drugs and/or the recovery of natural molecules that would allow consistent and proper control of pathogen-caused diseases. The antibiotic peptides are

powerful arsenal of molecules that could be the antimicrobial drugs of the new century as an innovative response to the increasing problem of medical research. Therefore, we will undertake further characterisation of the peptides from Rapana to reveal their full structures and to explain the antibacterial activity. Acknowledgements This work was supported by a research grant by the Bulgarian National Science Fund TK01-496/2009 and DFG-01/2008 (Germany). We thank to Ing. Yordan Peichev, Director of SYCOPHARMA, OOD, Sofia for his support and Ing. H. Stoyanov, Director of “Delta Industry” AD, Sozopol, for providing the animals. References [1] Abbassi F, Lequin O, Piesse C, Goasdoue N, Foulon T, Nicolas P, et al. TemporinSHf, a new type of phe-rich and hydrophobic ultrashort antimicrobial peptide. J Biol Chem 2010;22:16880–92. [2] Andrejko M, Mizerska-Dudka M, Jakubowicz T. Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa. Comp Biochem and Physiol Part B 2009;152:118–23. [3] Bolintineanu S, Kaznessis YN. Computational studies of protegrin antimicrobial peptides: a review. Peptides 2010;32(1):188–201. [4] Bulet P, Stöcklin R. Insect antimicrobial peptides: structures, properties and gene regulation. Protein Peptide Lett 2005;12:3–11. [5] Charlet M, Chernysh S, Philippei H, Hetru C, Hoffmann JA, Bulet P. Isolation of several cysteine-richn antimicrobial peptides from the blood of mollusc, Mytilus edulis. JBC 1996;271:21808–13. [6] Chernish S, Kim SI, Beckeer G, Pleskach VA, Filatova NA, Anikin VB, et al. Antiviral and antitumor peptides from insects. Proc Natl Acad Sci USA 2002;99(20):12628–32. [7] Chinchar VG, Wang J, Murti G, Carey C, Rollinss-Smith L. Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin. Virology 2001;288:351–7. [8] Corona M, Coronas FV, Merino E, Becerril B, Gutierrez R, Rebolledo-Antunez Garcia DE, et al. A novel class of peptide found in scorpion venom with neurodepressant effects in peripheral. Biochim Biophys Acta 2003;1649(June (1)):58–67. [9] Cosgrove SE, Vigliani GA, Campion M. Initial low dose gentamicin for Staphylococcus aureus bacteremia and endocarditis is nephrotoxic. Clin Infect Dis 2009;48:713–21. [10] Cytrynska M, Mak P, Suder P, Jakubowicz T. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides 2007;28:533–46. [11] Destoumieux-Garzon D, Saulnier D, Garnier J, Jouffrey C, Bulet P, Bachere E. Crustacean immunity: antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge. J Biol Chem 2001;275:47070–7. [12] Destoumieux D, Munoz M, Bulet P, Bachere E. Penaeidins, a family of antimicrobial peptides from penaeid shrimp. Cell Mol Life Sci 2000;57:1260–71. [13] Dimarcq JL, Keppei E, Dunabar B, Lambert J, Reichhart JM, Hoffman D, et al. Purification and characterisation of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino-acid sequence of the predominant member, diptericin A. Eur J Biochem 1988;171:17–22. [14] Dolashka-Angelova P, Velkova L, Shishkov S, Kostova K, Dolashki A, Dimitrov I, et al. Glycan structures of the structural subunit RvH2 of Rapana venosa hemocyanin and its effect on HSV type 1 virus. Carbohydr Res 2010;345:2361–7. [15] Dolashka-Angelova P, Schwarz H, Dolashki A, Beltramini M, Salvato B, Schick M, et al. Oligomeric stability of Rapana venosa hemocyanin (RvH) and its structural subunits. Biochim Biophys Acta 2003;1646:77–85. [16] Dolashka P, Genov N, Pervanova K, Voelter W, Geiger M, Stoeva S. Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits. Biochem J 1996;315:139–44. [17] Dolashka-Angelova P, Stefanovic S, Dolashki A, Devreese B, Tzvetkova B, Voelter W, et al. A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin. Arch Biochem Biophys 2007;459(1):50–8. [18] Dolashki A, Velkova L, Atanasov B, Hristova R, Voelter W, Stevanovic S, et al. Reversibility and “pH-T phase diagrams” of Rapana venosa hemocyanin and its structural subunits. BBA 2008;1784(11):1617–24. [19] Gonzalez M, Gueguen Y, Desserre G, Lorgeril J, Romestand B, Bachère E. Génome molecular characterization of two isoforms of defensin from hemocytes of the oyster Crassostrea gigas. Dev Comp Immunol 2007;31(4):332–9. [20] Hancock REW, Chapple D. Peptide antibiotics. Antimicrob Agents Chemother 1999:1317–23. [21] Hancock REW, Brown KL, Mookherjee N. Host defence peptides from invertebrates – emerging antimicrobial strategies. Immunobiology 2006;211:315–22. [22] Iijima R, Kisugi J, Yamazaki M. A novel antimicrobial peptide from the sea hare Dolabella auricularia. Dev Comp Immunol 2003;27:305–11.

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