Peptides 32 (2011) 14771483

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Peptides
journal homepage: www.elsevier.com/locate/peptides

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, Soa 1113, Bulgaria
SME SYCO-PHARMA, OOD, Ltd. 47 Bregalnitza Str., 1303 Soa, Bulgaria
c
Institute for Cell Biology, University of Tuebingen, Germany
d
Interfacultary Institute of Biochemistry, University of Tbingen, Hoppe-Seyler-Strasse 4, D-72076 Tbingen, 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 rst time
we have explored the isolation, identication and characterisation of 11 novel antimicrobial peptides
produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultraltration 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 identied 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 t 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, lamentous fungi, protozoan and metazoan parasites [21,32,33]. Antimicrobial peptides
(AMPs) are important components of the non-specic 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 classied 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: pda54@abv.bg (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 puried from scorpion Centruroides
limpidus limpidus [8], while a new type of short antimicrobial
peptides, designated temporin-SHf, were identied 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, houseies (Musca domestica), Galleria
mellonella, female thick Amblyomma hebraeum AMPs which are
strong inhibitors against gram-negative bacteria were identied
[3,10,3436]. Several novel AMPs with antimicrobial activity were
also isolated from the body wall of the sea hare Dolabella auric-

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P. Dolashka et al. / Peptides 32 (2011) 14771483

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 uid 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 benet for the treatment of cancer
and viral or parasitic infections is suggested [6,7,11]. There is also
a signicant 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, identication and isolation of the active substances
and determination of their primary structures or DNA sequences
are of enormous importance to understand non-specic immune
response mechanism of mollusks and arthropods against pathogen
invasion and for the development of new biopharmaceutical concept and nally products.
In this study, we report for the rst 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 purication the
hemolymph was centrifuged in two steps: rst 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. Purication of peptides
The supernatant was concentrated and separated into several fractions using Millipore lters 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, Dren, 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 ow rate of
1.5 ml min1 . 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 ow rate of 1 ml min1 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 AutoexTM 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 NdYAG 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.
24 l of the puried peptide solutions was applied to the thinlayer plate and air-dried, taking care to restrict the size of the spot
to 23 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 uorescence 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 lter or absorption effects, were analyzed by a
spectrouorimeter (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 uorescence 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 , Soa, Bulgaria. The concentrations of the peptides
were determined spectrophotometrically as: Peptide 2242 g/ml,
Peptide 3158 g/ml, Peptide 4113 g/ml, Peptide 5189 g/ml,
Peptide 6171 g/ml, Peptide 7598 g/ml, Peptide 8108 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. Briey, 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 denite 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.

P. Dolashka et al. / Peptides 32 (2011) 14771483

1479

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 rst peptidomic study on new AMPs
originated from the hemolymph of molluscan Rapana snail.
3.1. Purication 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
lters. The obtained fractions: Fraction 1 (310 kDa), Fraction 2
(1030 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 puried 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 purication 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
14, Fig. 1) and six fractions with 50% ACN (Peaks 511, Fig. 1).
In some of these fractions a higher content of sugar was identied (Fig. 1, inset). For further purication the pre-puried 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 (080% ACN, 0.8% TFA) after rechromatography
of Fractions no. 8 (Fig. 2), 9, 10 and 11. Totally eleven pure peptides, numbering Peptides 111, were obtained from the Fractions
811 and further studied. The preliminary analyses for antibacterial
activity of the isolated fractions showed that four (Fractions 811)
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. Identication of the peptides
To identify and analyze the isolated eleven peptides, several methods and techniques were applied. The peptides were
identied 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 14 have short chains with masses between 3 and 4 kDa, while
the masses of four of AMPs (Peptides 811) were found to be in the

Fig. 1. RP-HPLC purication 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, Dren, Germany), equilibrated with 0.1% (v/v) triuoroacetic
acid/water. Elution (1.5 ml min1 ) 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, Dren, Germany), equilibrated with 0.1% (v/v) triuoroacetic 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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P. Dolashka et al. / Peptides 32 (2011) 14771483

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 AutoexTM 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 puried
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 200600 nm; (Insert) uorescence spectrum of peptide 8 in 50 mM Tris/HCl buffer, pH 8.0 at ex 295 nm and
the uorescence 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, uorescence 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 210220 nm and a smaller one
between 250 and 285 nm (Fig. 5A). The absorption at 210220 nm
is mainly due to the peptide bond while the absorption at
250285 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 uorescence properties (quantum yield and max ) of Peptide 8 were analyzed upon excitation at 295 nm by the uorescence
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 355360 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 195260 nm and not Cotton effect was observed (Fig. 5B) as well

10

15

20

Peptide 3

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

Peptide 2

Peptide 4

S L P P T L E E

Peptide 5

P P S E Q L G K

Peptide 6

P P P

P P N

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) 14771483

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 Triuoroethanol (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 signicant
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 ndings 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-

1481

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 111, were isolated from
this fraction and their molecular masses, carbohydrate content and
N-terminal sequences were determined.
Some of the peptides, like Peptides 14 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 identied in the hemolymph of the Rapana snails (Peptides
811). 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 AMPs 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 312 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 15, 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 95100% 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. Thats 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 disulde 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 disulde 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 conrmation 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 specic 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, Soa for his support and Ing. H. Stoyanov, Director
of Delta Industry AD, Sozopol, for providing the animals.
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