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Originally published In Press as doi:10.1074/jbc.M602168200 on May 30, 2006

J. Biol. Chem., Vol. 281, Issue 30, 20983-20992, July 28, 2006
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Latarcins, Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) That Exemplify Biomolecular Diversity*

Sergey A. Kozlov1, Alexander A. Vassilevski, Alexei V. Feofanov, Andrey Y. Surovoy, Dmitry V. Karpunin, and Eugene V. Grishin

From the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya, 16/10, 117997 Moscow, Russia

Received for publication, March 7, 2006 , and in revised form, April 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Seven novel short linear antimicrobial and cytolytic peptides named latarcins were purified from the venom of the spider Lachesana tarabaevi. These peptides were found to produce lytic effects on cells of diverse origin (Gram-positive and Gram-negative bacteria, erythrocytes, and yeast) at micromolar concentrations. In addition, five novel peptides that share considerable structural similarity with the purified latarcins were predicted from the L. tarabaevi venom gland expressed sequence tag data base. Latarcins were shown to adopt amphipathic {alpha}-helical structure in membrane-mimicking environment by CD spectroscopy. Planar lipid bilayer studies indicated that the general mode of action was scaled membrane destabilization at the physiological membrane potential consistent with the "carpet-like" model. Latarcins represent seven new structural groups of lytic peptides and share little homology with other known peptide sequences. For every latarcin, a precursor protein sequence was identified. On the basis of structural features, latarcin precursors were split into three groups: simple precursors with a conventional prepropeptide structure; binary precursors with a typical modular organization; and complex precursors, which were suggested to be cleaved into mature chains of two different types.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the course of evolution, venomous animals developed highly specialized and sophisticated strategies that basically serve prey capture and/or aggressor deterrence. It is believed that elaboration of venom organs provided for drastic increase in hunting and defense efficiency. The molecular basis of such efficiency was formed by fine-tuning venom composition. A number of strategies were suggested to have been employed in the process of venom content optimization: increase in the number of functionally diverse compounds, usage of highly selective and effective molecules, and synergism between compounds (1).

Venoms of a vast number of animal species represent complex mixtures of compounds (ions, biogenic amines, polyamines, polypeptide neurotoxins, cytolytic peptides, enzymes, etc.) that exert various functions (ion misbalance, increased cellular and tissue barrier permeability, extracellular matrix and cell membrane disturbance, cytolysis, necrosis, and myo- and neurotoxicity), eventually leading to a common goal: fast paralysis/death or sharp pain. Synergism of action is thus an important characteristic inherent to venom components. Important clues to unraveling synergistic effects between different venom compounds have been provided (2-5).

We believe that another marked trend common to venomous animals from diverse taxonomic groups is the unique biomolecular diversity of venom polypeptide components. In some cases (Conus peptides are an obvious example), polypeptide molecules constitute what may be called naturally edited combinatorial libraries (6), i.e. a significant number of homologous molecules are simultaneously present in the same venom. Structurally important residues (mostly cysteines) that define the overall molecular fold remain conserved, whereas other residues are subject to change and selection so as to form different functional assemblies and provide functional diversity. In spiders, this trend is exemplified by neurotoxins belonging to the inhibitor cystine knot structural motif (7-9).

We suggest that the same strategy works with venom cytolytic and antimicrobial peptides (AMPs),2 i.e. a wide range of structurally related homologous and non-homologous molecules aimed to exert lytic action against cells of different origin are simultaneously present in the venom to ensure efficiency. Here, we report the isolation and characterization of seven short linear cysteine-free antimicrobial and cytolytic peptides designated latarcins from the venom of the spider Lachesana tarabaevi. We also report the results of L. tarabaevi venom gland expressed sequence tag (EST) data base analysis, which enabled us to identify five additional putative lytic peptides and to characterize precursor protein structures for every latarcin. We believe that latarcins are an example of the marked biomolecular diversity of venom cytolytic peptides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Chemicals were obtained from Fluka Chemie GmbH (Deisenhofen, Germany), Sigma, and Chimmed (Moscow, Russia); all solvents were analytical grade. N-(9-Fluorenyl)methoxycarbonyl (Fmoc)-protected amino acids were purchased from Novabiochem.

Isolation and Purification of Latarcins—The venom of the Central Asian spider L. tarabaevi (lyophilized powder) was purchased from Fauna Laboratories, Ltd. (Almaty, Republic of Kazakhstan). Crude venom (collected from several spiders) containing 1 mg of total protein was fractionated by reversed-phase high performance liquid chromatography (RP-HPLC) on a Jupiter C5 column (2 x 150 mm, 300 Å, 5 µm; Phenomenex). A short (35 min) linear gradient of acetonitrile (0-70%, v/v) in 0.1% (v/v) aqueous trifluoroacetic acid at a flow rate of 0.3 ml/min was used. Eluent absorbance was monitored at 210 nm. Fractions with antimicrobial activity were further separated on an Ascentis RP-amide column (2.1 x 100 mm, 3 µm; Supelco). The separation was carried out using a 40-min linear gradient of acetonitrile (20-60%, v/v) in 0.1% (v/v) aqueous trifluoroacetic acid at a flow rate of 0.3 ml/min. Final purification of active compounds was performed on a Luna C18 column (1 x 150 mm, 100 Å, 3 µm; Phenomenex), and peptides were eluted with a linear gradient of acetonitrile (20-60%, v/v) in 0.1% (v/v) aqueous trifluoroacetic acid for 40 min at a flow rate of 50 µl/min.

Mass Spectrometry—Separated fractions and crude venom composition were analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (MS). M@LDI LR (Micromass UK Ltd.) and Ultraflex TOF-TOF (Bruker Daltonik GmbH, Bremen, Germany) instruments were used. Calibration was performed using either a ProteoMass peptide and protein MALDI-MS calibration kit (mass range of 700-66,000 Da) or a ProteoMass peptide MALDI-MS calibration kit (mass range of 700-3500 Da) (both from Sigma). Molecular masses were determined in linear or reflector positive ion mode using samples prepared by the dried droplet method with a 2,5-dihydroxybenzoic acid (10 mg/ml in 70% acetonitrile with 0.1% trifluoroacetic acid) or {alpha}-cyano-4-hydroxycinnamic acid (10 mg/ml in 50% acetonitrile with 0.1% trifluoroacetic acid) matrix.

Amino Acid Sequence Analysis—N-terminal sequencing was carried out by automated stepwise Edman degradation using a Procise Model 492 protein sequencer (Applied Biosystems) according to the manufacturer's protocol.

Peptide Synthesis—Latarcins were synthesized in a stepwise manner by a solid-phase method using Fmoc/t-butyl chemistry. Fmoc-protected amino acids (10-fold excess) were coupled by in situ activation using N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazol for 2-4 h. The Fmoc-protecting group was removed by treatment with piperidine/N,N-dimethylformamide (1:4, v/v; twice for 20 min). The following side chain protections were used: t-butyl ether for Ser, Thr, and Tyr t-butyl ester for Asp and Glu; trityl for Asn, Gln, and His; t-butyloxycarbonyl for Lys and Trp; and 2,2,4,6,7-pentamethyldehydrobenzofuran-5-sulfonyl for Arg. Peptide carboxyls were synthesized on a trityl chloride resin (0.8 mmol/g), whereas peptide amides were synthesized on a Rink amide (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy) resin (0.51 mmol/g) (both from PepChem, Reutlingen, Germany). The resin was washed with N,N-dimethylformamide before usage as well as after each deprotection and coupling step. Peptide cleavage from the resin and side chain deprotection were carried out using trifluoroacetic acid/ethanedithiol/H2O (95:2.5:2.5) for 4 h at room temperature. The resin was then filtered, and the crude peptides were precipitated by adding cold diethyl ether (-20 °C). The precipitated peptides were collected by centrifugation and resuspended in cold diethyl ether (repeated twice). Finally, the peptides were dissolved in t-butyl alcohol/H2O (4:1, v/v) and lyophilized.

Synthetic peptides were purified by preparative RP-HPLC on a Diasorb C16 column (250 x 25 mm, 7 µm; BioChemMack, Moscow, Russia), which was eluted with linear gradient of acetonitrile (0-60%, v/v) in 0.1% (v/v) aqueous trifluoroacetic acid for 60 min at a flow rate of 10 ml/min. 99% purity of synthetic latarcins was achieved, which was verified by HPLC on a Luna C18 column (1 x 150 mm, 100 Å, 3 µm) and by MALDI-MS.

Circular Dichroism—CD spectra were obtained on a Jasco J-810 spectropolarimeter. The spectra were measured between 190 and 250 nm (0.2-nm step) at 20 °C. Latarcins were dissolved in water and 50% (v/v) trifluoroethanol; the concentrations of the peptides were 0.16 and 0.08 mM, respectively. A 0.01-cm path length cell (quartz SUPRASIL) with a detachable window (Hellma GmbH & Co. KG, Müllheim, Germany) was used. Base-line spectra were measured for water or 50% trifluoroethanol solution and subtracted from the corresponding peptide spectra. Data were analyzed using the CONTINLL program as described (10).

Antimicrobial Assays—Bacteria (Arthrobacter globiformis VKM Ac-1112, Bacillus subtilis VKM B-501, Escherichia coli DH5{alpha}, E. coli MH1, and Pseudomonas aeruginosa PAO1) were cultured in low salt LB broth, whereas yeast cells (Pichia pastoris GS115 and Saccharomyces cerevisiae Y190) were cultured in yeast extract/peptone/dextrose medium. Determination of minimal inhibitory concentrations (MICs) for latarcins was performed using a 2-fold microtiter broth dilution assay in 96-well sterile plates at a final volume of 100 µl. Mid-log phase cultures were diluted to a final concentration of 1 x 105 colonyforming units/ml. Dried HPLC fractions or pure peptides were dissolved in 10 µl of water and added to 90 µl of the bacterium/yeast dilution. The peptides, a non-treated control, and a sterility control were tested in triplicate. The microtiter plates were incubated for 24 h at 37 °C (bacteria) or for 48 h at 30 °C (yeast); growth inhibition was determined by measuring absorbance at 620 nm. MICs are expressed as the lowest concentration of peptides that caused 100% growth inhibition.

Hemolytic Assays—The hemolytic activity of latarcins was determined using fresh rabbit blood. 2 ml of blood was mixed with Hanks' balanced salt solution (8 ml) containing heparin (10 units/ml) and centrifuged at 200 x g for 5 min. The pellet was resuspended in Hanks' balanced salt solution to a final concentration of 2 x 107 cells/ml. 1 ml of diluted cells was incubated with peptides at various concentrations for 3 h at 37°C under gentle stirring. The samples were then centrifuged at 4000 x g for 5 min. Hemoglobin release was monitored by measuring the absorbance of the supernatant at 414 nm. The negative control (0% hemolysis) was 1 ml of diluted cells incubated without the peptides, and the positive control (100% hemolysis) was 1 ml of cells in distilled water.

Insecticidal Assays—Crude venom and synthetic peptides were assayed by injection into early third instar Musca domestica larvae (weight of ~60 mg). Samples were dissolved in physiological saline (140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 4 mM NaHCO3, and 5 mM HEPES (pH 7.2)), and a volume of 2 µl was injected into the fourth segment of the larva. Controls received pure saline. Paralytic and lethal effects were monitored for up to 24 h after the injection.

Planar Lipid Bilayer Membrane Assays—Latarcins were tested for ability to cause conductance changes in planar bilayer membranes. Bilayers were formed (painted) using the technique of Mueller et al. (11) across a 200-µm aperture in a horizontal Teflon septum separating two 2-ml chambers of the experimental cell. Membranes were formed from lipid solution in decane (20 mg/ml). Diphytanoylphosphatidylcholine (DPhPC) and a mixture of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG) (7:3, w/w) were used to mimic eukaryotic and prokaryotic plasma membranes, respectively. Electrical measurements were performed at room temperature using Ag/AgCl electrodes in 100 mM KCl solution buffered with 10 mM HEPES (pH 7.0). Peptides dissolved in distilled water (1 mg/ml) were added to the upper (cis) chamber. Current across the membrane was measured with an Axopatch 200B amplifier (Axon Instruments) and digitized with an L-780 ADC (L-Card Ltd., Moscow, Russia). The cis-chamber was referred to as ground, whereas the trans-chamber was clamped at holding potentials relative to the cis-chamber. For each of the peptides, the starting concentration was 1.5 µg/ml. Voltage across the membrane was clamped at 100 mV for 10 min, and then negative voltages were applied starting from -20 mV with 10-mV steps until -100 mV was reached. Each step lasted 30 s. If the membrane remained intact and no current was detected, higher peptide concentrations were tested.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AMP Identification and Separation—The venom of the spider L. tarabaevi3 from the Zodariidae family was initially shown to possess comparatively high antimicrobial activity against E. coli (MIC = 0.05 mg/ml). For this reason, it was chosen as the biological source of putative novel antimicrobials.

To isolate individual active components, crude venom was subjected to fractionation by RP-HPLC. The fractions obtained and the crude venom composition were analyzed by MALDI-MS (Fig. 1). L. tarabaevi venom was found to contain ~100 components that could be divided into two roughly equal groups according to their measured molecular masses (2-5 and 7-9 kDa).

During one-step RP-HPLC separation on a C5 column (Fig. 1), all antimicrobial components active against E. coli eluted with a relatively high percent of acetonitrile. Active polypeptide compounds with molecular mass in the 2-5-kDa range were further purified to homogeneity on an Ascentis RP-amide column (data not shown).

In total, seven AMPs were isolated from L. tarabaevi venom and named latarcins (Ltc 1, Ltc 2a, Ltc 3a, Ltc 3b, Ltc 4a, Ltc 4b, and Ltc 5). For each of the latarcins, chromatographic peak area was calculated, and its relative abundance in the venom was thus estimated. We found that latarcins could be ascribed to low content venom components (0.1-0.5% of total protein).

AMP Structure Determination and Synthesis—Purified latarcins were subjected to automatic Edman degradation, and their full amino acid sequences were determined (Table 1). The observed difference of 1 Da between theoretical and measured molecular masses allowed us to establish C-terminal amidation for Ltc 3a, Ltc 3b, Ltc 4a, Ltc 4b, and Ltc 5.


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  		TABLE 1
Amino acid sequences of latarcins isolated from the venom of L. tarabaevi

 
As seen from their amino acid sequences, latarcins represent short linear cationic amphiphilic polypeptides. Ltc 3a and Ltc 3b differ by one residue at position 3 (bearing Lys or Ala, respectively); Ltc 4a and Ltc 4b differ by two residues at positions 1 (Gly or Ser, respectively) and 6 (Phe or Val, respectively). Except for these two pairs, latarcins show negligible levels of homology between each other. Thus, five structurally different groups of AMPs were found to be simultaneously present in the venom of L. tarabaevi. Multiple sequence alignment using ClustalW (available at www.ebi.ac.uk/clustalw/) was performed (Fig. 2B) and revealed some degree of sequence similarity between Ltc 1 and Ltc 3a/3b (53% identity) and between Ltc 3a/3b and Ltc 4a/4b (~50% identity).

Latarcins are strongly cationic molecules: pI >10, and the net charge at pH 7 varies from +5 for Ltc 3b to +10 for Ltc 5. The distribution of positively charged lysine residues with intervening bulky hydrophobic groups found in latarcin sequences is common to other AMPs. Such distribution allows the peptides to adopt marked amphipathic structures in an {alpha}-helical conformation. Clusters of hydrophobic and positively charged residues become obvious when the peptides are plotted as a helical net or wheel.

All newly identified AMPs were chemically synthesized to provide sufficient material for detailed functional analysis. Synthetic and naturally occurring peptides were proven to be identical by comparing their molecular masses, RP-HPLC elution profiles, and biological activities.

Analysis of the L. tarabaevi Venom Gland EST Data Base— We performed analysis of the L. tarabaevi venom gland EST data base (obtained in collaboration with DuPont Agriculture & Nutrition) with latarcin sequences as query and identified full precursor protein sequences (named pLtc x) for every latarcin as well as four new structurally similar sequences (Fig. 2).

Latarcins represent secreted polypeptide molecules, and putative leader sequences were identified using the SignalP 3.0 program (available at www.cbs.dtu.dk/services/SignalP/) in every precursor protein. Mature peptides correspond to the C-terminal parts of the respective precursors. Ltc 3a, Ltc 3b, Ltc 4a, Ltc 4b, and Ltc 5, which were shown to be C-terminally amidated, all bear an additional C-terminal glycine in their precursor sequences, which provides additional evidence of the suggested post-translational modification. The Ltc 1 precursor has an additional C-terminal lysine residue that is suggested to be cleaved off post-translationally. Precursors of homologous peptides Ltc 3a/3b and Ltc 4a/4b share a high level of identity: pLtc 3a differs from pLtc 3b by one residue in the mature chain, and pLtc 4b differs from pLtc 4a-1 by 12 residues. For Ltc 4a, we found two related precursor sequences (pLtc 4a-1 and pLtc 4a-2) that differ by one repeat of a 28-residue block.


Figure 1
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  		FIGURE 1.
Mass spectrum and chromatographic separation profile of the venom of L. tarabaevi. The mass spectrum of crude venom recorded in linear mode using a 2,5-dihydroxybenzoic acid matrix is shown in the inset. The intensity of peaks above 3500 Da is magnified 10-fold. Peaks that correspond to short AMPs are indicated by asterisks. Crude venom was separated by RP-HPLC on a Jupiter C5 column using a linear gradient of acetonitrile. Fractions containing short AMPs are indicated by arrows.

 


Figure 2
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  		FIGURE 2.
Structures of latarcin precursors. A, sequence organization of simple, binary, and complex precursors; B, aligned sequences of mature latarcins; C, aligned sequences of RPEs. PQMs are underlined; similar amino acid residues in the aligned sequences are shown in boldface; and post-translationally cleaved fragments identified for latarcins and predicted for RPEs are enclosed in parentheses.

 
We also found a close homolog of Ltc 2a that differs only by the C-terminal residue (Asn instead of His) and three additional sequences that share the overall structural organization with latarcins. The peptides were by analogy named latarcins: Ltc 2b for the Ltc 2a homolog and Ltc 6a and Ltc 7 for the new unrelated structures (Fig. 2B). Full precursor protein sequences were also identified for the new peptides (Fig. 2). Note that pLtc 6-1 and pLtc 6-2 are close homologs (pLtc 6-2 differs from pLtc 6-1 by two residues) and that both serve as precursors for Ltc 6a.

The newly identified putative mature peptides Ltc 6a and Ltc 7 have negligible similarity (<20% identity) but share common characteristics with other latarcins, i.e. they represent short linear peptides with a net positive charge that may adopt amphipathic structures in an {alpha}-helical conformation. For this reason, Ltc 6a and Ltc 7 were chemically synthesized to investigate their possible functional activities.

On the basis of structural organization features, latarcin precursor proteins may be subdivided into three groups (Fig. 2). Simple precursors have only one motif that specifies the propeptide-processing site. This site (X1X2X3R, where any of Xn = E) was initially identified in precursors of spider ion channel-blocking toxins and was described previously as the processing quadruplet motif (PQM) (12). A simple latarcin precursor follows a structural organization scheme (prepropeptide) common to a wealth of secreted molecules and consists of a typical signal peptide, a rather extended acidic fragment (prosequence) that is removed post-translationally by cleavage at the PQM to finally yield the mature chain (latarcin) that corresponds to the C-terminal part of the precursor.

Binary precursors differ from simple precursors by having two PQMs. Therefore, we suggest that these proteins give rise to two mature peptides each. By analogy with simple precursors, we named the C-terminal putative mature chain common to both binary precursors Ltc 6a. The preceding sequences enclosed between two PQMs in pLtc 6-1 and pLtc 6-2 were found to share ~70% identity with Ltc 6a and were named Ltc 6b and Ltc 6c, respectively. Ltc 6c differs from Ltc 6b by one residue at position 30 (Lys instead of Arg). Thus, each of the binary precursors has a modular structure and seems to be processed post-translationally into two highly homologous mature peptides.

Complex precursors have five (pLtc 4a-1 and pLtc 4b) or six (pLtc 4a-2) PQMs and are suggested to be cleaved into five or six mature peptides, respectively. The C-terminal parts of precursor proteins correspond to mature latarcins. Sequences enclosed between PQMs are organized into highly homologous tandem repeats named repetitive polypeptide elements (RPEs), which do not share homology with mature latarcins (Fig. 2C). For this reason, complex precursors are suggested to be cleaved post-translationally to yield two types of mature chains that may have different functions. In total, 12 precursor protein sequences yielding 19 mature linear peptides (12 putative) were deduced from the L. tarabaevi venom gland EST data base.


Figure 3
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  		FIGURE 3.
CD spectra of synthetic latarcins in water (solid lines) and in 50% trifluoroethanol (dashed lines). A, Ltc 1 ({diamondsuit}) and Ltc 2a ({diamond}); B, Ltc 3a ({blacksquare}) and Ltc 3b ({square}); C, Ltc 4a (•) and Ltc 4b ({circ}); D, Ltc 5 ({triangleup}), Ltc 6a ({blacktriangleup}), and Ltc 7 ({star}). deg, degree.

 
CD Spectra—All synthetic latarcins were analyzed by CD spectroscopy to investigate their secondary structure in pure water and in membrane-mimicking 50% trifluoroethanol solution (Fig. 3). The results of secondary structure element content estimation by CD spectroscopy are summarized in Table 2.


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  		TABLE 2
Secondary structures of latarcins as estimated by CD spectroscopy

 
Generally, similar to other well documented linear AMPs, latarcins assume a random coil structure in water and form {alpha}-helices in 50% trifluoroethanol, which is viewed as an indication of their high helix-forming propensities (13). Ltc 3a and Ltc 3b were shown to be peculiar; they become partially spiralized in water (>30% {alpha}-helix), which is unusual for short linear peptides.

Biological Activities of Latarcins—Latarcins show pronounced antimicrobial effects against a number of Gram-positive (A. globiformis and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacterial strains in vitro. The corresponding MICs are listed in Table 3. In general, latarcins show MICs in the low micromolar range. Among the bacterial species tested, P. aeruginosa turned out to be the most resistant, which was frequently observed previously for other AMPs (14). The growth of these bacteria was still effectively inhibited by Ltc 1, Ltc 2a, and Ltc 5. We have also proved that a bactericidal and not simply a bacteriostatic effect is achieved, although usually at higher concentrations than the MIC (data not shown). Moreover, the antibacterial action was significantly lower (4-20 times) in the presence of divalent cations (20 mM CaCl2), which agrees with the proposed mechanism of cationic AMP action that implies electrostatic binding to the negatively charged target membrane surface (15). Latarcins were tested for antifungal activity against yeast cells (P. pastoris and S. cerevisiae), which were found to be much more resistant than bacteria, but their growth was still inhibited by higher concentrations of peptides (Table 3).


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  		TABLE 3
Antimicrobial and hemolytic activities of latarcins

 
Tests to determine the hemolytic activity of latarcins were performed and showed that erythrocytes are highly resistant to Ltc 3a, Ltc 3b, Ltc 4a, Ltc 4b, Ltc 6a, and Ltc 7 (Table 3). Ltc 2a exhibited comparatively strong hemolytic activity, whereas Ltc 1 and Ltc 5 were moderately hemolytic. Ltc 6a and Ltc 7 (identified as putative AMPs) showed neither antimicrobial nor hemolytic activity.

Latarcins did not cause lethal effects on house fly larvae when applied individually (10 mg/kg) or in a mixture (10 mg/kg for each of the peptides). However, paralysis, appearance of necrotic spots, and liquid flow out of the injection opening were observed.

Planar Bilayer Membrane Studies—Synthetic latarcins were studied for effects on the permeability of planar bilayer membranes that mimic zwitterionic eukaryotic (DPhPC) and anionic prokaryotic (DOPE/DOPG, 7:3) plasma membranes (Table 4). In general, the more biologically active peptides showed lower effective concentrations on the corresponding model membranes. Most of the peptides induced membrane breakdown at micromolar concentrations and negative potentials. However, Ltc 1 and Ltc 7 caused some conductance changes in membranes formed by DOPE/DOPG and DPhPC, respectively, without membrane rupture. We note that Ltc 6a and Ltc 7 were found to cause changes in model membrane permeability; therefore, we suggest a cytolytic function for these peptides with a biological target that might differ substantially from those tested in this work.


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  		TABLE 4
Disruption of planar bilayer membranes by latarcins Minimal peptide concentrations that caused conductance changes under negative voltages (-80/-100 mV) are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Biomolecular Diversity of Latarcins—This study represents a vivid example of an arguably common evolutionary trend of venom-producing animals to select for biomolecular diversity of venom polypeptide components. The newly identified L. tarabaevi venom peptides named latarcins form seven new structurally unrelated groups of membrane-active molecules. Seven latarcins from five structural groups that show pronounced antimicrobial and cytolytic effects against cells of different origin were found to be simultaneously present in the same venom. We note that the highly homologous peptides Ltc 2a/2b, Ltc 3a/3b, Ltc 4a/4b, and Ltc 6b/6c may either be coexpressed in one spider or represent an example of intraspecific differences because a mixture of venom from several spiders was used in this study and venom glands from several spiders were used previously to obtain the EST data base.

The specificity profiles of latarcins can be presented graphically (Fig. 4). Ltc 3a, Ltc 3b, Ltc 4a, and Ltc 4b are more active against Gram-positive bacteria than against the other cell types tested. Ltc 3b shows reduced antimicrobial activity compared with Ltc 3a. This is due to a Lys-Ala substitution at position 3 that brings about positive charge reduction. Similar effects have been documented for other AMPs (16). In contrast, Ltc 4a and Ltc 4b show very similar MICs, indicating that the observed differences in their amino acid sequences (Gly and Ser at position 1, respectively, and Phe and Val at position 6, respectively,) play a minor, if any, functional role. Ltc 1, Ltc 2a, and Ltc 5 seem to be rather nonspecific and are active against cells of diverse origin; Ltc 2a is the least specific and the most active peptide identified so far. In effect, we suggest that application of a peptide mixture consisting of all identified latarcins would produce lytic action on virtually any cell type.

We believe that L. tarabaevi has evolved to produce an array of structurally similar but unrelated peptides that serve a common goal: target cell membrane destabilization and cell lysis. A close example of biomolecular diversity is demonstrated by the ant Pachycondyla goeldii venom peptides called ponericins (17). These 15 short linear cationic peptides show distinct antimicrobial and insecticidal activity spectra and can be classified into three unrelated structural groups. Another example is formed by linear peptides from the scorpion Buthus martensii (18). In total, at least three unrelated structural groups of AMPs are simultaneously present in this scorpion venom; although a number of these peptides have not been functionally characterized, their pronounced homology to other AMPs suggests antimicrobial and cytolytic functions.

We also speculate that a common trend of structural diversification was followed in the case of host defense peptides, both inducible and constitutively expressed. Induction of the honeybee and Drosophila immune response leads to production of an array of peptides that belong to four and eight structurally distinct families, respectively (19-21). At least four groups of AMPs are simultaneously present in frog skin (22), and seven families of AMPs were found in wheat seeds (23).

Latarcins as Venom Components—The active role of antimicrobial and cytolytic peptides as effector molecules in the innate immunity of plants and animals is universally accepted (24). Bacteria and fungi also produce this kind of molecule to ensure competitive advantages over their rivals (25). On the other hand, venomous animals developed highly effective arsenals of potent chemical weapons that serve both offensive and defensive needs. The exact functional role of antimicrobial and cytolytic peptides in venom is still under question, although a number of possibilities have been suggested (26-28).

One possible function is a direct toxic effect on prey. This was suggested for a number of venom peptides, although the effective concentrations vary greatly: lycotoxins from the spider Lycosa carolinensis (Lycosidae) (28), ponericins from the ant P. goeldii (17), cupiennins from the spider Cupiennius salei (Ctenidae) (29), oxyopinins from the spider Oxyopes kitabensis (Oxyopidae) (2), and (originally) melittin from the honeybee Apis mellifera (30). L. tarabaevi is a night-hunting spider of a rather large average size (2.5 cm). It has a fast acting venom that is injected into prey in comparatively high doses (~1 µl). We tested the possible toxic effect of latarcins on house fly larvae, taking the calculated content of latarcins in the venom into consideration. At concentrations that seem to be found in vivo, latarcins caused larval paralysis and the appearance of necrotic spots, which were attributed to a direct cytolytic action of the peptides (1, 2). We suggest that latarcins add to the overall paralyzing and toxic effect of the crude venom. However, their contribution seems to be minor compared with the neurotoxic compounds.4


Figure 4
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  		FIGURE 4.
Graphical representation of latarcin activity spectra. Activities against Gram-positive (A. globiformis (first bars) and B. subtilis (second bars)) and Gram-negative (E. coli DH5{alpha} (third bars) and P. aeruginosa (fourth bars)) bacteria are represented as 1/MIC. Hemolytic activities (fifth bars) are represented as 10/(peptide concentration causing 20% erythrocyte lysis). Activities on planar bilayer membranes (DPhPC (sixth bars) and 7:3 DOPE/DOPG (seventh bars)) are represented as 1/(minimal effective concentration). The higher bars indicate stronger activity. See Tables 3 and 4 for exact values.

 
Another possibility is positive cooperativity with neurotoxins. By exerting a lytic effect, venom antimicrobial and cytolytic peptides may act as a spreading agent that facilitates and accelerates the passage of neurotoxin through cellular barriers and eventually ensures its access to the target neurons. Positive cooperativity with neurotoxins has been reported for cupiennins (29) and oxyopinins (2). In case of latarcins, we did not test this possibility because neurotoxic compounds from L. tarabaevi venom had not been isolated and biochemically characterized.

A direct antiseptic role has also been suggested for lycotoxins (28), ponericins (17), and cupiennins (29). L. tarabaevi spiders often form colonies with high population density; therefore, control of possible infection seems critical. Our calculations show that the quantities of latarcins injected into prey seem to be high enough to clear possible infectious microorganisms. Thus, we suggest that latarcins play a disinfecting and possibly conserving role in paralyzed prey. An additional function in venom gland protection is also under question.

Mode of Action—It is believed that the primary target of cationic antimicrobial and cytolytic peptides is the plasma membrane, although this turns out not to be true for at least some of the peptides (24). Three basic mechanisms of target cell membrane permeabilization have been proposed: barrel-stave pore formation, toroidal pore formation, and the so-called carpet-like mechanism (24). Whereas alamethicin was shown to form pores according to the barrel-stave model (31), cecropins were suggested to act in a detergent-like manner (carpet-like mechanism) (32); other antimicrobial and cytolytic peptides such as magainin and melittin were shown to form toroidal pores lined by both the peptides and lipid head groups (33, 34).

Our results obtained in planar bilayer experiments correlate with the lytic effects of latarcins produced on living cells and suggest potential-dependent detergent-like membrane disorganization consistent with the carpet-like mechanism for most latarcins. However, Ltc 1 and Ltc 7 seem to form pores in DOPE/DOPG and DPhPC membranes, respectively. Latarcins need negative potentials that closely resemble those found in living cells (up to -100 mV) to perform their membrane-destabilizing action. In contrast, at positive potentials (up to 100 mV), latarcins do not cause any membrane permeability changes. We may thus conclude that the mechanism of latarcin-induced membrane rupture is not simply a detergent-like facilitated breakdown, which is polarity-independent (35), but includes one or more crucial stages of electric field-driven peptide-membrane interactions.


Figure 5
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  		FIGURE 5.
Sequence alignments of latarcins with similar AMPs. CAP7 (NCBI accession number P25230) is from rabbit (37); dermaseptin B2 (DS B2), also known as adenoregulin (NCBI accession number P31107), is from leaf frog (38); and CRAMP-18 (NCBI accession number P51437) is from mouse (36). Identical residues are shaded, and similar residues are shown in boldface.

 
Structural Peculiarities—No significant homology to known sequences and only limited similarity to other AMPs were identified for latarcins (Fig. 5). Two of the three similar peptides are members of the cathelicidin family: an 18-residue fragment of the cathelicidin-related antimicrobial peptide (CRAMP-18) that retains antimicrobial activity from mouse (36) and CAP7, the primary antibacterial domain of CAP18 from rabbit (37). Dermaseptin B2, also known as adenoregulin, was purified from the arboreal frog Phyllomedusa bicolor and belongs to the dermaseptin family of AMPs (38). Ironically, the similarity of latarcins to AMPs from spiders (lycotoxins, cupiennins, and oxyopinins) was found to be considerably lower than that to cathelicidins and dermaseptins. We suppose that this is an illustration of convergent evolution of functional peptide structures. On account of the negligible homology to known sequences, we claim that 12 latarcins represent seven new structural groups of lytic peptides.

Although latarcins share a low level of homology with other AMPs from spiders, they can be described by similar motifs of repeated positively charged and hydrophobic residues. The distribution of lysine residues observed in latarcin sequences is similar to that found in oxyopinins, lycotoxins, and lycocitins (2, 28, 39) but is different from the repeats of four amino acid residues in cupiennins (29). On the basis of their structural traits, latarcins are placed in the fifth structural class of spider toxins according to the classification proposed previously (40).

As shown in Fig. 2, latarcin precursors can be split into three structurally diverse groups. Simple latarcin precursors represent conventional prepropeptide sequences. This type of precursor structure is usual for many gene-encoded antimicrobial and cytolytic peptides as well as for a wealth of other secreted peptide molecules, including most spider neurotoxins. The specific type precursor with a rather extended acidic propiece followed by the mature chain is also common to other AMPs such as insect defensins, mammalian {alpha}-defensins, dermaseptins, and hymenoptaecin (19, 22, 41, 42).

Each of the binary latarcin precursors contains two highly homologous putative mature chains separated by the cleavage sites. This type of modular structure is also found in precursors of apidaecins from the honeybee (up to 12 tandem repeats) (20), naegleriapores from the amoeboflagellate Naegleria fowleri (up to five repeats) (43), magainins from Xenopus laevis (six repeats) (44), and AMPs from the plant Impatiens balsamina (six repeats) (45).

Complex latarcin precursors have modular structures but seem to be processed into multiple mature peptides of two types because the tandem repeated sequences (RPEs) separated by the cleavage sites share no homology with latarcins. Examples of AMP precursors that are processed into mature chains of different types include precursors of cathelicidins (46), attacin C from Drosophila (47), and modular precursor proteins from the plants Macadamia integrifolia and Cucurbita maxima (48, 49).

In each case, a targeted endopeptidase cleavage is needed to remove the prosequence. Two positively charged residues usually serve as the signal for precursor cleavages (19). We have noticed that, in the case of prodermaseptins, however, the KR signal is always preceded by a glutamic acid residue (X1X2KR, where any of Xn = E) (22), whereas latarcin propeptides are processed at PQMs (X1X2X3R, where any of Xn = E). Thus, AMP propeptides from different organisms seem to have related processing sites.

All latarcin precursors have rather long prosequences that are extremely acidic (Asp and Glu residues account for up to 50% of the sequence length). The prosequences and mature latarcins are predicted to form {alpha}-helices according to Chou and Fasman (50) and Garnier et al. (51). Negatively charged residues in latarcin prosequence peptides tend to form clusters in the predicted {alpha}-helices, which may effectively interact with clusters of positively charged residues of the mature latarcin chains. We suppose that the functional role of the latarcin precursor prosequence is to neutralize the high positive charge of the mature peptide and thus prevent its interactions with proteins/lipids and inhibit its cytolytic activity during biosynthesis. These functions were also suggested previously for acidic prosequences in precursors of insect and mammalian defensins and plant thionins (42, 52, 53). Subsequent latarcin precursor processing in the venom gland results in mature chain separation and functional activation.

In contrast, the non-latarcin putative mature peptides (RPEs) derived from the same precursor proteins show a different kind of structure. RPEs may exhibit functions analogous to or different from those of latarcins. In conclusion, we believe that further insight into the biomolecular diversity of venom antimicrobial and cytolytic peptides will help to decipher AMP structure-function relationships and biogenesis and provide important clues to understanding the biology of venomous animals.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AM232689 [GenBank] -AM232700 [GenBank] .

* This work was supported in part by Russian Federation Federal Agency for Science and Innovations State Contract 02.467.11.3003 [EC] (4/20/2005), the Russian Foundation for Basic Research, and the Program of Cellular Biology Russian Academy of Sciences Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 7-495-336-6540; Fax: 7-495-330-7301; E-mail: serg{at}ibch.ru.

2 The abbreviations used are: AMP, antimicrobial peptide; EST, expressed sequence tag; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RP-HPLC, reversed-phase high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MICs, minimal inhibitory concentrations; DPhPC, diphytanoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; DOPG, dioleoylphosphatidylglycerol; PQM, processing quadruplet motif; RPEs, repetitive polypeptide elements. Back

3 By classification of Zonstein and Ovtchinnikov, 1999. Back

4 S. A. Kozlov, A. A. Vassilevski, A. V. Feofanov, A. Y. Surovoy, D. V. Karpunin, and E. V. Grishin, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank the DuPont Agriculture & Nutrition staff and especially Maureen Dolan, Will Krespan, Bill McCutchen, and Rafi Herrmann for cDNA library construction and sequencing.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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