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J. Biol. Chem., Vol. 281, Issue 30, 20983-20992, July 28, 2006
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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 |
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-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 |
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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 |
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Isolation and Purification of LatarcinsThe 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 SpectrometrySeparated 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
-cyano-4-hydroxycinnamic acid (10 mg/ml in 50% acetonitrile with 0.1% trifluoroacetic acid) matrix.
Amino Acid Sequence AnalysisN-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 SynthesisLatarcins 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 DichroismCD 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 AssaysBacteria (Arthrobacter globiformis VKM Ac-1112, Bacillus subtilis VKM B-501, Escherichia coli DH5
, 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 AssaysThe 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 AssaysCrude 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 AssaysLatarcins 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 |
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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 SynthesisPurified 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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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
-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.
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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
-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.
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-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%
-helix), which is unusual for short linear peptides. Biological Activities of LatarcinsLatarcins 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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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 StudiesSynthetic 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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| DISCUSSION |
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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 ComponentsThe 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
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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 ActionIt 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.
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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
-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
-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
-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 |
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* 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. ![]()
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. ![]()
3 By classification of Zonstein and Ovtchinnikov, 1999. ![]()
4 S. A. Kozlov, A. A. Vassilevski, A. V. Feofanov, A. Y. Surovoy, D. V. Karpunin, and E. V. Grishin, unpublished data. ![]()
| ACKNOWLEDGMENTS |
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