ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 4, pp. 376–382. © Pleiades Publishing, Inc., 2007. Original Russian Text © A.A. Vassilevski, S.A. Kozlov, M.N. Zhmak, I.A. Kudelina, P.V. Dubovskii, O.Ya. Shatursky, A.S. Arseniev, E.V. Grishin, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 4, pp. 405–412.
Synthetic Analogues of Antimicrobial Peptides from the Venom of the Central Asian Spider Lachesana tarabaevi A. A. Vassilevskia,1, S. A. Kozlova, M. N. Zhmaka, I. A. Kudelinaa, P. V. Dubovskiia, O. Ya. Shaturskyb, A. S. Arsenieva, and E. V. Grishina a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine ul. Leontovicha 9, Kiev, 01601 Ukraine Received June 26, 2006; in final form, October 31, 2006
Abstract—Analogues of latarcins Ltc1 and Ltc3b, antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi capable of formation of amphiphilic structures in membranes without involvement of disulfide bonds, were synthesized. The amino acid sequences of the analogues correspond to immature forms of these peptides, each of them containing an additional C-terminal amino acid residue. It is concluded from the study of the biological activity of the synthesized peptides that the posttranslational C-terminal amidation of Ltc3b is a functionally important modification that ensures a high activity of the mature peptide. The lipid composition was shown to affect the interaction of synthesized peptides with artificial membranes. The analogue of Ltc3b manifested the highest activity on cholesterol-containing membranes. The mechanism of action of the studied antimicrobial peptides on membranes is discussed. Key words: amphiphilic helix, antimicrobial peptides, bilayer lipid membranes, liposomes, posttranslational modifications DOI: 10.1134/S1068162007040024
INTRODUCTION Living organisms of various systematic positions produce peptides with antimicrobial properties that are referred to as AMPs.2 AMPs have now been found practically in all the organisms studied [1, 2]. A large body of experimental data supports a concept that AMPs may serve as universal and evolutionally ancient defensive agents in protection of higher organisms against infection and as a weapon in competitive struggle between microorganisms [3]. The study of AMPs is of great practical importance, since they have a number of advantages over other antibiotics: the lack of toxicity to macroorganism cells, a broad spectrum of action including the strains resistant to other antibiotics, a low probability of appearance of AMP-resistant mutants. The latter fact, along with a 1
Corresponding author; phone: +7 (495) 336-6540; fax: +7 (495) 330-7301; e-mail:
[email protected]. 2 Abbreviations: AMP, antimicrobial peptide; BLM, bilayer lipid membrane; CBF, carboxyfluorescein; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; DOPG, dioleoylphosphatidylglycerol; MIC, minimal inhibiting concentration; TBTU, O-(benzotriazol-1-yl)-N,N,N ',N '-tetramethyluronium tetrafluoroborate; and TFE, trifluoroethanol.
decrease in the efficiency of conventional antibiotics with time, allows one to consider AMPs as a basis for designing drugs of a new generation [4]. Most AMPs have small molecular sizes and do not contain cysteine residues. These structural features significantly facilitate their production by chemical or biotechnological methods. Linear AMPs were isolated from intestines and blood of mammals, skin glands of amphibians, and hemolymph and venoms of arthropods [2, 5, 6]. The greatest diversity is characteristic of the linear AMPs from the venom of the Central Asian spider Lachesana tarabaÂvi. Seven AMPs called as latarcins have been found among them, and five more have been identified when analyzing translated nucleotide sequences [7]. The full amino acid sequences of mature chains of all the latarcins as well as the structures of protein precursors are known and have been published in the UniProt Database (http://www.pir.uniprot.org/) under the accession numbers Q1ELT9, Q1ELU0–Q1ELU5, Q1ELU7–Q1ELU9, and Q1ELV0. A comparison of the structures of mature latarcins isolated from the venom with those of protein precursors (obtained from the translated nucleotide sequences
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signal peptide
prosequence
377
mature chain
pLtc1 pLtc2a pLtc3b
–K –G
Fig. 1. A comparison of the amino acid sequences of protein precursors of latarcins 1, 2a, and 3b (pLtc1, pLtc2a, and pLtc3b, respectively) [7]. Similar amino acid residues are shown in boldface; identical residues, by grey color. An arrow indicates C-terminal amino acid residues (Lys in Ltc1 and Gly in Ltc3b) cleaved off on maturation but preserved in the sequences of synthesized analogues (Ltc1-K and Ltc3b-G).
and containing an N-terminal fragment cleaved off during processing) identified posttranslational modifications were found in some peptide molecules. The protein precursors of latarcins 3a, 3b, 4a, 4b, and 5 contain an additional C-terminal Gly residue, which is cleaved during maturation with simultaneous amidation of the preceding residue. Latarcin 1 in the form of protein precursor contains an additional C-terminal Lys residue, which is also split off during maturation. Both types of posttranslational modifications are widely occurring in the secreted polypeptides, including AMPs [8–10]. For studying the functional importance of the posttranslational modifications, we synthesized analogues of latarcins 1 and 3b with the sequences corresponding to the unprocessed forms of natural molecules and tested their biological activities. RESULTS AND DISCUSSION Chemical Synthesis of Latarcin Analogues Three amino acid sequences were chosen for the synthesis based on the information on posttranslational modifications of latarcins (Fig. 1). The sequence Ltc1-K corresponded to the structure of mature latarcin 1 elongated by a ë-terminal Lys residue. The peptide Ltc3b-G corresponded to latarcin 3b without an amide group at the ë-terminal Ala and elongated by a ë-terminal Gly. The third amino acid sequence Ltc2a totally corresponded to latarcin 2a and was chosen as a control. The chosen peptides were synthesized by the solidphase method in quantities of several milligrams; according to HPLC data, the content of impurities in the synthetic peptides did not exceed 1%. The correctness of amino acid sequences of synthesized peptides was confirmed by MS analysis: Ltc3b-G was characterized by average M 2483.7 Da (calculated value 2484.0 Da); Ltc1-K, 3201.7 Da (calculated value 3202.0 Da); and Ltc2a, 2902.2 Da (calculated value 2902.6 Da). Study of the Secondary Structure The secondary structures of all the synthesized peptides were characterized by CD spectroscopy to deterRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
mine the relative content of the secondary structure elements (Fig. 2 and Table 1). The CD spectra were measured in water and 50% TFE, which mimicked the membrane environment. The peptides had predominantly disordered structures in water. The α-helical structure (~40%) was found only in the case of Ltc3b-G, which is unusual for the linear peptides. The use of TFE for mimicking the membrane environment is a standard practice in AMP studies [11–14]. The spatial organization of most linear AMPs is significantly changed when they contact lipid structures (micelles, liposomes, membranes, etc), so that the peptides adopt the ordered conformation of α-helix [2]. All the synthesized peptides predominantly formed ordered α-helical structures in 50% TFE (Table 1). A comparison of the results for the synthesized peptides with the data available for respective mature latarcins [7] shows no significant differences in their secondary structures. This means that the posttranslational modifications under consideration do not produce significant changes in the secondary structures of latarcins. Table 1. The content (%) of secondary structure elements in peptides Ltc3b-G, Ltc1-K, and Ltc2a determined from the data of CD spectroscopy Peptide Conditions α-Helix β-Structure β-Turn Coil Ltc3b-G Water 50% TFE Ltc1-K
Water 50% TFE
Ltc2a
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40
13
21
26
76
2
4
18
7
28
22
43
67
4
7
22
6
30
23
41
69
4
7
20
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[Θ], degree cm2/dmol 50000 (a) 25000
0
1 2
–25000 40000 (b) 20000
0
1 2
–20000
(c)
50000 25000 0 1
2
–25000 –50000 180
190
200
210
220
230
240
250 nm
Fig. 2. CD spectra of peptides: a, Ltc3b-G; b, Ltc1-K; and c, Ltc2a: 1, in water; 2, in 50% aqueous TFE. Peptide concentration 0.1 mM. Cell width 0.01 cm.
Interaction of Peptides with Artificial Membranes It is assumed that most AMPs affect the cells due to their ability to interact with the cellular membranes and to cause their destabilization and breakdown. Several models were proposed to describe the mechanism of the action of AMPs on membrane [1]: formation of peptide pores by oligomerization of peptide molecules specifically oriented in the membrane; formation of toroidal lipid–peptide pores with involvement of not only
peptide but also lipid molecules; global perturbation of the membrane structure due to multiple surface binding of the peptide molecules and a so-called carpet-like mechanism. According to the previously published data, the mode of action of latarcins agrees with the carpet-like mechanism [7]. The membrane activity of synthesized peptides was tested on liposomes containing CBF in the internal aqueous cavity of liposomes at the concentration of 50 mM, which is optimal for the self-quenching of this dye. The leakage of CBF from liposomes into surrounding aqueous solution resulted in an enhancement of the sample fluorescence. The leakage was characterized by the parameter R (see the Experimental section). Figure 3 shows the experimental values of R as a function of peptide concentration. The ability of AMPs to bind to membranes depends on the membrane lipid composition [1, 2]. Therefore, each peptide was tested on liposomes of three different compositions. The liposomes prepared from zwitterionic DOPC mimicked the membranes of eukaryotic cells. The liposomes consisting of anionic DOPG represented the cell membrane of Gram-positive bacteria. The liposomes composed of a mixture of DOPG with zwitterionic DOPE modeled the cytoplasmic membrane of Gram-negative bacteria. One can see from Fig. 3 that, in fact, the effect of the peptides greatly depends on the liposome composition. All the peptides exhibited the highest activity on neutral zwitterionic DOPC liposomes. The activity of Ltc3b-G and Ltc2a decreased along with increase in the content of negatively charged lipids (DOPG) in liposomes. Unusual behavior demonstrated Ltc1-K, which was completely inactive in mixed DOPG–DOPE liposomes, showed a moderate activity in the liposomes from pure DOPG and was higly active on the liposomes consisting of pure DOPC. Note that the data obtained on liposomes do not correlate in some cases with the results of biological tests (see below), which may be caused by insufficient adequacy of the model system used. The AMP mechanism of action was studied in the experiments carried out on BLMs. Since the most pronounced effect of the peptides under investigation was observed on DOPC liposomes (Fig. 3), we chose the BLM composed of a 2 : 1 phosphatidylcholine–cholesterol mixture. In this model system, the peptides taken at a concentration range of 0.1–1 µM destabilized the membrane, which became apparent in membrane rupture in 5−20 min, as a rule, without preceding changes in ionic conductivity (Fig. 4‡, curve 1, 4b, and 4c; shown for the peptide concentrations of 0.5 and 1 µM). An increase in the peptide concentration resulted in a reduction of the BLM lifetime. For example, the membrane was ruptured under the action of 0.5 µM Ltc2a on average in 15 min and at 1 µM, in 7 min (Fig. 4c). On the other hand, the membrane retained its integrity at lower pep-
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tide concentrations (
