Latarcins, Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) That Exemplify Biomolecular Diversity

Sergey A Kozlov,Alexander A Vassilevski,Alexei V Feofanov,Andrey Y Surovoy,Dmitry V Karpunin,Eugene V Grishin

doi:10.1074/jbc.m602168200

Sergey A Kozlov, Alexander A Vassilevski + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m602168200

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Abstract

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.

Highlights

Venoms of a vast number of animal species represent complex mixtures of compounds that exert various functions, eventually leading to a common goal: fast paralysis/death or sharp pain
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
We 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

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Chemicals were obtained from Fluka Chemie GmbH (Deisenhofen, Germany), Sigma, and Chimmed (Moscow, Russia); all solvents were analytical grade. 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 ϫ 150 mm, 100 Å, 3 ␮m; 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 ␮l/min. Synthetic peptides were purified by preparative RP-HPLC on a Diasorb C16 column (250 ϫ 25 mm, 7 ␮m; 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. If the membrane remained intact and no current was detected, higher peptide concentrations were tested

RESULTS

Mass difference

DISCUSSION

Minimal effective conc