Imaging the membrane lytic activity of bioactive peptide latarcin 2a

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is a short linear antimicrobial and cytolytic peptide extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a adopts a helix-hinge-helix structure in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid structure, demonstrates stronger nonspecific interaction with the zwitterionic membrane, and is potentially more toxic against eukaryotic cells. In this work, interactions of these two ltc2a derivatives with supported "raft" lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholin/egg sphingomyelin/cholesterol 40/40/20mol%) were studied by in situ atomic force microscopy in order to investigate the potential anticancer activity of the peptides since some breast and prostate cancer cell lines contain higher levels of cholesterol-rich lipid rafts than non-cancer cells. Both peptides induced reorganization of the raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A induced membrane thinning likely due to membrane interdigitation. Formation of large pores by the peptides in the bilayer was observed. Cholesterol was found to attenuate membrane disruption by the peptides. Finally, leakage assay showed that both peptides have similar membrane permeability toward various model membrane vesicles.     

Synthetic analogues of antimicrobial peptides from the venom of the Central Asian spider <Emphasis Type="Italic">Lachesana tarabaevi</Emphasis>

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.     

Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid

Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin Ltc1K. These data suggest that the peptides, due to their high conformational lability, can vary structural and amphiphilic properties in the presence of PSA. As a result, various scenarios of the interaction of the peptides with membranes, whose surface is abundant with anionic polysaccharides, can take place. This can account for difficulties in understanding the structure-functional relationships in interactions of linear cationic peptides with biological membranes.     

Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.     

Haemolytic and cytotoxic action of latarcin Ltc2a

Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC₅₀ = 3.4 μM), leukocytes (EC₅₀ = 19.5 μM) and erythroleukemia K562 cells (EC₅₀ = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.     

Chaperone over-expression in Escherichia coli: Apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates

The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.     

Synthetic analogues of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi

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.     

Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity

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.     

Functional expression of spider neurotoxic peptide huwentoxin-I in E. coli

The coding sequence of huwentoxin-I, a neurotoxic peptide isolated from the venom of the Chinese spider Ornithoctonus huwena, was amplified by PCR using the cDNA library constructed from the spider venom glands. The cloned fragment was inserted into the expression vector pET-40b and transformed into the E. coli strain BL21 (DE3). The expression of a soluble fusion protein, disulfide interchange protein (DsbC)-huwentoxin-I, was auto-induced in the periplasm of E. coli in the absence of IPTG. After partial purification using a Ni-NTA column, the expressed fusion protein was digested using enterokinase to release heteroexpressed huwentoxin-I and was further purified using RP-HPLC. The resulting peptide was subjected to gel electrophoresis and mass spectrometry analysis. The molecular weight of the heteroexpressed huwentoxin-I was 3750.69, which is identical to that of the natural form of the peptide isolated from spider venom. The physiological properties of the heteroexpressed huwentoxin-I were further analyzed using a whole-cell patch clamp assay. The heteroexpressed huwentoxin-I was able to block currents generated by human Na_v1.7 at an IC₅₀ of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.     

Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

Summary A novel approach to the production of a human glucagon in E. coli is described. The 29 amino acids of human glucagon and pentapeptide linker containing enzyme processing site were fused at the amino terminus to a 57 residue N-terminal portion of the human tumor necrosis factor-alpha (hTNF-). The fusion protein was expressed in the E. coli cytoplasm at levels up to 30% of the total cell protein. Precipitation of the fusion protein near its isoelectric point, specific enterokinase cleavage at the linker site and subsequent HPLC purification makes this approach suitable for the production of glucagon as well as other relatively small peptides with therapeutic interests.     

Spatial structure and activity mechanism of a novel spider antimicrobial peptide

Latarcins (Ltc), linear peptides (ca. 25 amino acid long) isolated from the venom of the Lachesana tarabaevi spider, exhibit a broad-spectrum antibacterial activity, most likely acting on the bacterial plasmatic membrane. We study the structure-activity relationships in the series of these compounds. At the first stage, we investigated the spatial structure of one of the peptides, Ltc2a, and its mode of membrane perturbation. This was done by a combination of experimental and theoretical methods. The approach includes (i) structural study of the peptide by CD spectroscopy in phospholipid liposomes and by (1)H NMR in detergent micelles, (ii) determination of the effect on the liposomes by a dye leakage fluorescent assay and (31)P NMR spectroscopy, (iii) refinement of the NMR-derived spatial structure via Monte Carlo simulations in an implicit water-octanol slab, and (iv) calculation of the molecular hydrophobicity potential. The molecule of Ltc2a was found to consist of two helical regions (residues 3-9 and 13-21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. This is also supported by the analysis of hydrophobic/hydrophilic characteristics of Ltc2a and homologous antimicrobial peptides. These peptides exhibiting a helix-hinge-helix structural motif are characterized by a distinct and feebly marked amphiphilicity of their N- and C-terminal helices, respectively, and by a hydrophobicity gradient along the peptide chain. The approach we suggested may be useful in studying not only other latarcins but also a wider class of membrane-active peptides.     

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A₆₀₀  8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the ¹³CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.     

Preparation of cell-permeable Cre recombinase by expressed protein ligation

Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.     

High expression of a recombinant human calcitonin precursor peptide in Escherichia coli

Human calcitonin (hCT) is a C-terminus -amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus--amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli -galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.     

蜘蛛抗菌肽研究进展

蜘蛛活性多肽研究主要集中于蜘蛛毒液中作用于离子通道的神经毒素多肽。但近年来,一些蜘蛛抗菌肽不断被分离纯化,其结构和抗菌活性也被广泛深入研究,这将成为蜘蛛活性多肽研究领域的一个新热点。在蜘蛛毒液和血液中,存在不同种类的抗菌肽,其多肽长度、结构、抗菌作用各不相同。而且,有些抗菌肽甚至具有抗肿瘤作用。概述了蜘蛛抗菌肽在结构和功能方面的研究进展。     

Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli

A recombinant glutaryl-7-aminocephalosporanic acid acylase (GLA) from Pseudomonas N176 has been over-expressed in BL21(DE3)pLysS Escherichia coli cells. By alternating screenings of medium components and simplified factorial experimental designs, an improved microbial process was set up at shake-flask level (and then scaled up to 2L-fermentors) giving a 80- and 120-fold increase in specific and volumetric enzyme productivity, respectively. Under the best expression conditions, 1380 U/g cell and 16,100 U/L of GLA were produced versus the 18 U/g cell and the 140 U/L obtained in the initial standard conditions. Osmotic stress caused by the addition of NaCl, low cell growth rate linked to high biomass yield in the properly-designed rich medium, optimization of the time and the amount of inducer’s addition and decrease of temperature during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is significantly higher than any previously described production of GLAs. High volumetric production, cost reduction and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, makes this GLA an economic tool to be used in the 7-ACA industrial production.     

A general strategy for the bacterial expression of amyloidogenic peptides using BCL‐XL‐1/2 fusions

Biophysical studies on amyloidogenic and aggregation‐prone peptides often require large quantities of material. However, solid‐phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding ～50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL‐XL‐1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.     

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

Fusion expression of cecropin B-like antibacterial peptide in <Emphasis Type="Italic">Escherichia coli</Emphasis> and preparation of its antiserum

Antibacterial peptides have a broad range of antibacterial properties that makes them highly toxic for expression in Escherichia coli. For prepare an antiserum to detect these peptides, we developed a cecropin B mutant with a green fluorescent protein fusion partner resulting in high expression of a 37 kDa fusion peptide in E. coli with a yield of 7.9 mg/l culture medium after purification on Ni-IDA resin. Guinea pigs when immunized with the fusion peptide produced a specific antiserum which titers in excess of 1:25,600.