Structure–activity relationship of mastoparan analogs: Effects of the number and positioning of Lys residues on secondary structure,interaction with membrane-mimetic systems and biological activity

In this study, a series of mastoparan analogs were engineered based on the strategies of Ala and Lys scanning in relation to the sequences of classical mastoparans. Ten analog mastoparans, presenting from zero to six Lys residues in their sequences were synthesized and assayed for some typical biological activities for this group of peptide: mast cell degranulation, hemolysis, and antibiosis. In relation to mast cell degranulation, the apparent structural requirement to optimize this activity was the existence of one or two Lys residues at positions 8 and/or 9. In relation to hemolysis, one structural feature that strongly correlated with the potency of this activity was the number of amino acid residues from the C-terminus of each peptide continuously embedded into the zwitterionic membrane of erythrocytes-mimicking liposomes, probably due to the contribution of this structural feature to the membrane perturbation. The antibiotic activity of mastoparan analogs was directly dependent on the apparent extension of their hydrophilic surface, i.e., their molecules must have from four to six Lys residues between positions 4 and 11 of the peptide chain to achieve activities comparable to or higher than the reference antibiotic compounds. The optimization of the antibacterial activity of the mastoparans must consider Lys residues at the positions 4, 5, 7, 8, 9, and 11 of the tetradecapeptide chain, with the other positions occupied by hydrophobic residues, and with the C-terminal residue in the amidated form. These requirements resulted in highly active AMPs with greatly reduced (or no) hemolytic and mast cell degranulating activities.     

Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor

The potency of venom from Bracon hebetor against lepidopterous larvae has been known for over 40 years, but previous attempts to purify and characterize individual protein toxins have been largely unsuccessful. Three protein toxins were purified from venom of this small parasitic wasp and the amino acid sequences of 22–31 consecutive residues at the amino-terminus were determined. These relatively large toxins (apparent molecular mass 73 kDa) were labile under many isolation techniques, but anion-exchange chromatography allowed purification with retention of biological activity. Two purified toxins were quite insecticidal (LD₅₀ < 0.3μg/g) when injected into six species of lepidopterous larvae. On a molar basis, one toxin (Brh-I) has the highest known biocidal activity against Heliothis virescens (LD₅₀ = 2 pmol/g).     

The mastoparanogen from wasp

Mastoparans are a family of small peptides identified from the venom of hymenopteroid insects. Although they have been characterized as early as 1979, and so far are recognized as a leading biomolecule in potential drug therapy, their precursors, mastoparanogen, have still not been determined. In this paper, several mastoparans from the venom of the wasp Vespa magnifica (Smith) are reported. The cDNA of mastoparanogen is 236 base pairs in length, and encodes 40 amino acid residues, including a N-terminal acidic fragment and a C-terminal mature basic mastoparan, which contain multiple acidic amino acid residues and a tetradecapeptide with three lysines, INLKAIAALAKKLLG, respectively. The glycine at the tetradecapeptide end is the donator of –NH₄ for the amidation of the leucine at the C-terminal. As far as we know, this is the first report of the precursor of animal mastoparan.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.     

Mammalian l-to-d-amino-acid-residue isomerase from platypus venom

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.     

Kinetic models for peptide-induced leakage from vesicles and cells

In this article analytical expressions for peptide-induced membrane leakage are presented. Two different models for time-dependent leakage have been developed. In the first, the leakage is assumed to be coupled by pores formed by the peptides. In the second model the peptide is assumed to induce a stress/perturbation in the membrane, and in order to reduce the stress, rearrangements in the membrane are induced. The leakage is coupled to these rearrangements, and when equilibrium is achieved no more leakage occurs. From the kinetic models simple fitting routines have been developed involving only two fitting parameters, and these have been used to fit experimental data for two prion protein-derived peptides as well as the honey bee toxin melittin in both vesicles and erythrocytes with good results. The fitted parameters provide both a quantitative and a qualitative basis for interpreting the experimental results. In addition a model for the peptide concentration-dependent leakage is presented, which was used to fit experimental data for leakage induced by the prion protein-derived peptides. The models presented in this article are compared with other models for peptide-induced membrane leakage.     

Effect of phospholipase A on actions of cobra venom cardiotoxins on erythrocytes and skeletal muscle

The actions of two phospholipase-free cardiotoxins from the venom of the cobra Naja naja siamensis were compared to phospholipase-contaminated cardiotoxins in terms of their ability to lyse human erythrocytes and to depolarize and contract skeletal muscle. The presence of 3–5% (w/w) phospholipase caused a 20–30-fold increase in the haemolytic activity of the two cardiotoxins, the pure cardiotoxins being virtually without haemolytic activity at 10^?7-10^?6 M. Phospholipase contamination did not enhance the ability of the cardiotoxins to cause contracture of chick biventer cervicis muscles and it caused less than a 2-fold increase in the depolarizing activity of the cardiotoxins on cultured skeletal muscle. Phospholipase-free cardiotoxins were about 10–20-times more active on cultured skeletal muscle fibres than on erythrocytes. These results support the hypothesis that some cardiotoxins have more affinity for the membranes of excitable cells than for those of other cells such as erythrocytes.     

SIGLOCPRED: an algorithm to predict bacterial signal peptides and OMPS

There is a growing interest in Biological investigation to determine the location of proteins, to identify new potentially accessible drug targets. Signal peptide directs the transport of the protein to its location. Bacterial OMPs are essential for their survival in the host organism. SIGLOCPRED a signal peptide predictor for the bacterial proteins as well as OMP prediction has been developed. The signal peptide prediction is done based on the influence of the flanking residues on the signal peptide cleavage. A dataset of proteins with confirmed outer membrane location has being created, and the probable OMP polypeptide sequence is predicted. Since the algorithm uses confirmed datasets the prediction is more reliable and efficient. SIGLOCPRED is as efficient as many of the existing signal peptide predictors and can also predict OMPs in addition.     

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH₂ (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH₂ (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the -amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.     

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides posses strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5–40 μM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt -helical conformation in such anisotropic environments.     

穴居狼蛛毒中抗菌活性物质的耐热及缔合性质

穴居狼蛛(Lycosa singoriensis)毒中的抗菌活性物质具有耐热性(在沸水浴中处理半小时活性不变)、耐酸性和耐碱性。但用透析袋(Visking 18/32)对毒腺提取物透析时,在中性及碱性条件下可全部被透析掉。当用Sephadex G-25柱对较少量样品进行层析时,在酸性条件下至少可被分出8个蛋白峰,其中峰4和峰8具抗菌活性,但随上柱样品量的增加,在层析图上峰4的面积增大,峰8的面积变小,甚至活性消失;反之,在中性或弱碱性条件下所得到的层析图上,则是峰8的面积大,抗菌活性明显,而峰4的面积小,甚至检不出抗菌活性。此峰8样品经热处理后,在酸性条件下经分子筛过滤,又可得到两个分子量不同的抗菌活性峰。这些结果提示,上述耐热和耐酸碱性强的抗菌物质在酸性环境中可能是以目身高聚体或与其它大分子蛋白非共价结合,随pH值的升高而解离成单体或低聚体。     

On “intercolonial” cannibalism in Japanese paper wasps,Polistes chinensis antennalis Pérez andP. Jadwigae dalla torre (hymenoptera: Vespidae)

Summary Foundresses of two species of Japanese paper wasps,Polistes chinensis antennalis andP. jadwigae, attacked other colonies of the same species. A foundress ofP. chinensis antennalis visited two nests of the same species, and ate larvae from them, while two foundresses ofP. jadwigae each visited a nest of the same species, eating larvae and pupae even when the foundress of the attacked nest was on her nest. In addition, a foundress ofP. jadwigae distributed flesh balls thus obtained among their larvae. Discussion was made on the adaptive significance of the inter-colonial cannibalism. It was considered that, at first, it increases the foraging efficiency and secondly it plays a role in regulating population density.     

Colony development and social structure in a subtropical paper wasp,Ropalidia fasciata (F.) (Hymenoptera: Vespidae)

More than 50% of nests of Ropalidia fasciata were founded by association of foundresses (multifemale nests). The multifemale nests were generally initiated earlier and grew faster than the single-female nests. The survival rate of the multifemale nests was significantly higher than that of single-female nests, and the productivity as measured by the number of cells produced per foundress had a peak at a foundress-group size from 6 to 10. The number of marked foundresses which were seen on their original nest decreased as the colony cycle proceeded, but some of them continued to coexist on the original nests after emergency of many female progeny. Except in the case where a large number of foundresses attended a young nest so that some foundresses could not sit on the nest, the dominance interactions among cofoundresses were mild. More than twothirds (71.4%) of nests (including those at the post-emergence stage) had multiple egg-layers. The foundress association in this species is considered to be beneficial for every foundress because it raises ability to avoid predation or to reconstruct their nests when the nests are destroyed by typhoons.     

Ecological studies ofRoparidia fasciata in Okinawa island I. Distribution of single- and multiple-foundress colonies

To compare between a single-foundress colony and a multiple-foundress colony at the pre-emergence state of a social wasp, R. fasciata, nest distributions and colony terminations were investigated in 8 sites with different environmental conditions. Marking experiments were also conducted in two sites at high wasp density.

1. Foundress populations were composed of single-foundress colonies in sites C, D and E, new environments where have recently suited for inhaviting, at low wasp density. In sites like A and B which were used year after year, at high wasp density, coexistence of multiple-and single-foundress colonies was observed.
2. From the marking experiment, nests initiated by a single foundress were more distant away from the nest where the original foundress emerged the fall before, compared to multiple-foundress nests which were initiated by multiple foundress.
3. Greater percentage of colony termination was observed in single-foundress nests than in multiple-foundress nests, and the colony termination in single-foundress colonies increased with the nest density.
4. Ant predation was the key factor causing the variation of the percentage of colony termination.

     

Multipurpose isothiocyanyl alanine/lysine: Use as solvatochromic IR probes and in site specific labeling/ligation of short peptides

The solvatochromic IR responsivity of small side chain –NCS in two unexplored unnatural amino acids, isothiocyanyl alanine (^NCSAla = Ita) and lysine (^NCSLys = Itl), without perturbing the conformation is demonstrated in two designed short tripeptide (BocAla-^NCSAla-Ala-OMe) and hexapeptide (BocLeu-Val-Phe-Phe-^NCSLys-Gly-OMe). Demonstration of site specific fluorescent labeling in both the peptides and ligation type reaction in ^NCSLys indicates the novelty of these two amino acids as alternative to the available canonical amino acids.