New methods of isolating bee venom peptides

The three major peptides of bee venom, melittin, apamin, and peptide 401 (MCD peptide), can be readily separated on columns of Heparin Sepharose CL-6B at pH 6.8 (phosphate buffer) using a linear salt gradient. Contamination by melittin of the other two components can be assessed by fluorescence spectrophotometry.     

Isolation of modified allergens from bee venom

To obtain modified bee venom (BV) allergens, covalent binding of BV with previously carboxylated polyethylene glycol (PEG) has been used. The conjugation of BV and PEG has been achieved by means of carbodiimide. Thus 4 kinds of the conjugate with BV/PEG ratio ranging from 30:1 to 63:1 have been obtained. The study has shown that chemical treatment in the process of this reaction, dialysis and chromatography does not lead to a decrease in the specific activity of BV, while lyophilization produces such an effect. The above method for the modification of BV allergen, used with a view to obtaining high molecular compositions, is reproducible, ensures sufficient yield (about 30%), and permits obtaining conjugates with specified BV/PEG ratios.     

Assessment of hemolytic activity of bee venom against some physicochemical factors

Bee venom (BV) is a biotoxin with biologically active peptides which have cell lysis and hemolytic activity properties. These properties can be affected under different storage conditions or during the production process. In present study, we investigated effects of a number of physicochemical factors, including temperature, pH, UV radiation, ultrasound waves and storage time on hemolytic activity of BV. Maximum absorption and melting temperature of BV solution were obtained as 280 nm and ~70 °C, respectively. Cell hemolysis 50 (CH₅₀) -concentration of BV that can lyse 50% of red blood cells- was determined as 0.94 μg/ml at ambient temperature. CH₅₀ was shown not to be importantly varied at temperature up to 60 °C, pH value 2 to 13 and under UV/ ultrasound radiation. Storage at ?20, 6 and 25 °C for 6 months made about 2.5, 35 and 1000 times increase in CH₅₀. From the results, it may be concluded that BV is a relatively resistant hemolytic agent and can be used in a variety of laboratory research and product manufacturing methods.     

Histamine-liberating action of MCD-peptide from bee venom]

Histamine release (HR) from the isolated rat mast cells induced by MCD-peptide, mellitin (from the Apis mellifica venom) and compound 48/80 was studied. The dose-response curve, the latent period and temperature dependence of HR induced by MCD-peptide were similar to those of HR induced by compound 48/80. The HR induced by MCD-peptide proved to be an energy-dependent process that was independent of cyclic adenosine-3',5'-monophosphate.     

Spectroscopic investigations of peptide 401 from bee venom

The circular dichroism (CD) and ¹H-nmr properties of peptide 401, a bee venom component with 22 amino acid residues and two disulfide bridges, have been studied under a variety of conditions and compared with those of the structurally related octadecapeptide apamin. The major component of the relatively intense CD signal in the 200–230-nm region in both cases probably arises from the rigid asymmetric ring structures of the disulfide bridges. CD spectra are practically unaffected by pH (in the region 1–7), solvent (water, trifluoroethanol, dioxane/water mixtures), concentration of peptide, or additions of salt (guanidinium chloride, KCl). Temperature changes (in the range 20–59°C) have only a modest influence. For both apamin and peptide 401, reduction of the two disulfide bridges results in a dramatic change of the CD spectrum, which acquires the characteristic form of a random coil. Preliminary ¹H-nmr data are presented for both the reduced and the oxidized form. Several resonance peaks could be assigned on the basis of the theoretical random-coil spectrum. In the oxidized forms, six slowly exchangeable amide protons could be found in a spectrum taken at low pH, which are ascribed to intramolecular hydrogen bonds. Each of the four protons of the two histidine residues of peptide 401 appears as two distinct resonance peaks in the oxidized form but not in the reduced form. This is interpreted as arising from conformational heterogeneity of peptide 401.     

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells

Recently, we have isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. These peptides (melectin, lasioglossins, halictines and macropin) and their analogs display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity and low or moderate hemolytic activity. Here we describe cytotoxicity of the above-mentioned AMPs and some of their analogs toward two normal cell lines (human umbilical vein endothelial cells, HUVEC, and rat intestinal epithelial cells, IEC) and three cancer cell lines (HeLa S3, CRC SW 480 and CCRF-CEM T). HeLa S3 cells were the most sensitive ones (concentration causing 50% cell death in the case of the most toxic analogs was 2.5-10 μM) followed by CEM cells. For the other cell lines to be killed, the concentrations had to be four to twenty times higher. These results bring promising outlooks of finding medically applicable drugs on the basis of AMPs. Experiments using fluorescently labeled lasioglossin III (Fl-VNWKKILGKIIKVVK-NH(2)) as a tracer confirmed that the peptides entered the mammalian cells in higher quantities only after they reached the toxic concentration. After entering the cells, their concentration was the highest in the vicinity of the nucleus, in the nucleolus and in granules which were situated at very similar places as mitochondria. Experiments performed using cells with tetramethylrhodamine labeled mitochondria showed that mitochondria were fragmented and lost their membrane potential in parallel with the entrance of the peptides into the cell and the disturbance of the cell membrane.     

Allergens of honey bee venom.

The allergenic activities of four purified components of honeybee venom were studied by using histamine release from leukocytes of bee sting-allergic patients. The components studied were hyaluronidase, phospholipase A₂, melittin and apamin with molecular weights, respectively, of about 50,000, 15,800, 2840 and 2038 d. In six of the seven patients studied, hyaluronidase and phospholipase were, respectively, on the average about two and eight times more active by weight than the venom. The situation was reversed in one patient in that hyaluronidase and phospholipase A₂ were, respectively, 90 and 0.5 times more active than the venom. With this single exception, hyaluronidase and phospholipase were about equally active on a molar basis as allergens. Melittin was on the average about one-tenth as active as the venom, and apamin was inactive as an allergen.Chemical modifications of phospholipase A₂ were carried out. Succinylation of eight of its eleven amino groups yielded a derivative that retained 4% of the enzymic activity of the native enzyme. Reduction and carboxymethylation of its four disulfide bonds or cyanogen bromide cleavage of its three methionyl bonds yielded enzymatically inactive derivatives. These derivatives showed varying decreases of allergenic activities when compared to the native enzyme. The results indicate that the antigenic determinants of phospholipase depend on the charge, the amino acid sequence and the conformation of the molecule.     

The antifungal effect of peptides from hymenoptera venom and their analogs

As the occurrence of Candida species infections increases, so does resistance against commonly-used antifungal agents. It is therefore necessary to look for new antifungal drugs. This study investigated the antifungal activity of recently isolated, synthesized and characterized antimicrobial α-helical amphipathic peptides (12–18 amino acids long) from the venom of hymenoptera (melectin, lasioglossins I, II, and III, halictines I and II) as well as a whole series of synthetic analogs. The minimal inhibitory concentrations (MICs) against different Candida species (C. albicans, C. krusei, C. glabrata, C. tropicalis and C. parapsilosis) of the natural peptides amounted to 4–20 μM (7–40 mg/l). The most active were the synthetic analog all-D-lasioglossin III and lasioglossin III analog KNWKK-Aib-LGK-Aib-IK-Aib-VK-NH₂. As shown using a) colony forming unit determination on agar plates, b) the efflux of the dye from rhodamine 6B-loaded cells, c) propidium iodide and DAPI staining, and d) fluorescently labeled antimicrobial peptide (5(6)-carboxyfluorescein lasioglossin-III), the killing of fungi by the peptides studied occurs within minutes and might be accompanied by a disturbance of all membrane barriers. The peptides represent a promising lead for the development of new, effective antifungal drugs.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Synthesis of apamin, a neurotoxic peptide from bee venom.

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physiochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 mug).