Lytic activity of monomeric and oligomeric melittin

The haemolytic activities of melittin and melittin tetramer as induced by high phosphate counterion concentration, were monitored. Monomeric melittin was found to be fully lytic, whilst tetrameric melittin lacked such activity. Under conditions where melittin was fully tetrameric attempts were made to covalently cross-link the native tetramer using a series of different chain length bifunctional imido esters. The cross-linked oligomers were fully lytic under conditions where melittin was demonstrated to lack such activity. This finding, together with molecular weight determinations and circular dichroism studies, indicated that the cross-linked melittin was quite different to the native tetramer. The haemolytic activity of melittin-containing solutions was related to the concentration of monomeric melittin. The effect of reduced dielectric constant (?) on the aggregation behaviour of melittin and its derivatives was found to favour monomeric melittin.     

蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造。将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu。用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA。该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg／ml。     

Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change

The (Na+ + K+)ATPase is inhibited by the bee venom polypeptide, melittin. KCl and NaCl protect the enzyme from melittin inhibition. Analysis of the K+ and Na+ protection against melittin inhibition suggested a kinetic model which was consistent with slowly reversible melittin binding, and mutually exclusive binding of melittin with K+ and Na+. Accordingly, in the absence of salt, the KI for melittin inhibition = 1.2 microM, and the protection by KCl occurs with a KA,KCl = 0.6 mM. The protection by NaCl occurs with a KA,NaCl = 15 mM. Melittin inhibition of enzyme activity is due to direct interactions with the (Na+ + K+)ATPase, as demonstrated by photolabeling with [125I]azidosalicylyl melittin, which labeled the alpha subunit, but not the beta subunit of the (Na+ + K+)ATPase. Melittin and KCl reduced the extent of labeling. In non-covalent binding studies using [125I]azidosalicylyl melittin, the stoichiometry of binding was 1.6 melittin per (Na+ + K+)ATPase. Ligand-induced conformational changes of FITC-labeled (Na+ + K+)ATPase were examined in the presence and absence of melittin. K+ alone or melittin alone caused a fluorescence intensity quenching consistent with formation of an E2 form of the enzyme. The NaCl-induced (E2----E1) fluorescence intensity changes were maximal when the enzyme was treated with K+. NaCl-induced fluorescence changes did not occur when the enzyme was treated with melittin in the absence of K+. However, when K+ was present before the addition of melittin, NaCl-induced fluorescence intensity increases were observed, which were dependent upon the concentration of K+ in the preincubation mixture. The results of the labeling and conformational studies support the kinetic model and suggest a mechanism for inhibition of ion pumps by (poly)peptides.     

Conformational Studies of a Melittin—Inhibitor Complex

The conformation of a melittin—inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in ¹H T ₁ relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in α-helical structure for increasing ratios of inhibitor bound to melittin.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.     

改造的蜂毒素基因在毕赤酵母中分泌表达及培养条件研究

对蜂毒素基因进行改造后,通过PCR方法获得新蜂毒素基因(MEA),将其克隆到表达载体pPICZa—A,而后将重组表达载体pPICZa-A-MEA转化GS115,筛选获得重组酵母菌．对GS115-ME经甲醇诱导表达并对培养条件进行优化探讨．对改造的蜂毒素进行了溶血活性、热稳定性及酸碱稳定性测定．结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造的蜂毒素在保留了抗菌活性的同时溶血活性降低20倍左右,同时还具有良好的热稳定性和酸碱稳定性．     

Phospholipase A2 activation by melittin causes amylase release from exocrine pancreas

Phospholipase A2-induced deacylation of membrane phospholipids is associated with changes in membrane fluidity. The importance of this reaction in the pancreatic amylase secretory process was tested using melittin, a phospholipase A2 stimulating peptide. Phospholipase A2 activity (using [3H]arachidonic acid release as an index) and amylase secretion were both increased in a time- and concentration-dependent manner by melittin. Phospholipids prelabelled with [3H]oleic acid or [14C]linoleic acid also released radioactive free fatty acids in response to melittin. Prostaglandin synthesis was not involved in the melittin response, since inhibitors of arachidonic acid oxidation (indomethacin, 5,8,11,14-eicosatetraynoic acid) did not alter the ability of melittin to release [3H]arachidonic acid or amylase. When melittin was co-applied with carbachol, cholecystokinin octapeptide, or vasoactive intestinal peptide, amylase secretion was additive. The effect of melittin on both fatty acid and amylase release was dependent on extracellular calcium, though melittin's effects were not dependent on the intracellular accumulation of second messengers such as calcium or cAMP. The data suggest that activation of phospholipase A2 by melittin results in the triggering of the secretory process in exocrine pancreas by a different mechanism than that for other pancreatic secretagogues.     

The influence of melittin on the rotation of band 3 protein in the human erythrocyte membrane

The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilise is reduced. Succinylated melittin, however, is devoid of immobilising activity.The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergise with phospholipase A₂, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin > acetyl melittin > succinyl melittin = 0, the same as their order of effectiveness in immobilising band 3.A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A₂ to gain access to the membrane bilayer and commence catalysis.     

Protein rotational diffusion measurements on the interaction of bee venom melittin with bacteriorhodopsin in lipid vesicles

The rotational diffusion of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine vesicles was measured by the technique of flash-induced transient dichroism. In the presence of melittin, a cell lysing peptide from honey bee (Apis mellifera) venom, dose-dependent loss of rotational mobility was observed. Chemically modified melittin derivatives, in which free amine groups were either acetylated or succinylated, were impaired in their ability to induce immobilisation of bacteriorhodopsin. Bacteriorhodopsin reconstitutions of differing lipid/protein ratio were tested and it was found that the bacteriorhodopsin immobilisation phenomena depended on the melittin/protein ratio, not the melittin/lipid ratio. This suggests that melittin produces its effect via direct interaction with bacteriorhodopsin. A mechanism is proposed in which the aggregation of bacteriorhodopsin is induced by electrostatic attraction between its anionic surface moieties and the highly cationic C-terminal segment of melittin.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Apoptotic Effect of Melittin Purified from Iranian Honey Bee Venom on Human Cervical Cancer HeLa Cell Line

Melittin, an amphipathic 26-residue peptide, is the main component of honey bee venom. Studies have been demonstrated that melittin has an inhibitory effect on proliferation of cancer cells. However, the precise mechanism of action is not completely understood. In the present study we have shown that purified melittin from Iranian honey bee venom shows anti-cancer effects on human cervical cancer cell line through induction of apoptosis. The venom was collected from Iranian honey bee (Apis mellifera meda) and melittin isolated using reversed phase HPLC. Biological activity of melittin was analyzed by hemolytic test on human red blood cells. In order to investigate whether melittin inhibits proliferation of cervical cancer cells, the viability of the melittin treated HeLa cell line was measured via MTT assay. Finally, cell death analysis was performed using Propidum iodide and Annexin V-FITC dual staining. The results showed that the half hemolytic concentration (HD50) induced by mellitin was 0.5 µg/ml in free FBS solution. IC50 obtained after 12 h at 1.8 µg/ml by MTT assay. According to flow cytometric analysis, melittin induced apoptosis at concentrations more than 1 µg/ml. These results suggest that melittin induces apoptotic cell death in cervical cancerous cells as observed by flow cytometric assay. It is concluded that melittin could be regarded as a potential candidate in future studies to discovery of new anticancer agents.     

Ultrastructural observation on sterilization of melittin

The effects of melittin on growth and bacteriostasis of four pathogens were extensively investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results indicated that the melittin had a marked bacteriostatic effect on the four pathogenic bacteria. Among these, E. cacotowora was influenced most powerfully and quickly, the yeast and F. fulva were the second, and the S. aureus was inhibited by a low concentration but was killed by a high concentration. It was observed in the experiments that melittin killed pathogenic bacteria in three ways. One was by pore formation. After integrating melittin into the plasma membrane, a vacuole was formed then penetrated, resulting in bacterial content leakage. The vacuole also experienced plasmolysis and the growing cavity destroyed the membrane. A third effect was the formation of vacuoles in the cells which induced the pycnosis of the cytoplasm resulting in a cell death. The mechanism of melittin bacteriostasis was the result of integrating melittin with phospholipod double layers of the plasmalemma and the endomembranes.     

Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.     

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.     

Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C

To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy

We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.     

Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels.

Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

The aggregation state of mellitin in lipid bilayers. An energy transfer study

We used fluorescence non-radiative energy transfer to measure the self-association of melittin in solution and when bound to lipid bilayers. Energy transfer occurred from the tryptophan residue of unlabeled melittin to an N-methyl anthraniloyl residue covalently bound to a basic lysine residue on melittin. The extent of energy transfer from tryptophan to the label was found to increase severalfold upon the salt-induced tetramerization of melittin. When bound to vesicles of dimyristoyl-L-alpha-phosphatidylcholine, the extent of energy transfer was found to be equivalent to that of monomeric melittin, irrespective of the presence of monomeric or tetrameric melittin in the aqueous phase. We conclude that membrane-bound melittin is monomeric.     

Conformational studies of a melittin-inhibitor complex

The conformation of a melittin—inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in ¹H T ₁ relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in -helical structure for increasing ratios of inhibitor bound to melittin.