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PubMed2002

Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells.

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.

Amino Acid Sequence↗
PubMed1995

Melittin cardiotoxicity in cultured mouse cardiac myocytes and its correlation with calcium overload.

Venom from the honey bee Apis mellifera induces cardiovascular dysfunction. We studied which constituent(s) of the venom induces cardiotoxicity and how, using cultured cardiac myocytes from mouse fetuses. Among the venom constituents, only melittin caused contractile and morphological effects; other peptides, such as apamin and mastparan; enzymes, such as phospholipase A2; and low-molecular-weight compounds, such as histamine and dopamine, did not. Treatment with 4.5 micrograms/ml melittin, which accounts for about half the dry weight of the venom, induced the same cardiotoxic effects as treatment with 9.0 micrograms/ml whole venom; these effects were a transient increase in the spontaneous beating rate, then a decrease, then cessation of beating, and finally, morphological degeneration. The cardiotoxicity of whole bee venom was completely destroyed by pretreatment of the venom with antimelittin antibody. These results suggest that bee venom cardiotoxicity is attributable to melittin. When spontaneous beating ceased following the addition of melittin or whole venom, an increase in systolic [Ca2+]i, was observed. On further incubation with melittin or bee venom, morphological injury, such as balloon degeneration, occurred concomitant with a further increase in the [Ca2+]i. An extracellular Ca2+ concentration of more than 10(-6) M was necessary for morphological injury. Melittin depolarized the maximum diastolic potentials, inhibited the generation of action potentials, and induced an increase in [Na+]i. Cells were protected against the melittin-induced increase in [Ca2+]i by pretreatment with bepridil, an inhibitor of Na(+)-Ca2+ exchange, but not by Ca2+ channel blockers such as verapamil. These observations suggest that the melittin-induced increase in [Ca2+]i was due to entry of extracellular Ca2+ via the sarcolemmal Na(+)-Ca+ exchange system.

Animals↗
PubMed1993

Role of the Na+,K+ pump in herpes simplex type 1-induced cell fusion: melittin causes specific reversion of syncytial mutants with the syn1 mutation to Syn+ (wild-type) phenotype.

To evaluate the importance of the Na+,K+ pump and ionic gradients in virus-induced cell fusion, we investigated the effects of melittin, a 26 amino acid bioactive peptide found in honey bee venom, on cell fusion caused by HSV-1 syncytial mutants. Melittin inhibited fusion of Vero cells caused by HSV-1 mutant viruses mP(MP), KOS (syn20) and KOS (FFV3) containing the syncytial mutation syn1 in glycoprotein K. However, it did not affect cell fusion caused by mutants HFEM(tsB5) or KOS amb1511-7 with mutations in glycoprotein B. Melittin caused specific reversion of syn1 mutant virus plaques to syn+ (wild-type) plaque morphology, and inhibited virus adsorption and penetration. It also inhibited the Na+,K+ pump activity, and the binding of 3H-ouabain to the Na+,K+ pump of infected Vero cells. The Na+,K+ pump activity of infected Vero cells in comparison to mock-infected cells was significantly decreased. Ouabain, a specific inhibitor of the Na+,K+ pump, inhibited fusion of Vero cells caused by all syncytial virus strains.

Animals↗
PubMed1994

Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences.

The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. However, deletion of the HIV-1 gp120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells. Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120. Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion. However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120. These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion. Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus.

Amino Acid Sequence↗
PubMed1996

Neutron diffraction studies of amphipathic helices in phospholipid bilayers.

The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.

Amino Acid Sequence↗
PubMed1994

Light-evoked arachidonic acid release in the retina: illuminance/duration dependence and the effects of quinacrine, mellitin and lithium. Light-evoked arachidonic acid release.

Arachidonic acid (AA) is the precursor molecule of a variety of cellular lipid mediators that interact with retinal physiology. In this study, we investigated the time- and illuminance-dependence of the release of AA in the rat retina in vitro in control and lithium-pretreated rats. We also studied the effects of the specific phospholipase A2 (PLA2) inhibitor quinacrine and the specific PLA2 stimulator mellitin on the release of AA. Isolated rat retinas were labelled with 3H-AA for 90 min in vitro in darkness and the incorporation of AA into retinal phospholipids was monitored by thin-layer chromatography. The release of 3H-AA in the incubation medium was determined under different illuminance and timing conditions, with the addition of quinacrine and mellitin, and after pretreatment of the animals with lithium. Light exposure of the prelabelled isolated retinas evoked up to a two-fold increase in AA release compared with retinas incubated for the same time in darkness. The AA release was dependent on illuminance time (10,000 1x white fluorescent light for 0.25, 2, 5 and 10 min) and illuminance level (0, 100, 1000, 5000, and 10,000 1x for 10 min). Complete rhodopsin bleaching occurred after 2 min at 10,000 1x. Quinacrine significantly suppressed the light-elicited AA release whereas mellitin increased the release of AA in dark-adapted and light-exposed retinas. Lithium pretreatment, which is known to potentiate light-evoked rod outer segment disruptions, significantly augmented the light-evoked AA release. Our results confirm a light-stimulated release of AA in the retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1991

Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa.

Melittin (0.5-2 micrograms ml-1) increased the short-circuit current (ISC) in mucosa-submucosa and mucosa preparations of the rat colon descendens in a dose-dependent manner. In the preparation with the submucosal plexus, quinacrine and indomethacin completely blocked the effect of melittin, indicating activation of phospholipase A2 and production of prostaglandins induced by the drug. The melittin response was also partially sensitive to the lipoxygenase inhibitor, nordihydroguaiaretic acid. Complete inhibition by tetrodotoxin and atropine gives evidence for the involvement of cholinergic neurons in the mediation of the response induced by melittin. In contrast, in the preparation without the submucosal plexus the effect of melittin was only partially inhibited by quinacrine, indomethacin, or by neuronal blockers, suggesting direct interactions of melittin with the epithelium in addition. The effect of melittin resembles to the action of bradykinin, which is neuronally mediated and quinacrine-sensitive in the mucosa-submucosa preparation, and quinacrine-resistant and not neuronally mediated in the mucosa preparation. In the mucosa-submucosa preparation, the melittin response is even partially sensitive to the bradykinin receptor antagonist [D-Phe7]-bradykinin. The results provide evidence for the presence of a quinacrine-sensitive phospholipase A2 in the preparation with and that without the submucosa.

Animals↗
PubMed1993

13C alpha-NMR assignments of melittin in methanol and chemical shift correlations with secondary structure.

Melittin is a naturally occurring hexacosa peptide which forms an amphiphilic helix in methanol, a random coil in water, and a tetramer of helices at basic pH or in the presence of a high salt concentration. The monomeric structure in methanol has been well characterized by proton NMR (Pastore et al. (1989) Eur. Biophys. J., 16, 363-367). In the present paper, chemical shifts of the backbone alpha-carbons of melittin in methanol were determined by mapping previously published alpha-proton shifts (Bazzo et al. (1988) Eur. J. Biochem., 173, 139-146), to natural abundance (1H)13C cross peaks appearing in the 2D heteronuclear multiple-quantum NMR spectrum. Changes in chemical shifts consequent to stepwise increases in the percentage of water in a mixed methanol/water solvent system were observed in similar spectra. The alpha-carbon shifts varied more smoothly than the corresponding alpha-proton shifts and were found to correlate with the transition from the helix to the random coil conformer in parallel with changes in the circular dichroism spectrum. Chemical shifts of this peptide are interpreted with regard to the current database of assignments in proteins of known 3D structure (Wishart et al. (1991) J. Mol. Biol., 222, 311-333). The N-terminal region of the peptide shows increased flexibility at lower methanol concentrations, as evidenced by the merger of the alpha-proton resonances of G1 (at 40% and 15% methanol) and G3 (at 15% methanol). Conformational exchange rates for G1 and G3 were estimated by comparison of the experimental spectra with simulated spectra and found to be as large as 4000 s-1 for G1 in 40% and 15% methanol and 600 s-1 for G3 in 15% methanol. Overall, these 1H and 13C chemical shift data support the description of monomeric melittin in methanol currently evolving in the literature and suggest a structure composed of a linked pair of helices with different structural stabilities, each of which experiences dynamical fraying at its free terminus.

Calcium Isotopes↗
PubMed1994

Activation of mechanonociceptors by pro-inflammatory peptides melittin and PLAP peptide.

Studies were conducted to characterize the chemical reactivity of A and C fiber mechanical and mechanoheat nociceptors that innervate the goat palatal mucosa. In mechanical nociceptors, no significant chemical reactivity to either serotonin, bradykinin, prostaglandin E1, or prostaglandin E2 was observed, regardless of whether substances were injected singly or in spaced sequential combinations. Weak reactivity was observed in mechanoheat nociceptors. In contrast, both mechanical and mechanoheat nociceptors were activated by novel proinflammatory peptides. Nineteen of 30 mechanonociceptors and 12 of 13 mechanoheat nociceptors were activated by the insect venom peptide, melittin or its endogenous mammalian homologue PLAP (phospholipase A2 activating protein) peptide. Low threshold mechanoreceptors were never activated by melittin or PLAP peptide.

Animals↗
PubMed1991

Modulation of the beta-adrenergic response in cultured rat heart cells. I. Beta-adrenergic supersensitivity is induced by lactate via a phospholipase A2 and 15-lipoxygenase involving pathway.

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A2-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A2 inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A2 in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclo-oxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A2-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.

Acetophenones↗
PubMed1992

Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin, synthetic analogs, and a potential intracellular regulatory protein.

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H+ + K+)ATPase. The site of interaction with the (H+ + K+)ATPase was shown to be the alpha subunit of the (H+ + K+)ATPase in studies using [125I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H+ + K+)ATPase, and prevented labeling by [125I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H+ + K+)ATPase. These studies help to establish that the (H+ + K+)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H+ + K+)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H+ + K+)ATPase. Using an antibody which recognizes this amphipathic helical ('melittin-like') motif, we have demonstrated that the gastric parietal cell contains a 67 kDa 'melittin-like' protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric 'melittin-like' protein appears to overlap with the melittin binding site on the alpha subunit of the (H+ + K+)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous 'melittin-like' protein on the (H+ + K+)ATPase to regulated HCl secretion by the parietal cell is presently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Multigranular exocytosis induced by phospholipase A2-activators, melittin and mastoparan, in rat anterior pituitary cells.

Effects of phospholipase A2-activators, melittin and mastoparan, on rat anterior pituitary cells were studied by use of the electron microscope. Rat anterior pituitaries were incubated in HEPES buffer containing 20 micrograms/ml of melittin or the same dose of mastoparan for 5 min, 10 min and 20 min. Features indicating discharge of granule contents by exocytosis were increased with time, and the simultaneous extrusion of a number of secretory granules, named "multigranular exocytosis", was often recognized in addition to single-granule exocytosis at 10 min and 20 min. Most membrane pits, where the multigranular exocytosis as well as the single-granule exocytosis occurred, were coated. Moreover, a large number of vesicles coated or non-coated were distributed near the trans side of the Golgi apparatus of melittin-treated or mastoparan-treated cells after 20 min. These vesicles might be related to membrane internalized from the excess surface membrane derived from the limiting membrane of exocytosed granules. These observations indicate that phospholipase A2-activators induce hormone release involving membrane fusion between limiting membranes of secretory granules, and between granule-limiting membrane and plasma membrane in rat anterior pituitary cells.

Animals↗
PubMed1984

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 immobilize is reduced. Succinylated melittin, however, is devoid of immobilizing 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 synergize with phospholipase A2, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin greater than acetyl melittin greater than succinyl melittin = 0, the same as their order of effectiveness in immobilizing 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.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1989

The dynamic properties of melittin in solution. Investigations by NMR and molecular dynamics.

Molecular dynamics simulations are described for the peptide melittin. The atomic trajectories are calculated both with normal potential energy functions and with additional distance restraints deduced from nuclear Overhauser effects observed in NMR experiments. The results are compared with NMR data on coupling constants and amide exchange rates and with B-factors from X-ray crystallography. The observed correlations between experiment and molecular dynamics simulations suggest a relatively mobile C-terminus and relatively high flexibility around residue 11. It is noted that the high conformational variation around residue 11 is due in part to the presence of a proline at position 14 which results in a "missing" H-bond in the largely alpha-helical structure. It is also noted that a proline is a common feature of many putative membrane spanning helices. A role for such prolines is suggested.

Bee Venoms↗
PubMed1985

Inhibition of growth of leukemic cells by inhibitors of calmodulin: phenothiazines and melittin.

Calmodulin, a ubiquitous calcium-binding protein, has recently been shown to play an important role in cellular proliferation. The calmodulin inhibitors melittin, trifluoperazine, and chlorpromazine inhibited the growth and clonogenicity of human and murine leukemic cells, and their potency reflected their activity as inhibitors of calmodulin. Melittin, which is a far more potent inhibitor of calmodulin activity, was also a more potent inhibitor of cell growth and clonogenicity. The less active phenothiazine metabolite, chlorpromazine sulfoxide, had much less potent cytotoxic activity.

Animals↗
PubMed1987

Different states of self-association of melittin in phospholipid bilayers. A resonance energy transfer approach.

Melittin is known to self-associate as tetramers in solutions of high ionic strength. Here, an N-bromosuccinimide oxidized-Trp19 melittin is prepared. This derivative can act as an acceptor of the fluorescence of native melittin and is used in order to observe a possible self-association of melittin in phospholipid bilayers. Resonance energy transfer was shown to occur in solutions of high ionic strength, showing that oxidized melittin can associate with native melittin. In phospholipid bilayers, no association is detected in the absence of NaCl. In its presence, an equilibrium between monomeric melittin and oligomeric species is observed. These species are not dimers, but any other degree of association may account for our experimental results. Significant differences in characteristic transfer efficiency reveal differences in the structure of these oligomers according to the length or state of phospholipids (fluid or at the transition temperature). These bound complexes are also different from the soluble hetero-oligomer. Some models of bound complexes are proposed which may explain the leakage and the further disruption of vesicles or cells induced by melittin.

1,2-Dipalmitoylphosphatidylcholine↗