AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.

Identification and characterization of novel sodium channel toxins from the sea anemone Anthopleura xanthogrammica.

Six new toxins from the sea anemone Anthopleura xanthogrammica were identified using a molecular biological approach. Five of these novel isoforms resemble the 47 residue type I long polypeptides native to Anthopleura elegantissima, Anthopleura fuscoviridis and Anemonia sulcata, while one appears to be chimera of the two previously identified 49 residue toxins native to A. xanthogrammica. Four of these toxins were expressed in bacteria, purified and characterized by ion flux assays in RT4-B and N1E-115 cell lines expressing the cardiac and neuronal Na channel isoforms, respectively. The novel 47 residue toxin isoforms form a new subclass within the A. xanthogrammica neurotoxin family, although they are related to previously described anemone toxins. One of the three 47 residue toxins characterized, PCR2-10, enhances veratridine-dependent sodium uptake, displaying a K0.5 of 329 nM and 1354 nM in RT4-B and N1E-115 cell lines, respectively. The novel 49 residue toxin, PCR3-7, interacts with the sodium channel with even higher affinity, enhancing sodium uptake with a K0.5 of 47 nM and 108 nM in RT4-B and N1E-115 cells, respectively.

A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B.

The polypeptide neurotoxin anthopleurin B (ApB) isolated from the venom of the sea anemone Anthopleura xanthogrammica is one of a family of toxins that bind to the extracellular face of voltage-dependent sodium channels and retard channel inactivation. Because most regions of the sodium channel known to contribute to inactivation are located intracellularly or within the membrane bilayer, identification of the toxin/channel binding site is of obvious interest. Recently, mutation of a glutamic acid residue on the extracellular face of the fourth domain of the rat neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers, J. C., Qu, Y. S., Tanada, T. N., Scheuer, T., and Catterall, W. A. (1996) J. Biol. Chem. 271, 15950-15962). A negative charge at this position is highly conserved between mammalian sodium channel isoforms. We have constructed mutations of the corresponding residue (Asp-1612) in the rat cardiac channel isoform (rH1) and shown that the lowered affinity occurs primarily through an increase in the toxin/channel dissociation rate koff. Further, we have used thermodynamic mutant cycle analysis to demonstrate a specific interaction between this anionic amino acid and Lys-37 of ApB (DeltaDeltaG = 1.5 kcal/mol), a residue that is conserved among many sea anemone toxins. Reversal of the charge at Asp-1612, as in the mutant D1612R, also affects channel inactivation independent of toxin (-14 mV shift in channel availability). Binding of the toxin to Asp-1612 may therefore contribute both to toxin/channel affinity and to transduction of the effects of the toxin on channel kinetics.

Structure and function of Cerebratulus lacteus neurotoxin B-IV: tryptophan-30 is critical for function while lysines-18, -19, -29, and -33 are not required.

The Cerebratulus lacteus B-toxins are a family of polypeptide neurotoxins known to bind to crustacean voltage-sensitive sodium channels. We have previously shown that in the most abundant homolog, toxin B-IV, Arg-17 in the N-terminal helix and a positive charge at position 25 in the loop region are essential for function. In this report, we target a tryptophan residue at position 30, as well as lysine residues found in both the N-terminal helix and loop regions by polymerase chain reaction mutagenesis, to determine their contributions to toxin activity. Substitution of Trp-30 with a serine causes a more than 40-fold reduction in specific toxicity, whereas replacement by tyrosine and phenylalanine is well tolerated. The secondary structures of both these muteins are identical to that of the wild-type toxin as determined by circular dichroism spectroscopy. Thermal denaturation experiments also show that their conformational stabilities are intact. These results demonstrate that an aromatic residue at this position is required for toxin function. Charge neutralizing substitutions of Lys-18 and Lys-19 located in the N-terminal helix have very little effect on toxicity, suggesting the nonessentiality of these residues. Similar results are also obtained for the charge neutralizing muteins for Lys-29 and Lys-33 in the loop region. Interestingly, reduction experiments demonstrate that both K29N and W30S are more sensitive to reducing agent than wild-type B-IV, raising the possibility that the loop sequence may modulate toxin stability.

High affinity dimerization by Ski involves parallel pairing of a novel bipartite alpha-helical domain.

c-Ski protein possesses a C-terminal dimerization domain that was deleted during the generation of v-ski, and has been implicated in the increased potency of c-ski in cellular transformation compared with the viral gene. The domain is predicted to consist of an extended alpha-helical segment made up of two motifs: a tandem repeat (TR) consisting of five imperfect repeats of 25 residues each and a leucine zipper (LZ) consisting of six heptad repeats. We have examined the structure and dimerization of TR or LZ individually or the entire TR-LZ domain. Using a quenched chemical cross-linking method, we show that the TR dimerizes with moderate efficiency (Kd = 4 x 10(-6) M), whereas LZ dimerizes poorly (Kd > 2 x 10(-5) M). However, the entire TR-LZ domain dimerizes efficiently (Kd = 2 x 10(-8) M), showing a cooperative effect of the two motifs. CD analyses indicate that all three proteins contain predominantly alpha-helices. Limited proteolysis of the TR-LZ dimer indicates that the two helical motifs are linked by a small loop. Interchain disulfide bond formation indicates that both the LZ and TR helices are oriented in parallel. We propose a model for the dimer interface in the TR region consisting of discontinuous clusters of hydrophobic residues forming "leucine buttons."

Role for Pro-13 in directing high-affinity binding of anthopleurin B to the voltage-sensitive sodium channel.

Anthopleurin A (ApA) and B (ApB) are 49-amino acid polypeptide toxins from the Pacific sea anemone Anthopleura xanthogrammica that interfere with inactivation of voltage-gated sodium channels. ApA, which differs from ApB in seven of the 49 amino acids, displays markedly enhanced isoform selectivity compared with ApB, acting preferentially on cardiac over neuronal sodium channels. Previous studies in this lab have indicated the importance of two unique charged residues in ApB, Arg-12 and Lys-49, in this toxin's ability to discriminate between neuronal and cardiac sodium channels. Likewise, a double mutant (R12S/K49Q) recently characterized in this lab (Khera et al., 1995) displays a greatly reduced affinity for neuronal channels, essentially restoring the discriminatory ability of ApA. When the remaining five residues unique to ApB are individually converted to those of ApA, only ApB (Pro-13) shows a major effect, reducing the affinity of the new mutant toxin (P13V) against both channel isoforms approximately 10-fold. This effect is most likely the result of a conformational rearrangement within the surrounding cationic cluster which includes Arg-12 and -14, as well as Lys-49. However, when placed into the context of the double mutant R12S/K49Q a unique effect is observed: the new triple mutant (R12S/P13V/K49Q) is no longer able to discriminate effectively between channel isoforms. Its affinity for the neuronal sodium channel is significantly enhanced compared to either P13V or to the double mutant R12S/K49Q. These results are consistent both with our proposed model (Khera et al., 1995) and with the recently reported solution structure of ApB, which implicate the cationic cluster in both affinity and channel isoform selectivity. We suggest that the P13V mutation results in a shift in the relative orientation of cationic residues within the large flexible loop between residues 9-18, thus strengthening their interactions with target sequences of the neuronal sodium channel.

Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus.

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.

The role of exposed tryptophan residues in the activity of the cardiotonic polypeptide anthopleurin B.

Scorpion and sea anemone venoms contain several polypeptides that delay inactivation of voltage-sensitive sodium channels via interaction with a common site. In this report, we target exposed hydrophobic residues at positions 33 and 45 of anthopleurin B (ApB) by polymerase chain reaction mutagenesis to ascertain their contribution to toxin activity. Nonconservative replacements are not permitted at position 33, indicating that Trp-33 may play an important structural role. Strikingly, the relatively conservative substitution of Trp-33 by phenylalanine results in major reductions in binding affinity for both the cardiac and neuronal channel isoforms as measured by ion flux, whereas substitution with tyrosine is tolerated and exhibits near wild-type affinities, suggesting that either the ability to form a hydrogen bond or the amphiphilic nature of the side chain are important at this position. Electrophysiological analysis of W33F indicates that its diminished affinity is primarily due to a decreased association rate. Analysis of a panel of mutants at Trp-45 shows only modest changes in apparent binding affinity for both channel isoforms but significant effects on Vmax. In neuronal channels, the maximal levels of uptake for W45A/S/F are about 50% those seen with ApB. This effect is also observed for W45A and W45S in the cardiac model, wherein W45F is normal. These results suggest that a hydrophobic contact is involved in toxin-induced stabilization of the open conformation of the cardiac sodium channel. We conclude that Trp-33 contributes significantly to apparent affinity, whereas Trp-45 does not appear to affect binding per se. Furthermore, W33F is the first ApB mutant that displays a significantly altered association rate and may prove to be a useful probe of the channel binding site.

Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltage-sensitive sodium channel.

Anthopleurin B is a potent anemone toxin that binds with nanomolar affinity to the cardiac and neuronal isoforms of the voltage-gated sodium channel. A cationic cluster that includes Arg-12, Arg-14 and Lys-49 has been shown previously to be important in this interaction. In this study, we have used site-directed mutagenesis to determine the contribution to activity of two aliphatic residues, Leu-18 and Ile-43, that have previously been experimentally inaccessible. Leu-18, a residue proximal to the cationic cluster, plays a critical role in defining the high affinity of the toxin. In ion flux studies, this is exemplified by the several hundredfold loss in affinity (231-672-fold) observed for both L18A and L18V toxins on either isoform of the sodium channel. When analyzed electrophysiologically, L18A, the most severely compromised mutant, also displays a substantial loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms. This difference in affinities may reflect an increased preference of the L18A mutant for the closed state of the neuronal channel. In contrast, Ile-43, a residue distal to the cationic cluster, plays at most a very modest role in affinity toward both isoforms of the sodium channel. Only conservative substitutions are tolerated at this position, implying that it may contribute to an important structural component. Our results indicate that Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date. These results have extended the binding site beyond the cationic cluster to include Leu-18 and broadened our emphasis from the basic residues to include the crucial role of hydrophobic residues in toxin-receptor interactions.

Importance of highly conserved anionic residues and electrostatic interactions in the activity and structure of the cardiotonic polypeptide anthopleurin B.

Several polypeptide toxins from sea anemones caused delayed inactivation of mammalian voltage-dependent sodium channels, resulting in a positive inotropic effect on the heart. Anthopleurin B (ApB), a toxin produced by the sea anemone Anthopleura xanthogrammica, is the most potent of all known anemone toxins. Previous studies in this laboratory have both defined and revealed an important role for the cationic cluster of Arg-12, Arg-14, and Lys-49 in the expression of ApB's biological activity. In the present investigation, we explore the role of all remaining charged residues by producing and characterizing mutants of ApB at Asp-7, Asp-9, Lys-37, His-39, and His-34. Recombinant toxins have been purified to homogeneity and their abilities to enhance veratridine-dependent sodium uptake in cell lines expressing either the neuronal or cardiac isoform of the sodium channel evaluated. Replacement of Asp-7 results in a product that fails to fold, while muteins H39A and H34A have activities very similar or identical to wild-type ApB. In contrast, the D9N and K37A muteins are 7-12-fold less active that wild-type ApB, and truncation of the side chain in D9A results in a further decrease in activity, especially in the cardiac model. We conclude that although a negative charge per se is not essential at position 9, the presence of a hydrogen-bond forming side chain is critical both for appropriate folding and for interaction with the sodium channel. Because the K37A and H39A mutant toxins can fold normally, neither Lys-37 nor His-39 seem to participate in an intramolecular salt bridge, in contrast to suggestions arising from NMR studies of ApA and ApB. However, Lys-37 may play a role in channel interaction.

Multiple cationic residues of anthopleurin B that determine high affinity and channel isoform discrimination.

Site 3 sea anemone toxins modify inactivation of mammalian voltage-gated Na channels. One variant, anthopleurin A (ApA), effectively selects for cardiac over neuronal mammalian isoforms while another, anthopleurin B (ApB), which differs in 7 of 49 amino acids, modifies both cardiac and neuronal channels with high and approximately equal affinity. Previous investigations have suggested an important role for cationic residues in determination of toxin activity, and our single-site mutagenesis studies have indicated that isoform discrimination can be partially explained by the unique cationic residues Arg-12 and Lys-49 of anthopleurin B (ApB). Here, we have further investigated the role of cationic residues by characterizing toxin mutants in which two such residues are replaced simultaneously. The ApB double mutants R14Q-K48A (cationic residues identical in both ApA and ApB), R12S-K49Q (cationic residues unique to ApB), and R12S-R14Q (cationic residues located in the unstructured loop shared among anemone toxins) were constructed by site-directed mutagenesis and their biological activities characterized by sodium uptake assays in cell lines expressing the neuronal (N1E-115) or cardiac (RT4-B) isoform of the Na channel. Each double mutant displayed reduced activity compared with wild type, but none were completely inactive. Neutralization of the proximal cationic residues (R12 and R14) was the most effective, reducing affinity 72-fold (neuronal) and 56-fold (cardiac). Substitution of cationic residues that differed between ApB and ApA (R12S-K49Q) reduced affinity of the toxin for neuronal channels to a much greater extent than for cardiac channels, producing affinities only slightly lower than for ApA in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of the unique cationic residues arginine 12 and lysine 49 in the activity of the cardiotonic polypeptide anthopleurin B.

Sea anemone toxins are potentially important tools for understanding the pharmacology of voltage-sensitive sodium channels. We have previously described a bacterial expression system capable of producing large amounts of one such toxin, anthopleurin B (ApB), which delays channel repolarization (Gallagher, M. J., and Blumenthal K. M. (1992) J. Biol. Chem. 267, 13958-13963). It has been suggested that cationic residues are a major determinant of anemone toxin binding. In this paper, we describe characterization of three mutants at each of two unique cationic sites of ApB, Arg-12 and Lys-49. The activities of all mutants on cardiac and neuronal sodium channels have been compared with that of wild-type ApB. Mutation of Lys-49 has relatively minor effects on toxicity, whereas the mutant R12A, but not R12S or R12K, is severely impaired. These results indicate that cationic residues per se are not absolutely required at either position, but that polar side chains at position 12 contribute significantly to binding affinity. Furthermore, Arg-12 appears to be involved in the toxin's ability to discriminate between neuronal and cardiac sodium channels.

Role of the cationic residues arginine 14 and lysine 48 in the function of the cardiotonic polypeptide anthopleurin B.

Polypeptide neurotoxins from sea anemones have been useful biological probes for sodium channel function. Cationic residues, specifically Arg-14, which is conserved in essentially all known sea anemone toxins, have been generally thought to be important determinants of their binding affinity and/or efficacy. In the present study, we have constructed site-directed mutants of the Anthopleura xanthogrammica toxin anthopleurin B (ApB) at Arg-14 and Lys-48 to characterize the roles played by these cationic residues in the biological activity of the toxin. Using a bacterial expression system developed in this laboratory, we have produced recombinant proteins having three substitutions at each of these positions. The proteins produced have been purified to homogeneity and have structures and conformational stabilities identical to wild-type ApB. We have assayed the mutants by determining their ability to enhance the veratridine-dependent uptake of sodium by both N1E-115 neuroblastoma cells and RT4-B cells, which express the cardiac/denervated skeletal muscle sodium channel. All mutants showed activities only slightly reduced from that of wild-type ApB, with the greatest reductions (4- and 6-fold) being observed for the mutants R14A and K48A, respectively. We conclude that contrary to results from chemical modification studies, Arg-14 is not essential for the biological activity of the toxin. Our data also indicate that Lys-48 plays a small but discernible role in the toxin-receptor interaction.

Cloning and expression of wild-type and mutant forms of the cardiotonic polypeptide anthopleurin B.

Venom of the sea anemone Anthopleura xanthogrammica contains a minimum of three polypeptide toxins capable of prolonging the repolarization phase of the action potential. A synthetic gene for the most toxic of the Anthopleura toxins, anthopleurin B (ApB), has been designed, synthesized, and expressed as a fusion protein with the gene 9 product of bacteriophage T7 in Escherichia coli. The fusion protein has been purified and its disulfide bonds reoxidized using glutathione redox couples. Upon cleavage with staphylococcal protease, this protocol yields approximately 1 mg of native ApB/liter of original culture. The recombinant protein has been shown to be identical to natural ApB with respect to amino acid composition, amino-terminal sequence, secondary structure, high pressure liquid chromatographic mobility, and biological activity. A second form of ApB containing additional residues of glycine and arginine at its amino terminus has also been characterized. This protein, termed GR-ApB, is identical in specific activity to the wild-type form. This work lays the groundwork for a detailed analysis of ApB structure and action by site-directed mutagenesis.

Mutagenesis of Cerebratulus lacteus neurotoxin B-IV identifies NH2-terminal sequences important for biological activity.

We have previously demonstrated that a synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV can be expressed in bacteria, and the recombinant toxin purified and refolded (Howell, M. L., and Blumenthal, K. M. (1989) J. Biol. Chem. 264, 15268-15273). This toxin, which contains an NH2-terminal methionine residue not present in authentic B-IV, has a specific toxicity 35-40% that of the naturally occurring form. Deletion of the codon for the NH2-terminal methionine allows expression of fully active recombinant B-IV, demonstrating that hydroxylation of Pro-10 is not important for biological activity. Site-directed mutagenesis of the des-Met(-1) form has been employed to analyze the contribution of NH2-terminal sequences of this toxin to its activity. We have emphasized replacement of helix-favoring residues by helix-destabilizing ones which are otherwise sterically similar. When Ala-3 or Ala-8 is replaced by serine, little or no effect on specific toxicity is observed. However, the double mutant in which both alanines are substituted with serine is more than twice as active as natural B-IV, although the secondary structures and conformational stabilities of the wild-type and mutant forms are the same. When Ala-3 and 8 are simultaneously replaced with glycine, the resulting toxin displays an activity similar to that of the wild-type form. The conformational properties of this mutant are unchanged from that of either wild-type or the serine double mutant. These data indicate that insertion into the NH2-terminal region of toxin B-IV of residues which can participate in hydrogen bond formation enhances biological activity of the protein.

Identification of oleic acid binding sites in cytolysin A-III from the heteronemertine Cerebratulus lacteus.

We previously demonstrated that binding of oleic acid to Cerebratulus lacteus cytolysin A-III results in aggregation of this monomeric protein to a tetramer, concomitant with an increase in hemolytic activity. In the present study, incubation of cytolysin A-III with [14C]-oleic acid in the presence of a water-soluble carbodiimide results in covalent incorporation of a maximum of two molecules of the fatty acid into the protein. Labeling is restricted to two large tryptic peptides. Sequence analysis of peptide mixtures derived from the labeled protein reveals that the predominant sites of labeling are Lys-31 and Lys-71; the latter site is part of the C-terminal amphipathic helix previously shown to be important for hemolytic activity while the former lies in the other significant hydrophobic region of this largely hydrophilic protein.

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin.

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.

Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV.

A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli. Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E. coli beta-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient. The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture. The recombinant protein is identical with B-IV isolated from Cerebratulus with respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10 by Pro. Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity. The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10 may also contribute.

Functional interaction between Cerebratulus lacteus cytolysin A-III and phospholipase A2. Implications for the mechanism of cytolysis.

A study on the interaction between bee venom phospholipase A2 and Cerebratulus lacteus cytolysin A-III, a major hemolysin secreted by this organism has been carried out. The hemolytic activity of A-III in phosphate-buffered saline is increased 5-fold in the presence of phospholipase A2 from bee venom. Dansylphosphatidylethanolamine (DPE) labeled, phosphatidylcholine-containing liposomes and human erythrocyte membranes were employed to study the interaction between these two proteins. In DPE-liposomes, A-III alone had no effect on DPE fluorescence nor did it enhance either the phospholipase A2-dependent fluorescence increase or blue shift in emission maximum, indicating that the cytolysin is not a major phospholipase A2-activator. However, when DPE was incorporated into erythrocyte membranes, A-III alone induced a 40% fluorescence increase and a 5 nm blue shift, implying a transient activation of an endogenous phospholipase A2. Further studies using synthetic lysophosphatidylcholine and free fatty acids demonstrated that the hemolytic activity of A-III is potentiated by free fatty acids, a product of phospholipid degradation catalyzed by phospholipase A2. Together, sublytic concentrations of A-III and nonlytic concentrations of oleic acid cause extensive cell lysis. Subsequent analysis of this phenomenon by gel filtration chromatography, analytical ultracentrifugation, chemical cross-linking, and measurement of [14C]oleic acid binding by the cytolysin demonstrated that binding of oleic acid to A-III causes aggregation of the toxin molecules to a tetrameric form which has a higher alpha-helix content and a greater activity than the monomer.

Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity.

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.