Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.     

Membrane-Pore Forming Characteristics of the Bordetella pertussis CyaA-Hemolysin Domain

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.     

The Crystal Structure of Bacillus cereus HblL1

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL₁ component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL₁ and HblB, and suggests a head to tail dimer might form, burying the HblL₁ beta tongue region.     

Isolation and biological characterization of neurotoxic compounds from the sea anemone Lebrunia danae (Duchassaing and Michelotti, 1860)

This paper describes two neurotoxic proteins obtained from the Caribbean sea anemone Lebrunia danae. To assess the neurotoxic activity of the venom of L. danae, several bioassays were carried out, and to evaluate the effect of the toxin, Median Lethal Doses (LD₅₀) were determined in vivo using sea crabs (Ocypode quadrata) and Artemia salina nauplii with the crude extract of the proportion of 2.82 mg/m. The proteins with neurotoxic effects were isolated using low-pressure liquid chromatography. The fractions containing the neurotoxic activity were analyzed by SDS-PAGE and showed protein bands with an apparent molecular weight of 62.50 kDa (LdNt1) and 58 kDa (LdNt2). To demonstrate that these proteins were indeed responsible for the neurotoxic activity observed, we injected a small fraction of the purified protein into the third walking leg of a crab and observed the typical convulsions, paralysis and death provoked by neurotoxins. Hemolytic activity was also tested for 0.238 mg of crude extract; the hemolytic value was 39.5, 49.6 and 50.1% for cow, sheep and pig erythrocytes, respectively.     

P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca²⁺-sensitive K⁺ (K_Ca3.1) and Cl⁻ channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca²⁺-dependent activation of K_Ca3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of K_Ca3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.     

Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-Terminal Domain to Interact with a Target Receptor—Aedes aegypti Membrane-Bound Alkaline Phosphatase

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa high-purity isolated DIII fragment derived from the Cry4Ba mosquito-specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor—Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP). Binding analysis via dotblotting revealed that the Cry4Ba-DIII truncate was capable of specific binding to nitrocellulose-bound Aa-mALP, with a binding signal comparable to its 65 kDa Cry4Ba-R203Q full-length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba-DIII exhibited a rather weak binding to Aa-mALP with a dissociation constant (K_d) of ≈1.1 × 10⁻⁷ M as compared with the full-length toxin. Intermolecular docking between the Cry4Ba-R203Q active toxin and Aa-mALP suggested that four Cry4Ba-DIII residues, i.e., Glu⁵²², Asn⁵⁵², Asn⁵⁷⁶, and Leu⁶¹⁵, are potentially involved in such toxin–receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A-impaired toxin also exhibited a reduction in binding capability to the surface-immobilized Aa-mALP receptor, while two bio-inactive DII-mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C-terminal domain of Cry4Ba for serving as a potential receptor-binding moiety in which DIII-Leu⁶¹⁵ could conceivably be exploited for the binding to Aa-mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.     

DAMGO recognizes four residues in the third extracellular loop to discriminate between μ- and κ-opioid receptors

Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of κ-opioid receptor with the corresponding region of μ-opioid receptor gives high affinity for [

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-Ala², N-MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), a μ-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between μ- and κ-opioid receptors by DAMGO. In the present study, we constructed further six μ/κ-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between μ- and κ-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu²⁹⁷ of κ-opioid receptor and Lys³⁰³ of μ-opioid receptor in one region, and the difference between Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor and Val³¹⁶, Trp³¹⁸ and His³¹⁹ of μ-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, κ (E297K+Y313H+Y312W+S310V), in which the Glu²⁹⁷, Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (K_d=8.7±1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between μ- and κ-opioid receptors by DAMGO.     

Functional preference of the constituent amino acid residues in a phage‐library‐based nonphosphorylated inhibitor of the Grb2‐SH2 domain

Abstract: A nonphosphorylated disulfide‐bridged peptide, cyclo(Cys‐Glu¹‐Leu‐Tyr‐Glu‐Asn‐Val‐Gly‐Met‐Tyr⁹‐Cys)‐amide (termed G1) has been identified, by phage library, that binds to the Grb2‐SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2‐SH2 domain association (IC₅₀ of 15.5 µm ) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2‐SH2 domain in a pTyr‐independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side‐chains of amino acids Glu¹, Tyr³, Glu⁴, Asn⁵, and Met⁸. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2‐SH2 domain in a β‐turn‐like conformation. Replacement of Tyr³ or Asn⁵ with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y‐X‐N consensus sequence similar to that found in natural pTyr‐containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu¹, i.e. the amino acid N‐terminal to Y³, remarkably reduces the binding affinity. The position of the Glu¹ side‐chain is confirmed to provide a complementary role for pTyr³, as demonstrated by the low micromolar inhibitory activity (IC₅₀ = 1.02 µm ) of the nonphosphorylated peptide 11 , G1(Gla¹), in which Glu¹ was replaced by γ‐carboxy‐glutamic acid (Gla).     

Mechanism for the Potential Inhibition Effect of Microcystin-LR Disinfectant By-Products on Protein Phosphatase 2A

The secondary contamination of microcystin disinfection by-products (MC-DBPs) is of concern due to the residual structure similar to their original toxin. Based on identification and preparation, the potential inhibition effect of typical MCLR-DBPs (associated with the oxidation of Adda⁵) on PP2A was confirmed in the sequence of MCLR > P1 > P4 > P3 ≈ P2 > P7 ≈ P6 ≈ P5 > P8. To elucidate the molecular mechanism underlying the inhibition effect, the interaction models for typical MCLR-DBPs and PP2A were constructed using a modeling-based-on-ligand-similarity approach, and the candidate interaction parameters between typical MCLR-DBPs and PP2A were obtained by molecular docking. By analyzing the correlation between inhibition data and candidate interaction parameters, the key interaction parameters were filtered as hydrogen bonds “Adda⁵”←Asn₁₁₇, “Adda⁵”←His₁₁₈, MeAsp³←Arg₈₉, Arg⁴←Arg₂₁₄, Arg⁴→Pro₂₁₃; ionic bonds Glu⁶-Arg₈₉, Asp₈₅-Mn₁²⁺, Asp₅₇-Mn₂²⁺; and metal bonds Glu⁶-Mn₁²⁺, Glu⁶-Mn₂²⁺. With the gradual intensification of chlorination, Adda⁵ was destroyed to varying degrees. The key interactions changed correspondingly, resulting in the discrepant inhibition effects of typical MCLR-DBPs on PP2A.     

Inhibition of the hemolytic action of and lysins of Staphylococcus pyogenes by Corynebacterium hemolyticum, C. ovis and C. ulcerans

Extracellular products of Corynebacterium hemolyticum, C. ovis and C. ulcerans were purified by methanol precipitation and ion exchange chromatography in CM-Sephadex. Three different activities were determined i.e. dermonecrotoxin, phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) and inhibition of staphylococcal β lysin. All these activities were found in the same fraction. Sheep and rabbit erythrocytes were treated with partially purified toxin and the hemolytic activities of staphylococcal α and β lysins and phospholipase C (phosphatidylcholine cholinephosphohydrolase EC 3.1.4.3) of Clostridium perfringens were estimated quantitatively. Sheep erythrocytes were completely protected against the hemolytic action of staphylococcal α and β lysins. Excess of the other hemolytic agents was needed to cause hemolysis in treated erythrocytes, namely 4 HU of α lysin in rabbit erythrocytes, 256 HU of phospholipase C in sheep erythrocytes and 16 HU of amboceptor in immune hemolysis. Sphingomyelins form the major part of phospholipids in the cell membrane of sheep erythrocytes and are hydrolyzed by the corynebacterial phospholipase D which is probably the reason for the protective action of the toxin.     

Isolation of a hemolytic,toxic phospholipase from the venom of the Australian red-bellied black snake (Pseudechis porphyriacus)

A weak toxin was isolated from the venom of the Australian red-bellied black snake, Pseudechis porphyriacus, by ion-exchange chromatography on Bio-Rex 70, followed by gel filtration on Sephadex G-50. The toxin, pseudexin, accounts for 25% of the venom and has an ld₅₀ (i.p.) of 480 μg/kg mouse. It is a polypeptide of 143 amino acid residues and has a formula weight of 16,659. Pseudexin has phospholipase A (phosphatidate acyl-hydrolase, EC 3.1.1.4) activity. Modification of the toxin with p-bromophenacyl bromide resulted in a 99·9% loss of phospholipase activity and a reduction of its toxicity. The toxin, like other phospholipases, caused indirect hemolysis of washed erythrocytes. In addition, pseudexin directly hemolysed erythrocytes under conditions in which phospholipases are normally non-hemolytic.     

New indolicidin analogues with potent antibacterial activity*

Abstract: Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH₂, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH₂ in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe^4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.     

Purification of a cytotoxic protein from Pseudomonas aeruginosa.

F. Lutz. Purification of a cytotoxic protein from Pseudomonas aeruginosa. Toxicon17, 467–475, 1979.—A method for preparation of the cytotoxin from Pseudomonas aeruginosa, strain 158, is presented. The procedure described by Scharmann (1976b) was improved by a precipitation step with ammonium sulphate and by subsequent chromatography of the dissolved precipitate on acrylamide-agarose. The recovery of purified toxin was 1 mg from 100 mg protein in supernatant (autolysate). The purity of the preparations was tested by sodium dodecyl sulphate gel electrophoresis, immunodiffusion and enzymatic methods for the presence of other known pseudomonal components.The molecular weight of the toxin was estimated by sodium dodecyl sulphate gel electrophoresis (25,100) and by gel chromatography (22,300–22,800). Amino acid analysis revealed a ratio of acidic to basic amino acids of 4:1. Cysteine was absent. The protein contains neither carbohydrate nor lipids. By disc electrophoresis the toxin separated into 4–6 fractions with the same toxicity and with isoelectric points between 5·0 and 6·4. In a slide adhesion test with human granulocytes the ed₁₀₀ of cell swelling was 9 μg/ml. The ed₅₀ for growth inibition of chicken embryo fibroblasts was 11 μg/ml. At 44 μg/ml the toxin liberates half of the hemoglobin in rabbit red cells, but did not hemolyse erythrocytes of sheep, horse and cow. The ld₅₀ on mice was 25 ng/g body weight. In postmortem histological examination of mice, fatty liver necrosis was observed.     

Properties of synthetic analogs of gramicidin S containing L-serine or L-glutamic acid residue in place of L-ornithine residue

In order to investigate the biological role of the δ-amino groups of the Orn residue in gramicidin S, the four analogs, [Ser²]-, [Ser^2,2′]-, [Glu²]- and [Glu^2,2′]-gramicidin S were synthesized by a solution method. Except for the last one, these analogs show antibiotic activity to a certain extent against gram-positive micro-organisms, but the activities are weaker than that of gramicidin S. These results indicate that the δ-amino group of the Orn residue is quite important for exhibiting of the activity of gramicidin S, but is not essential to the activity. NMR and CD studies of these analogs indicate that these analogs possess four intramolecular hydrogen bonds between the Val and Leu residues similar to those of GS, and the removal or diminution of the δ-amino group of ornithine residues give the considerable influence to the dihedral angle of N^αH-C^αH of D-Phe residues and consequently affect the type II′β-turn structure of the cyclic peptides. © Munksgaard 1996.     

Insight into the Molecular Mechanism for the Discrepant Inhibition of Microcystins (MCLR,LA, LF,LW, LY) on Protein Phosphatase 2A

Microcystins (MCs) exhibit diversified inhibition effects on protein phosphatases (PPs) due to their structural differences. To fully evaluate the potential mechanism for the discrepant inhibition effects, the five most frequent MCs with varying residues at position Z⁴ were selected as the tested toxins. Their inhibition sequence on PP2A was detected as follows: MCLR > MCLW > MCLA > MCLF > MCLY. Combined with homology modeling and molecular docking technology, the major interaction parameters between the MCs and PP2A were obtained. The correlation analysis for the major interaction parameters and inhibition effects showed that the hydrophobicity of Z⁴ had an important influence on the interaction of the MCs to PP2A. The introduction of hydrophobic Z⁴ directly weakened hydrogen bonds Z⁴→Pro₂₁₃ and Z⁴←Arg₂₁₄, indirectly weakened hydrogen bonds Adda⁵←Asn₁₁₇, Glu⁶←Arg₈₉, and MeAsp³←Arg₈₉, but indirectly enhanced ionic bonds Glu⁶←Arg₈₉, Glu⁶-Mn₁²⁺, and Glu⁶-Mn₂²⁺. In this way, the combination of the MCs with PP2A was blocked, and thus, the interactions between PP2A and the Mn²⁺ ions (in the catalytic center) were further affected; metal bonds Asp₈₅-Mn₁²⁺ and Asp₈₅-Mn₂²⁺ were weakened, while metal bond His₂₄₁-Mn₁²⁺ was enhanced. As a result, the interactions in the catalytic center were inhibited to varying degrees, resulting in the reduced toxicity of MCs.     

New biologic functions — Selenium-dependent nucleic acids and proteins

Selenium occurs normally in living things as a highly specific component of certain enzymes and amino acid transfer nucleic acids (tRNAs). In bacteria, biosynthesis of essential selenoenzymes has been shown to be unaffected by wide variations in sulfur levels. The naturally occurrring selenoenzymes so far identified from bacterial sources include glycine reductase, certain formate dehydrogenases, a hydrogenase, nicotinic acid hydroxylase, xanthine dehydrogenase and thiolase. The selenoenzyme, glutathione peroxidase, and three other selenoproteins of unknown function have been isolated from animals. In certain enzymes, e.g. glycine reductase, formate dehydrogenase, hydrogenase and glutathione peroxidase, the chemical form of selenium has been identified as selenocysteine. One enzyme, a bacterial thiolase, contains selenomethionine rather than selenocysteine. A labile, unidentified form of selenium is present in nicotinic acid hydroxylase, and by inference, xanthine dehydrogenase. The seleno-tRNAs serve as examples of a different type of biological macromolecule that is specifically modified with selenium. The major seleno-tRNAs in Clostridium sticklandii and Escherichia coli have been identified as glutamate and lysine isoaccepting species. The selenium-modified nucleoside is 5-methyl-aminomethyl-2-selenouridine (mnm⁵Se²U), which is the chemical analog of 5-methylaminomethyl-2-thiouridine, a previously identified minor base of E. coli tRNA₂^Glu. The seleno-tRNA^Glu of C. sticklandii contains one gram atom of Se per mole of biologically active tRNA. Loss of Se from the modified nucleoside, mnm⁵Se²U, in this tRNA results in concomitant loss of glutamate charging activity suggesting that selenium is essential for interaction of the synthetase and its cognate tRNA.     

Shiga toxin type 2 (Stx2), a potential agent of bioterrorism, has a short distribution and a long elimination half-life, and induces kidney and thymus lesions in rats

Shiga toxin type 2, a major virulence factor produced by the Shiga toxin?Cproducing Escherichia coli, is a potential toxin agent of bioterrorism. In this study, iodine-125 (¹²⁵I) was used as an indicator to describe the in vivo Stx2 biodistribution profile. The rats were injected intravenously (i.v.) with ¹²⁵I-Stx2 at three doses of 5.1?C127.5???g/kg body weight. Stx2 had a short distribution half-life (t _1/2??, less than 6?min) and a long elimination half-life in rat. The toxicokinetics of Stx2 in rats was dose dependent and nonlinear. Stx2 concentrations in various tissues were detected at 5-min, 0.5-h, and 72-h postinjection. High radioactivity was found in the lungs, kidneys, nasal turbinates, and sometimes in the eyes, which has never been reported in previous studies. In a preliminary assessment, lesions were found in the kidney and thymus.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

The incubation of a solution of the human growth hormone releasing factor analog, [Leu²⁷] hGRF(1-32)NH₂ at pH 7.4 and 37°, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ and [α-Asp⁸, Leu²⁷] hGRF(1-32)NH₂, produced by deamidation of the Asn⁸ residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was estimated to be 400-500 times less potent than the parent Asn⁸ peptide, while [2-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was calculated to be 25 times less potent than the parent Asn⁸ peptide. Three additional analogs of [Leu²⁷] hGRF(1-32)NH₂ containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37°). Based on disappearance kinetics, [Leu²⁷] hGRF(1-32)NH₂ had a half-life of 202 h while the other analogs had the following half-lives: [Leu²⁷, Asn²⁸] hGRF(1-32)NH_: (150h); [Ser⁸, Leu²⁷, Asn²⁸] hGRF(l-32)NH₂ (746h); and [Ser⁸, Leu²⁷] hGRF(1-32)NH₂ (1550 h). After 14 days, incubated samples of the Asn⁸ analogs lost GH releasing potency, while the Ser⁸ analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg⁵² in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu², Phe¹⁵) did not modify the toxin’s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation.     

Detection of a cytolytic toxin in the venom of the stonefish (Synanceia trachynis)

The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an Mr of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.