Effect on sphingomyelin-containing liposomes of phospholipase D from Corynebacterium ovis and the cytolysin from Stoichactis helianthus

The toxic, sphingomyelin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from Corynebacterium ovis was purified to near homogeneity. It has a molecular weight of 31 000 and a pI of approx. 9.8. Although not cytolytic itself, it protected red cells from hemolysis by staphylococcal sphingomyelinase (beta-hemolysin) and helianthus toxin. The apparently non-enzymatic cytolysin (helianthus toxin) from the sea anemone Stoichactis helianthus also interacts with membrane sphingomyelin. C. ovis and helianthus toxins were compared with regard to their effects on liposome model membranes, and they were found both to produce changes analogous to those in erythrocytes. Only helianthus toxin caused release of trapped glucose marker, but liposomes could be protected from release by pretreatment with C. ovis toxin. Both toxins demonstrated binding to sphingomyelin-containing liposomes, but only the bacterial sphingomyelinase catalyzed the release of choline from these vesicles.     

Two-dimensional crystallization on lipid monolayers and three-dimensional structure of sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers.

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17,500.     

Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus

Summary When present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10^–10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K⁺ and Na⁺ than to Cl^– and SO ₄ ⁼ , but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K⁺ with respect to SO ₄ ⁼ , whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added.     

Primary structure of porcine muscarinic acetylcholine receptor III and antagonist binding studies

The complete amino acid sequence of porcine muscarinic acetylcholine receptor III has been deduced by cloning and sequencing the genomic DNA. The antagonist binding properties of muscarinic acetylcholine receptor III expressed from the cloned DNA in Xenopus oocytes correspond most closely to those of the pharmacologically defined M2 glandular (III) subtype.     

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Molecular cloning of rat cytolysin

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.     

Three-dimensional structure of the detergent-solubilized Vibrio cholerae cytolysin (VCC) heptamer by electron cryomicroscopy

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that inserts a lytic water-filled channel into susceptible host membranes. Assembly of the toxin on cell surfaces may be enhanced by two tandem lectin domains, in addition to direct interactions with lipids and cholesterol within the membrane itself. We used single-particle electron cryomicroscopy (cryoEM) to generate a low-resolution molecular structure of the detergent-solubilized VCC oligomer to 20 Å resolution. After confirming a heptameric arrangement of individual protomers, sevenfold averaging around the central pore was utilized to improve the structure. Docking of the previously determined VCC protoxin crystal structure was possible with rigid-body rearrangements between the cytolytic and lectin domains. A second cryoEM reconstruction of a truncated VCC mutant supported the topology of our model in which the carboxyl-terminal lectin domain forms “spikes” around the toxin core with the putative carbohydrate receptor-binding site accessible on the surface of the oligomer. This finding points to an assembly mechanism in which lectin domains may remain bound to receptors on the cell surface throughout assembly of the cytolytic toxin core and explains the hemagglutinating activity of purified toxin. Our model provides an insight into the structural rearrangements that accompany VCC-mediated cytolysis and may aid in the engineering of novel pore-forming toxins to attack specific cells towards therapeutic ends.     

The immunologic properties of Legionella cytolysin

Immunogenic properties of cytolysin were studied in experiments on guinea pigs. Preliminary immunization with cytolysin led to the suppression of response to ConA in lymphocytes not adhering to nylon wool and to the stimulation of response to Legionella antigens in lymphocytes adhering to nylon wool. For a month after infection with L. pneumophila the suppression of the proliferative activity of lymphocytes in the spleen of the immunized animals in response to ConA and Legionella antigens was observed, while in the lungs transitory suppression of response to ConA and Legionella antigens was followed by the restoration and then stimulation of proliferation in response to T-cell mitogen and specific antigens. The data obtained in these experiments indicate the capacity of cytolysin for modulating the development of immune response.     

Primary structure of a DNA- and heparin-binding domain (Domain III) in human plasma fibronectin

The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.     

Primary structure of streptococcal proteinase. III. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain.

The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.     

Characteristics of the cytolysin of Legionella pneumophila

A thermolabile cytolysin was purified from liquid culture of L. pneumophila. Its homogenicity was determined by the Ouchterlony double immunodiffusion and SDS-electrophoresis in polyacrylamide gel. The molecular weight of cytolysin was ca. 37 kDa. Analysis of amino acid composition revealed a high proportion of aromatic, dicarbonic amino acids, and methionine. The minimal cytolytic concentration for CHO cells and erythrocytes was ca. 1 microgram/ml: Purified cytolysin in doses of 10-60 micrograms caused haemorrhage and necrosis when injected i. c. into guinea pigs.     

Diterpenes of helianthus rigidus and H. Salicifolius

The chloroform extract of Helianthus rigidus gave ciliaric acid and 16-hydroxy-11-kauren-19-oic acid. H. salicifolius furnished these compounds as well as (?)-kauranol.     

Monoclonal antibodies to Legionella pneumophila cytolysin

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.