Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.     

Lipocortin-like anti-phospholipase A2 activity of endonexin

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAF-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid-labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner.     

Immunochemistry of scorpion toxins. Immunogenicity of peptide 19-28 a model of an accessible and relatively rigid region

Peptide 19-28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid alpha-helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti-(peptide-BSA) and the anti-(peptide-resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19-28 suggests a specificity difference in the two antipeptides. Anti-(peptide-BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti-(peptide-resin) preferentially recognizes the N-terminal extremity. Finally we showed that the alpha-helix region remains accessible to antipeptide 19-28 when the toxin is bound to its receptor.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.     

Photoaffinity labeling of scorpion toxin receptors associated with insect synaptosomal Na+ channels

Photoreactive and radioiodinated derivatives of several scorpion toxins acting on insect Na+ channels were prepared without loss of their pharmacological activities. Photoaffinity experiments were carried out on a synaptosomal fraction from the nerve cord of the cockroach Periplaneta americana: with all toxin derivatives, a single specifically labeled band was obtained with a molecular weight of 188,000 +/- 12,000 (n = 17). These results indicate for the first time the molecular weight of the scorpion toxin receptor from the insect nervous system which is probably associated with voltage sensitive Na+ channels. One of these toxins, toxin VII from Tityus serrulatus venom, has been previously shown to be active both in mammals and in insects, in rat brain synaptosomes this toxin labeled a Mr = 31,000 +/- 4,000 band in contrast, to observations in the insect preparation.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin