Classification of scorpion toxins according to amino acid composition and sequence

Summary Scorpion venom toxins were systematically classified according to amino acid composition, insertion/deletion events and sequence. The significance of each comparison method and its outcome is discussed in relation to known immunological and structural properties. A general classification of the toxins is proposed that accounts for both the immunological groupings and the differences in mode of action.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector

Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.     

Immunochemistry of scorpion toxins. Synthesis and antigenic properties of a model of a loop region specific to alpha-toxins

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee-Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti-toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.     

Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid.

The amino-terminal amino acid sequences of the pili proteins from four antigenically dissimilar strains of Neisseria gonorrhoeae, from Neisseria meningiditis, and from Escherichia coli were determined. Although antibodies raised to the pili protein from a given strain of gonococcus cross-reacted poorly or not at all with each of the other strains tested, the amino-terminal sequences were all identical. The meningococcal protein sequence was also identical with the gonococcal sequence through 29 residues, and this sequence was highly homologous to the sequence of the pili protein of Moraxella nonliquifaciens determined by other workers. However, the sequence of the pili protein from E. coli showed no similarity to the other sequences. The gonococcal and meningococcal proteins have an unusual amino acid at the amino termini, N-methylphenylalanine. In addition, the first 24 residues of these proteins have only two hydrophilic residues (at positions 2 and 5) with the rest being predominantly aliphatic hydrophobic amino acids. The preservation of this highly unusual sequence among five antigenically dissimilar Neisseria pili proteins implies a role for the amino-terminal structure in pilus function. The amino terminus may be directly or indirectly (through preservation of tertiary structure) important for the pilus function of facilitating attachment of bacteria to human cells.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution, and computer-simulated sequence generation

Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured. The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution,and computer-simulated sequence generation

Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured.The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.     

Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1

Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as muramyldipeptide; N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram-positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide-binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and beta-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, LL-DAP, and DD-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity.     

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation. The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins. Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins. In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number. The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects.     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

Porcine guanylin and uroguanylin: cDNA sequences, deduced amino acid sequences, and biological activity of the chemically synthesized peptides.

Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber.