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To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the [alpha]-cobratoxin-[alpha]7 receptor complex. This model was achieved by using (i) a three-dimensional model of the [alpha]7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the [alpha]-cobratoxin and [alpha]7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin-receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists.

An expression cDNA library was constructed with poly(A)-rich RNA extracted from the collar of the giant African snail, Achatina fulica Férussac. A 1.9-kbp cDNA clone encoding a precursor of antibacterial glycoprotein of the snail, achacin, was isolated from the cDNA expression library. The cDNA sequence contains an open reading frame with 1593-nucleotide residues. The deduced amino acid sequence of this achacin precursor starts with a 29-residue leader peptide followed by a 502- residue mature peptide (56 kDa) with four possible N-glycosylation sites, Asn-Xaa-Ser or Asn-Xaa-Thr. The Northern-blot analysis proved that the achacin precursor was specifically expressed in the tissue of snail collar and processed to mature achacin, cDNA inserts encoding achacin precursor were subcloned into expression plasmids. Three kinds of expressed polypeptides were cross-reacted with rabbit antiserum raised against achacin. The largest polypeptide (Mr 63000) should be the achacin precursor. [ABSTRACT FROM AUTHOR]

Phospholipase A2 (PLA2) genes expressed in the venom glands of the sea snake, Laticauda semifasciata, were investigated. Both mRNAs, encoding group IA (without a pancreatic loop) and group IB (with pancreatic loop), were detected from venom glands by Northern blot hybridization analysis and RT-PCR. The results of quantitative PCR analysis indicated that the expression amount of group IA genes was around 100–300 times greater than that of group IB genes. Sequence analysis of 5′-upstream regions and a reporter gene assay of the genes (groups IA and IB) previously cloned showed that the functional sequence (411 bp) was inserted in the 5′-flanking region of the group IA PLA2 genes. It seemed that the contribution of the inserted sequence to the amount of transcribed mRNAs was greater than that of number of genes present in the genome. Comparative analysis of the 5′-flanking sequences from several snake genes encoding toxic PLA2s revealed that this sequence was probably inserted into an ancestral gene of PLA2 with a pancreatic loop. After the duplication of the gene, which contained the inserted sequence, the PLA2 gene without a pancreatic loop evolved from one of the duplicate genes. This inserted sequence might determine the future of the genes expressed in the venom glands. [Copyright &y& Elsevier]

We have cloned two phospholipase A2 (PLA2) DNA complementary to RNA that contained nucleotide sequences encoding pancreas loop by the reverse transcription-polymerase chain reaction cloning procedure using messenger RNA isolated from Laticauda semifasciata pancreas. Additionally, a gene clone encoding PLA2 with the pancreatic loop sequence was isolated from a L. semifasciata genomic library. Subsequent sequence analysis revealed that PLA2 clones encoding group IB″ PLA2. Comparative analysis of group IA and IB″ PLA2 genes revealed that the exon-intron organization is conserved in the genes of both groups. The invaded sequences in the second intron were very similar to those of the L. semifasciata group IA gene. This observation suggested that the integration of the invaded sequences occurred before the divergence of groups IA and IB″ during the evolution of PLA2 gene. The comparative analysis revealed that the arising of group IA PLA2 occurred by the deletion and substitution of nucleotide sequences in exon III region during the process of accelerated evolution. [Copyright &y& Elsevier]