Shellfish poisons.

In our ongoing search for bioactive metabolites from marine organisms, novel shellfish poisons have been isolated. Pinnatoxins, which are amphoteric polyether compounds, were purified from the Okinawan bivalve Pinna muricata. Pinnatoxins show acute toxicity against mice and activate Ca2+ channels. Two novel alkaloids, pinnamine and pinnaic acid, were also obtained from P. muricata. Pinnaic acid inhibits cytosolic phospholipase (cPLA2). Pteriatoxins, which are pinnatoxin analogs, were isolated from the Okinawan bivalve Pteria penguin. A nanomole-order structure determination of pteriatoxins was achieved by the detailed analysis of 2D-NMR and ESI-TOF MS/MS. This review covers the isolation, structure determination, bioactivity, synthesis, and biogenesis of these shellfish poisons and related compounds.

Production of recombinant Conkunitzin-S1 in Escherichia coli.

Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channels of the Shaker family. The peptide was expressed in insoluble form in fusion with an N-terminal intein. Refolding in the presence of glutathione followed by pH shift-induced cleavage of the fusion protein resulted in a functional toxin as demonstrated by voltage-clamp measurements.

Anatomical correlates of venom production in Conus californicus.

Like all members of the genus, Conus californicus has a specialized venom apparatus, including a modified radular tooth, with which it injects paralyzing venom into its prey. In this paper the venom duct and its connection to the pharynx, along with the radular sac and teeth, were examined using light and transmission electron microscopy. The general anatomy of the venom apparatus resembles that in other members of the genus, but several features are described that have not been previously reported for other species. The proximal (posterior) quarter of the venom duct is composed of a complex epithelium that may be specialized for active transport rather than secretion. The distal portion of the duct is composed of a different type of epithelium, suggestive of holocrine secretion, and the cells display prominent intracellular granules of at least two types. Similar granules fill the lumen of the duct. The passageway between the lumen of the venom duct and pharynx is a flattened branching channel that narrows to a width of 10 micro m and is lined by a unique cell type of unknown function. Granular material similar to that in the venom duct was also found in the lumen of individual teeth within the radular sac. Mass spectrometry (MALDI-TOF) demonstrated the presence of putative peptides in material derived from the tooth lumen, and all of the more prominent species were also evident in the anterior venom duct. Radular teeth thus appear to be loaded with peptide toxins while they are still in the radular sac.

Latent specificity of molecular recognition in sodium channels engineered to discriminate between two "indistinguishable" mu-conotoxins.

mu-Conotoxins (mu-CTX) are potent oligopeptide blockers of sodium channels. The best characterized forms of mu-CTX, GIIIA and GIIIB, have similar primary and three-dimensional structures and comparable potencies (IC(50) approximately 30 nM) for block of wild-type skeletal muscle Na(+) channels. The two toxins are thus considered to be indistinguishable by their target channels. We have found mutations in the domain II pore region (D762K and E765K) that decrease GIIIB blocking affinity approximately 200-fold, but reduce GIIIA affinity by only approximately 4-fold, compared with wild-type channels. Synthetic mu-CTX GIIIA mutants reveal that the critical residue for differential recognition is at position 14, the site of the only charge difference between the two toxin isoforms. Therefore, engineered Na(+) channels, but not wild-type channels, can discriminate between two highly homologous conotoxins. Latent specificity of toxin-channel interactions, such as that revealed here, is a principle worthy of exploitation in the design and construction of improved biosensors.

A new Ig-superfamily member, molluscan defence molecule (MDM) from Lymnaea stagnalis, is down-regulated during parasitosis.

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to approximately 21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper function of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling on non-self recognition.

Structural and biosynthetic properties of peptides in cone snail venoms.

Venoms of the predatory cone snails Conus textile, Conus striatus, and Conus magus were subjected to comprehensive analysis of peptide content. With the fish-eating cone snails C. magus and C. striatus, the most abundant venom peptides were of > 30-50 residues, whereas the predominant peptides in the venom of the mollusc-eating snail, C. textile, were of 20-35 residues. Amino acid sequencing revealed an identical but unusual amino acid in a conserved position in four novel omega-type peptides from the C. textile venom. Two conserved amino acid sequences were obtained from the venoms of both C. magus and C. striatus. The amino acid compositions of the isolated C. textile peptides and the expected processing products of the propeptides (42) were compared. Despite the recovery in abundance of the carboxyl-terminal omega-type peptides, none of the isolated peptides had compositions expected from the propeptide amino-terminal fragments. We conclude that there are likely mechanisms for excluding the amino-terminal propeptide fragments from this venom, resulting in a venom with greater potency. Amounts of the different omega-type peptides in the venom vary widely, suggesting a distinct mechanism that results in the selective synthesis of different bioactive carboxyl-terminal propeptide fragments at elevated levels.