William R. Gray

Isolation and structure of a peptide toxin from the marine snail Conus magus

Abstract A 14-residue peptide toxin has been isolated from the venom of the marine snail Conus magus. Its amino acid sequence, GlyArgCysCysHisProAlaCysGly LysAsnTyrSerCysNH2, is homologous with those of the previously described conotoxins GI, GII, and GIA from Conus geographus. The new peptide, conotoxin MI, is two to three times more active than the others, and is presumed to act as they do at the acetylcholine receptor of vertebrate neuromuscular junctions.

μ-Conotoxin PIIIA, a New Peptide for Discriminating among Tetrodotoxin-Sensitive Na Channel Subtypes

We report the characterization of a new sodium channel blocker, μ-conotoxin PIIIA (μ-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: where Z = pyroglutamate andO = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, μ-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized μ-conotoxin GIIIA (μ-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of μ-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to μ-PIIIA and μ-conotoxin GIIIA, (2) sensitive to μ-PIIIA but not to μ-GIIIA, and (3) resistant to μ-PIIIA and μ-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, μ-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

KAPPA -CONOTOXIN PVIIA IS A PEPTIDE INHIBITING THE SHAKER K+CHANNEL

κ-Conotoxin Pviia(κ-Pviia), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. κ-Conotoxin Pviia has the following structure. This is the first Conus peptide known to target K+channels. Although the Shaker K+ channel is sensitive to κ-Pviia, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K+ channels were constructed and expressed inXenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that κ-Pviia interacts with the external tetraethyl-ammonium binding site on the Shaker channel. Although both κ-Pviia and charybdotoxin inhibit theShaker channel, they must interact differently. The F425GShaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes κ-Pviiasensitivity. The precursor sequence of κ-Pviia was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the κ-Pviia prepropeptide exhibits striking homology to the ω-, μO-, and δ-conotoxins. Thus, at least four pharmacologically distinct superfamilies ofConus peptides belong to the same “O” superfamily, with the ω- and κ-conotoxins forming one branch, and the δ- and μO-conotoxins forming a second major branch.

A New Family of Conus Peptides Targeted to the Nicotinic Acetylcholine Receptor

In this work, a new family of Conus peptides, the αA-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, αA-conotoxin PIVA, is as follows: where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, αA-conotoxin PIVA and [Pro]αA-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with α-bungarotox- in demonstrate that αA-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

Contryphan Is a D-Tryptophan-containing Conus Peptide

In this report, we document for the first time the occurrence of D-tryptophan in a normally translated polypeptide, contryphan. The peptide, isolated from the venom of the fish-hunting marine snail Conus radiatus, produces the “stiff-tail” syndrome in mice. Characterization of the octapeptide gave the following sequence, where Hyp = 4-trans-hydroxyproline. The presence of D-tryptophan in position 4 of contryphan was confirmed by chemical synthesis. The post-translational epimerization in all other D-amino acid-containing small peptides characterized previously from vertebrates and molluscan systems is in position 2.

A toxic thionin from Pyrularia pubera: Purification, properties, and amino acid sequence☆

A low-molecular-weight cytotoxic protein has been purified from Pyrularia pubera Michx. (Santalaceae). By comparison with the behavior of proteins of known molecular weight during Sephadex G-75 gel filtration and denaturing electrophoresis, a molecular weight of somewhat less than 6000 is indicated. Purification involves ammonium sulfate fractionation followed by either gel filtration on Sephadex G-75 or separation on a carboxymethyl cellulose CM52 column. At concentrations of 0.04 mg/ml the protein causes visible disruption of cultured mouse B16 melanoma cells. The complete amino acid sequence has been determined. The toxin contains 47 amino acids arranged as follows:Lys-Ser-Cys-Cys-Arg-Asn-Thr-Trp-Ala-Arg-Asn-C ys-Tyr-Asn-Val-Cys-Arg-Leu-Pro-Gly-Thr-Ile-Ser-Arg-Glu-Ile-Cys-Ala-Lys- Lys-Cys-Asp-Cys-Lys-Ile-Ile-Ser-Gly-Thr-Thr-Cys-Pro-Ser-Asp-Tyr-Pro-Ly s-OH. The protein is clearly a thionin, as shown by its close resemblance to the thionins from wheat and barley, to the viscotoxins from mistletoes, and to crambin.

Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus

The role of copper metallothionein (CuMT) in copper metabolism and metalloenzyme activation is poorly understood. We have chosen marine crustaceans, in which a direct correlation exists between levels of Cu(I)MT and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-251, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homarus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclear-copper sites, which subsequently slowly return to their native stereochemical configuration. Finally, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.

Purification and properties of a myotoxin from Conus geographus venom

Abstract Previous studies (R. Endean et al. (1974), Toxicon12: 131–138) indicate that whole venom from the marine mollusc Conus geographus directly inhibits skeletal muscle, leaving peripheral nerves, cardiac and smooth muscle unaffected. A quantitative bioassay has been used to detect and measure biologically active myotoxin. By using chromatography on phosphocellulose, purified myotoxin is obtained which has the same physiological effects as whole venom. The LD50 of purified toxin is 12 μg/kg in mice, death occurring as a result of flaccid paralysis and respiratory failure. A biochemical characterization of the purified myotoxin indicates that it is a peptide of 13 amino acids containing two disulfide bonds. This and related peptide myotoxins from Conus should be of exceptional interest in muscle physiology.

Differential targeting of nicotinic acetylcholine receptors by novel alphaA-conotoxins.

We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12–18 residue, 4 Cys-containing α-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named αA-conotoxins EIVA and EIVB, block both Torpedo and mouse α1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to α-bungarotoxin, αA-EIVA is inactive at α7-containing nAChRs even at micromolar concentrations. In this regard, αA-EIVA is similar to the previously described α-conotoxins (e.g. α-MI and α-GI) which also selectively target α1- versus α7-containing nAChRs. However, α-MI and α-GI discriminate between the α/δversus α/γ subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, αA-conotoxin EIVA blocks both the α/γ site and α/δ site with equally high affinity but with distinct kinetics. The αA-conotoxins thus represent novel probes for the α/γ as well as the α/δ binding sites of the nAChR.

Elastin and Elastic Tissue

Elastic fibers have been shown to contain two proteins, insoluble elastin and the elastic fiber microfibril, a glycoprotein. The microfibril has been suggested to playa morphogenetic role in determining the presumptive shape and direction of the forming elastic fiber. The principal alteration seen in individuals with the disease Pseudoxanthoma Elasticum is in insoluble elastin which loses its amorphous appearance and affinity for anionic stains, and takes on a finely granular appearance and shows increased affinity for cationic stains. Normal elastic fiber microfibrils are sometimes associated with this material; although, in general, these structures are not present in the elastic fibers that are markedly altered in this disease. Recent work on the nature of the elastic fiber has demonstrated that unlike the fibrous protein collagen, the elastic fiber consists of two morphologic entities that represent two discreet proteins (1-3). In mature elastic fibers the bulk of the elastic fiber consists of the well known protein, elastin, which has been recognized by histologists for over 100 years due to its characteristic staining features, its morphology and its marked insolubility. In the electron microscope this material has, until recently, demonstrated an amorphous appearance in which the elastic fiber takes different shapes, depending upon where the fiber is located, and