An O-glycosylated neuroexcitatory conus peptide.

We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.

Novel alpha- and omega-conotoxins from Conus striatus venom.

Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.

Elucidation of the role of arginine-244 in the turnover processes of class A beta-lactamases.

The highly conserved arginine-244 of beta-lactamases has been postulated to play a role in their initial recognition of substrates, presumably through ion pairing interactions [Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P., & Frère, J. M. (1990) Proteins: Struct., Funct., Genet. 7, 156-171]. However, in the Michaelis enzyme-substrate complex, no direct function has been attributed to this residue. Two mutants with substitutions of this residue in the TEM-1 beta-lactamase (lysine-244 and serine-244) have been prepared to explore whether the guanidinium group of arginine-244 plays a critical role in the turnover processes. The mutant enzymes are effective catalysts for the hydrolysis of both penicillins and cephalosporins, and the lysine mutant enzyme behaves virtually identically to the wild-type beta-lactamase. Comparative kinetic characterization of the serine mutant and wild-type enzymes attributed apparent binding energies of 1.3-2.3 kcal/mol for the penicillins and 0.3-1.0 kcal/mol for the cephalosporins to the transition-state species by arginine-244. Furthermore, it was shown that arginine-244 also contributes equally well to ground-state binding stabilization. These results were interpreted to indicate the involvement of a long hydrogen bond between arginine-244 and the substrate carboxylate, both in the ground and transition states. A reassessed picture for substrate anchoring involving interactions of the substrate carboxylate with the side chains of Ser-130, Ser-235, and Arg-244 is proposed to accommodate these observations.

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors.

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.

Phylogenetic specificity of cholinergic ligands: alpha-conotoxin SI.

The alpha-conotoxins are small peptide neurotoxins from the venom of fish-hunting cone snails which block nicotinic acetylcholine receptors (nAChRs). We describe the purification, characterization, and chemical synthesis of a new alpha-conotoxin from Conus striatus, alpha-conotoxin SI. In contrast to other AChR ligands, alpha-SI discriminates between different vertebrate nAChRs. The sequence of alpha-conotoxin SI is Ile-Cys-Cys-Asn-Pro5-Ala-Cys-Gly-Pro-Lys10-Tyr-Ser-Cys-NH2. This sequence was confirmed by chemical synthesis. A des-Ile-alpha-SI derivative was also synthesized and is biologically active. Although alpha-conotoxin SI is highly homologous to previously described alpha-conotoxins, it has one noteworthy sequence feature which may account for its novel biological specificity. In all other alpha-conotoxins, there is a positively charged amino acid at residue 9; in alpha-conotoxin SI, this is replaced by proline. The discovery that different alpha-conotoxins can vary by orders of magnitude in their apparent affinity for different vertebrate receptors demonstrates that alpha-conotoxins will be useful probes for investigating phylogenetic differences between vertebrate nAChRs.

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms.

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.