John Dykert

Contulakin-G, an O-glycosylated invertebrate neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide β-d-Galp-(1→3)-α-d-GalpNAc-(1→) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(β→3) GalNAc(α→)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakı́n-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10contulakin-G was approximately an order of magnitude lower than that of neurotensin1–13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

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.

A Novel Post-translational Modification Involving Bromination of Tryptophan IDENTIFICATION OF THE RESIDUE, L-6-BROMOTRYPTOPHAN, IN PEPTIDES FROM Conus imperialis AND Conus radiatus VENOM

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = γ-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Isolation of Lys-conopressin-G from the venom of the worm-hunting snail, Conus imperialis

Vasopressin homologs have previously been isolated from the venom of fish-hunting cone snails. We investigated whether a vasopressin-like peptide is found in the worm-hunter, Conus imperialis. Using i.c. injections in mice, we isolated a peptide from the venom of C. imperialis which induces scratching and grooming behavior characteristic of the conopressins. Biochemical characterization showed that this peptide is identical to Lys-conopressin-G. The results led us to speculate that the vasopressin-like peptides in Conus venoms may be examples of an evolving conversion of endogenous peptides for specialized venom uses.

Pharmacological Characterization of FE 202158, a Novel, Potent, Selective, and Short-Acting Peptidic Vasopressin V1a Receptor Full Agonist for the Treatment of Vasodilatory Hypotension

FE 202158, ([Phe2,Ile3,Hgn4,Orn(iPr)8]vasopressin, where Hgn is homoglutamine and iPr is isopropyl), a peptidic analog of the vasoconstrictor hormone [Arg8]vasopressin (AVP), was designed to be a potent, selective, and short-acting vasopressin type 1a receptor (V1aR) agonist. In functional reporter gene assays, FE 202158 was a potent and selective human V1aR agonist [EC50 = 2.4 nM; selectivity ratio of 1:142:1107:440 versus human vasopressin type 1b receptor, vasopressin type 2 receptor (V2R), and oxytocin receptor, respectively] contrasting with AVPs lack of selectivity, especially versus the V2R (selectivity ratio of 1:18:0.2:92; human V1aR EC50 = 0.24 nM). This activity and selectivity profile was confirmed in radioligand binding assays. FE 202158 was a potent vasoconstrictor in the isolated rat common iliac artery ex vivo (EC50 = 3.6 nM versus 0.8 nM for AVP) and reduced rat ear skin blood flow after intravenous infusion in vivo (ED50 = 4.0 versus 3.4 pmol/kg/min for AVP). The duration of its vasopressor effect by intravenous bolus in rats was as short as AVP at submaximally effective doses. FE 202158 had no V2R-mediated antidiuretic activity in rats by intravenous infusion at its ED50 for reduction of ear skin blood flow, in contrast with the pronounced antidiuretic effect of AVP. Thus, FE 202158 seems suitable for treatment of conditions where V1aR activity is desirable but V2R activity is potentially deleterious, such as vasodilatory hypotension in septic shock. In addition to the desirable selectivity profile, its short-acting nature should allow dose titration with rapid onset and offset of action to optimize vasoconstriction efficacy and safety.

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (α1β1γδ-subunits). Although this conotoxin, αA-conotoxin OIVB (αA-OIVB), is a high-affinity antagonist (IC50 of 56 nm) of the fetal muscle nAChR, it has >1800-fold lower affinity for the adult muscle nAChR (α1β1ϵδ-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC50 > 100 μm in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown.αA-OIVB is a potentially important tool for delineating the functional roles ofα1β1γδ receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, αA-OIVB may have some direct biomedical applications.

Synthesis, purification and characterization of rat histone H2A (1–53)-NH2

Abstract Synthetic peptides (up to 40 residues in length) are routinely purified and characterized by reversed-phase liquid chromatography (RP-LC). However, as the peptide size is further increased, this technique becomes less efficient in separating micro-heterogeneities despite the extensive use of columns or buffer systems known to exhibit different selectivities. As a result, alternative analytical and preparative methodologies were sought for the purification and characterization of synthetic histone H2A (1–53)-NH 2 . This 53-peptide was synthesized on a 2,4-dimethoxybenzhydrylamine resin using the fluorenylmethyloxycarbonyl strategy. Interestingly, the crude histone, after trifluoroacetic acid cleavage from the resin and deprotection, appeared to be remarkably pure. This result being unexpected, this sample was also analyzed by cation-exchange chromatography using a recently developed narrow-bore Pharmacia Mono S Precision Column 1.6/5 and an organic modifier (acetonitrile) in the buffer. Under the experimental conditions that were used, the presence of the desired peptide was determined by mass spectrometry and accounted for ca. 7% of the total absorbance at 214 nm. Scale-up studies allowed the isolation of significant amounts of highly purified histone H2A (1–53)-NH 2 . This preparation was characterized using RP-LC, capillary zone electrophoresis, amino acid analysis, mass spectrometry and sequence analysis using automated Edman degradation.

Rat corticostatin R4: Synthesis, disulfide bridge assignment, and in vivo activity

We have synthesized significant amounts of the most potent member of the rat corticostatins that inhibits ACTH-induced corticosteroid and compared its structure to that of the natural hormone. The cystine bridging arrangement that corresponds to that reported for a human defensin (3-31, 5-20, 10-30) was determined. The in vitro corticostatic activity of the synthetic rat corticostatin R4 paralleled that of the natural R4. Biological studies in vivo showed that doses of 8 or 12 mg corticostatin/kg effectively interfered with corticosterone release in stressed rats. We conclude that in the assays that were used, the biological activity of the synthetic and natural molecules was identical. The availability of significant amounts of synthetic material will make possible studies investigating the physiological role played by corticostatins in modulating the activity of the hypothalamic-pituitary-adrenal axis.