Contulakin-G, an O-Glycosylated Invertebrate Neurotensin

Abstract

We have purified contulakin-G, a 16-amino acid Olinked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-SerAsn-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 ␤-D-Galp-(133)-␣-D-GalpNAc-(13) attached to Thr 10 . The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(␤33) GalNAc(␣3)Thr 10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated contulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr 10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr 10 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 Thr 10 contulakin-G was approximately an order of magnitude lower than that of neurotensin [1][2][3][4][5][6][7][8][9][10][11][12][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 Thr 10 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.

Conotoxins: Structure, Therapeutic Potential and Pharmacological Applications

“…They are reported to undergo O-linked glycosylation in which a carbohydrate group is attached to a hydroxyl group of serine, threonine or tyrosine amino acid residues of the peptide back bone. The first evidence for O-glycosylation in conopeptide was found in κA-conotoxin SIVA using electrospray-mass spectrometry [26,27] and the glycan moiety was identified to be Hex 3 HexNc 2 . Another example of O-linked glycosylation is observed in contulakin-G, which contains HexHexNAc, SO4(HexNAc), Hex3 and Hex2HexNAc2 glycoform sequences identified using combination of MALDI-TOF-MS, liquid secondary ion mass spectrometry (LSI-MS) and electrospray ionisation mass spectrometry (ESI-MS).…”

ConusVenoms: A Rich Source of Novel Ion Channel-Targeted Peptides

“…Additionally, a number of conopeptides have been shown to target G protein-coupled receptors. Interestingly, two of these are agonists: conopressin-G is an agonist for the vasopressin receptor (31), and contulakin-G is believed to be a subtype-specific agonist of a receptor for the neurotensin peptide family (29). Only one peptide so far, -conotoxin TIA, which has been shown to target the ␣ 1 -adrenergic receptor, is a G protein-coupled receptor antagonist (164).…”

“…Conantokin-G (that targets NMDA receptors; see sect. IIIE) and contulakin-G, likely a neurotensin receptor agonist (29), are being developed for epilepsy and pain, respectively; both have been through phase I human clinical trials. Another peptide, the nicotinic receptor antagonist vg1 (see Table 3), is being explored as a therapeutic agent for pain.…”

Two Toxins from Conus striatus That Individually Induce Tetanic Paralysis

“…O-linked glycosylation has been reported previously in Conus toxins (9,11,(29)(30)(31), but the functional significance of this PTM is not clear. Previous work with glycosylated conotoxins has focused on venoms derived from the venom duct, leaving open the possibility that the Olinked glycan may play some role in secretion within the venom duct but is lost in maturation of the injected toxin.…”