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 pyr oglutamate) from Conus geographus venom. The major glycosylated form of con tulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alp ha-D-GalpNAc-(1-->) attached to Thr(10). The C-terminal sequence of contula kin-G shows a high degree of similarity to the neurotensin family of peptid es. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr(10) c ontulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-f luorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synt hetic glycosylated contulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction obse rved for the native peptide, Contulakin-G was found to be active at 10-fold lower doses than the nonglycosylated Thr(10) contulakin-G analog. The bind ing 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 affi nity of the nonglycosylated Thr(10) contulakin-G was approximately an order of magnitude lower than that of neurotensin,,, for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited signif icantly weaker binding affinity for all of the receptors tested. However, b oth 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 str ategy for increasing the efficacy of toxins directed against neurotransmitt er receptors.