Contulakin-G, an O-glycosylated invertebrate neurotensin

A. Grey Craig, Thomas Norberg, David Griffin, Carl Hoeger, Mateen Akhtar, Karsten Schmidt, William Low, John Dykert, Elliott Richelson, Valérie Navarro, Jean Mazella, Maren Watkins, David Hillyard, Julita Imperial, Lourdes J. Cruz, Baldomero M. Olivera

Research output: Contribution to journalArticlepeer-review

164 Scopus citations

Abstract

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 contulakin-G, when administered intracerebroventricular into mice, was found to result in motor control- associated dysfunction observed for the native peptide. Contulakin-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 Thr10 contulakin-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.

Original languageEnglish (US)
Pages (from-to)13752-13759
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number20
DOIs
StatePublished - May 14 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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