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 beta-D-Galp-(1-->3)-alpha-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(beta-->3) GalNAc(alpha-->)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. Contulakín-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.
Highlights
We have purified contulakin-G, a 16-amino acid Olinked glycopeptide from Conus geographus venom
Upon icv injection of the fraction from C. geographus indicated in Fig. 1, panel A, the mice had to be poked with much more force before they got up at all, and after getting up, they would walk one or two steps and immediately sit down again
The “G” indicates that the peptide is from C. geographus; we have used an analogous nomenclature for other cysteine-sparse Conus peptides
Summary
Crude Venom—Conus geographus specimens were collected from Marinduque island in the Philippines. The RP-HPLC-purified sample, collected in 0.1% aqueous trifluoroacetic acid and acetonitrile, was diluted in methanol 1% acetic acid, transferred to a nanospray capillary, and analyzed. Co-elution—The native and synthetic contulakin-G were analyzed separately (Fig. 6, panels A and B, respectively) and co-eluted with RP-HPLC (panel C), using a 2.1 ϫ 150-mm Vydac C18 column and a 0.5%/min gradient from 0% to 40% B (where the A buffer was 0.55% trifluoroacetic acid in water, and the B buffer was 0.05% trifluoroacetic acid in 90% aqueous acetonitrile). Binding Experiments on Cell Membranes—Membranes (25 g for NTR2 and 10 g for NTR1) were incubated with 0.4 nM 125I-Tyr neurotensin (2000 Ci/mmol) and increasing concentrations of neurotensin, nonglycosylated Thr contulakin-G, or synthetic contulakin-G for 20 min at 25 °C in 250 l of 50 mM Tris-HCl (pH 7.5) containing 0.1% bovine serum albumin and 0.8 mM 1–10-phenanthroline. The nucleic acid sequence was determined according to the standard protocol for Sequenase version 2.0 DNA sequencing kit as described previously [4]
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