Functional analysis of a mutated analogue of the crustacean hyperglycaemic hormone from the crayfish Pontastacus leptodactylus.

The crustacean hyperglycaemic hormone (CHH), a pleiotropic neuropeptide, belongs to a family of structurally related peptides, having six cysteine residues in conserved positions forming three disulphide bridges, and regulating several physiological processes in crustaceans and insects. Structure-activity studies have shown that amidation of the C-terminus is important to confer biological activity to CHH. In this study we investigated the function of the d-Phe(3) of the N-terminal motif of Pontastacus leptodactylus CHH by a mutational analysis. The d-Phe in position 3 was substituted by a d-Ala and the functionality of the mutated analogue (Glp-d-A-CHH) was tested by in vivo biological assays. The mutated analogue resulted far less active than its wild-type counterparts, either in d- (Glp-d-CHH) or l- (Glp-l-CHH) configuration. These results suggest that Phe(3) is essential for the biological activity of P. leptodactylus CHH, demonstrating that also the N-terminus is involved in the binding with the receptor, and identifying in the Phe(3) a hot spot for the peptide-receptor binding.