Characterization of crustacean hyperglycemic hormone from the crayfish (Procambarus clarkii): multiplicity of molecular forms by stereoinversion and diverse functions

Abstract

The complete amino acid sequence of two forms of crustacean hyperglycemic hormone (CHH) from the X-organ sinus gland complex of crayfish (Procambarus clarkii) has been determined. There are two variants of P. clarkii CHH (Prc-CHH), I and II, which can be separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Each variant was oxidized by performic acid and then cleaved with lysyl endopeptidase. Intact hormone was also digested with trypsin and endoproteinase Asp-N, successively. The resulting fragments were separated by RP-HPLC and subjected to sequence analyses by a gas-phase sequencer and tandem mass spectrometry. Both variants contain 72 amino acid residues with three disulfide linkages, at positions 7-43, 23-39, and 26-52, and differ from each other by the D/L epimerization of phenylalanine at position 3; Prc-CHH-II contains D-amino acid. Injections of Prc-CHH-I and Prc-CHH-II at a dose of 12.5 pmol resulted in significant increase of hemolymph glucose levels in the crayfish. The hormones are also active in repressing ecdysteroid synthesis at concentrations of 250 mM (Prc-CHH-I) and 25 nM (Prc-CHH-II) in Y-organ culture. These results may indicate that the stereoinversion in the CHH molecule leads to an important alternation in hormonal functions during crustacean development.