Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproductionand molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure–function relationships of theseneuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptidemotifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHHand its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild typecHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHHantibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, whichconserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important forfunctional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identifyamino acid residues that are required for the hyperglycemic activity of cHH.D 2004 Elsevier B.V. All rights reserved.