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, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.
Mettulio
Romina
R
BRAIN Center, Department of Biology, University of Trieste, via Giorgieri 7, I-34127 Trieste, Italy.
Giulianini
Piero Giulio
PG
Ferrero
Enrico Antonio
EA
Lorenzon
Simonetta
S
Edomi
Paolo
P
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001681:Biological Assay; D003001:Cloning, Molecular; D033364:Decapoda / Q000235:genetics / Q000502:physiology*; D006943:Hyperglycemia / Q000378:metabolism; D007447:Invertebrate Hormones; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C015947:hyperglycemic hormone, crustacean