Mutational analysis of the N-terminus in Schistocerca gregaria ion-transport peptide expressed in Drosophila Kc1 cells.
The functions of the 6-7 amino acid N-terminal domain conserved in insect and crustacean members of the hyperglycemic hormone (CHH) family were assayed by site-directed mutagenesis of Schistocerca gregaria ion-transport peptide (SchgrITP). Mutant peptides were expressed in Drosophila Kc1 cells and tested in a biological assay measuring stimulation of active Cl(-) transport across the locust ileum. We exchanged the N-terminal domain of SchgrITP with that of the shrimp Penaeus japonicus hyperglycemic hormone leaving the remainder of SchgrITP intact. The chimeric peptide was completely inactive in the ileal bioassay, showing that the N-terminus of SchgrITP is essential and that the 2 amino acids (phenylalanine-3 and aspartate-4) conserved in the shrimp and locust peptides are not sufficient for function. We made all possible alanine substitutions in the SchgrITP N-terminal domain. Only phenylalanines 2 and 3 were essential for function in the locust ileal bioassay. All N-terminal mutations were cleaved correctly from the prepropeptide, and expressed in similar concentrations as wild-type ITP suggesting the specific amino acids are not essential for these functions. Post-translational modification may explain a minor ITP isomorph observed in Drosophila Kc1 cell expression. Alanine substitution at position 2 produced a weak ITP antagonist. These structure-function studies, the first for any member of the CHH family, show that both conserved and unconserved amino acids contribute to SchgrITP ion-transport function and that the conserved aspartate in position 4 is required for a yet uncharacterized function.
Zhao
Y
Y
Department of Zoology, University of British Columbia, Vancouver, B.C. V6T1Z4, Canada.
Meredith
J
J
Brock
H W
HW
Phillips
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004330:Drosophila; D005260:Female; D015870:Gene Expression; D006110:Grasshoppers / Q000737:chemistry* / Q000502:physiology; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016623:Ion Pumps / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D019476:Insect Proteins; D016623:Ion Pumps; D011994:Recombinant Proteins