Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system.
Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has previously been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria. We used the partial amino acid sequence of purified ITP to derive degenerate primers. These were used to amplify a cDNA from brain RNA using reverse transcription and the polymerase chain reaction (RtPCR). This sequence was extended using anchored PCR to yield a partial, 517bp cDNA clone. This cDNA encodes a putative ITP prohormone which could be cleaved at two dibasic amino acid sites to yield a 72 residue active amidated peptide. The deduced amino acid sequence from the cDNA agrees completely with the amino acid sequence and molecular mass (8564Da) derived from analysis of purified ITP. Relative to a family of crustacean hyperglycaemic hormones (CHH), all six cysteine residues and many other amino acid residues are conserved in ITP, establishing that ITP is a homologue. However, CHH, crab eyestalk and CC extracts from distantly related insects have no action, whereas CC extracts from closely related insects are active on the locust ITP assay, showing that the bioassay is selective. Insect Sf9 cells transfected with a baculovirus containing our partial cDNA secreted a potent stimulant of locust ileal transport, confirming that the peptide encoded by our ITP clone has biological activity. The mRNA for ITP is restricted to the brain and CC. Interestingly, a related mRNA is observed in other tissues which are not active on the ITP bioassay.
Meredith
J
J
Department of Zoology, University of British Columbia, Vancouver, Canada.
Ring
M
M
Macins
A
A
Marschall
J
J
Cheng
N N
NN
Theilmann
D
D
Brock
H W
HW
Phillips
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D003445:Crustacea; D018076:DNA, Complementary / Q000737:chemistry*; D004553:Electric Conductivity; D015870:Gene Expression; D006110:Grasshoppers; D019476:Insect Proteins; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D012313:RNA; D011994:Recombinant Proteins / Q000737:chemistry; D017385:Sequence Homology
D002352:Carrier Proteins; D018076:DNA, Complementary; D019476:Insect Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C079361:ion-transport peptide, Schistocerca gregaria; D012313:RNA