LOCUST ION-TRANSPORT PEPTIDE (ITP) - PRIMARY STRUCTURE, CDNA AND EXPRESSION IN A BACULOVIRUS SYSTEM

Ion transport peptide (ITP) purified from locust nervous corpus cardia cum (CC) has previously been shown to stimulate salt and water reabsor ption and inhibit acid secretion in the ileum of Schistocerca gregaria . We used the partial amino acid sequence of purified ITP to derive de generate primers. These were used to amplify a cDNA from brain RNA usi ng reverse transcription and the polymerase chain reaction (RtPCR). Th is sequence was extended using anchored PCR to yield a partial, 517 bp cDNA clone. This cDNA encodes a putative ITP prohormone which could b e cleaved at two dibasic amino acid sites to yield a 72 residue active amidated peptide. The deduced amino acid sequence from the cDNA agree s completely with the amino acid sequence and molecular mass (8564 Da) derived from analysis of purified ITP. Relative to a family of crusta cean 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 dist antly 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 ile al 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 a re not active on the ITP bioassay.