The structure-activity relationship study of the N-terminal domain in desert locust ion transport peptide (ITP) release_puhhc3bffzhglejb3mfyhkfhpy

by Ying Zhao

Published by The University of British Columbia.



The ileum and rectum of locust hindgut constitute the reabsorptive part of the excretory system. They are functionally analogous to a proximal and distal parts of convoluted tubule of the mammalian kidney tubule, respectively. Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria . The primary structure of ITP deduced from its cDNA suggests that it is a 72 amino acid peptide with C-terminal amidation and three disulfide bonds. Both synthetic and expressed ITP mimic the biological activity of ITP purified from S.gregaria CC. It has been demonstrated that ITP is a true member of the CHH (Crustacean Hyperglycemic Hormone) family. This study examines the structure-activity relationship of the N-terminal domain of ITP. Two questions are addressed: 1. Is the N-terminal domain of ITP consisting of the first six amino acids (SFFDIQ) important to bioactivity? 2. Which amino acids in the N-terminal domain of ITP are essential for ITP binding to the receptor and/or activating the receptor? Using site-directed mutagenesis and voltage-clamped locust ileum as bioassay, I found the ITP N-terminal domain (SFFDIQ) is important for its bioactivity. Among the six amino acid of ITP N-terminus, Phe2 and Phe3 are essential for ITP binding to the receptor, and Phe2 is also important to receptor activation. The other four amino acids SI, D4,15, and Q6 didn't contribute to the ITP bioactivity, even D4 is a highly conserved amino acid. Post-translational modification of conversion L- to D- amino acid probably occurs at Phe2 and Phe3 to yield two ITP isomorphs. Mutations on the ITP N-terminal domain didn't interfere with the dibasic cleavage site in spite of its immediate proximity to the dibasic cleavage site. Mutant F2A has the potential to be ITP antagonist.
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