Structure-Activity Analysis of C-Terminal Endothelin Analogues

Abstract

Several synthetic endothelin (ET) analogues of the C-terminal ET hexapeptide (ET16-21) were analyzed by radio-receptor competition binding assays and biologic activity using both ETA and ETB receptor subtypes. In addition, we produced a hybridoma monoclonal antibody, anti-ET15-21, that appeared to crossreact with the entire ET molecule and was able to neutralize its biologic activity. Antibody binding was measured with competition enzyme-linked immunosorbent assays and a surface plasmon resonance-based biosensor (BIA technology). The ET16-21 moiety was modified with systematic replacement of each residue by alanine (Ala-scan). Whereas the C-terminal residues (Asp18, Ile20, and particularly Trp21) were very important for both receptor binding and immunologic activity, Ala substitution in positions 16, 17, and 19 hardly affected such activities. Analysis of another series of synthetic ET16-21 analogues with the His16 residue replaced by a non-amino-acidic block confirmed that the last two C-terminal residues are essential for receptor and antibody binding, whereas the central region of this hexapeptide is much more tolerant to modification. However, a critical steric conformation of the active hexapeptide is necessary.

An Automated High-Performance Liquid Chromatography Fluorescence Method for the Analyses of Endothelins in Plasma Samples

“…Plasmatic levels of ET-1 and ET-3 were positively correlated with blood pressure and negatively correlated with blood nitrite (not statistically significant). The observations are in line with the known regulation of endogenous NO formation by endothelins and vice versa (11,12). We have recently verified that an increase in circulating levels of endothelins measured by the present method is related to a vasopressor response in rats (15).…”

“…The antibodies used in some of these studies were raised against the N-terminal region of endothelin-1 and can detect ET-2 and cross react with ET-3 and BET-1 (9,10). Also, with the C-terminal ET hexapeptide, it was observed that only the C-terminal residues Asp18, Ile20, and particularly Trp21 were important for immunologic activity (11), and since these three amino acid residues are similar in ET-1, ET-2, and ET-3, there exists a strong potential for equal cross reactivity with the antibodies raised against the common C-terminus of the peptide. Besides these methods, the HPLC method with mass detection using the atmospheric pressure electrospray ionization technique has been reported to date (10).…”

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

“…Functional studies of interactions of peptide ligands with chimera receptors combining ET A and ET B revealed that the C ‐terminal peptide region binds to TMH4 through TMH6, whereas the N ‐terminus interacts with TMH1, TMH2, TMH3 and TMH717. Already short peptide constructs of residues from 16 to 21 are sufficient to inhibit the endothelin receptors18–20. The hydrophobic C ‐terminal residues Ile20 and Trp21 as well as the negatively charged residue Asp18 and the carboxyl group at the C ‐terminus are pivotal points in signal transduction21, 22.…”

Endothelin immunocytochemistry: indications of false-positive labeling patterns and non-detectable antigen concentrations

“…Since these epitopes are also freely accessible in big ET, N-terminal ET antibodies AB I, AB III, and AB IV showed crossreactivity with big ET. Concerning C-terminal ET antibodies residues 18, 20, and, particularly, 21 (i.e., tryptophan) are important for both receptor binding and immunoreactivity (Frosco et al 1993;Rovero et al 1998). Absence of C-terminal ET antibody crossreactivity with big ET (AB V, AB VI, and AB VII) can be explained by an epitope that is not accessible in big ET.…”