Biological Activity and Ferric Ion Binding of Fragments of Glycine-Extended Gastrin

Publisher Website
Google Scholar
Abstract
Nonamidated gastrins such as progastrin and glycine-extended gastrin17 (Ggly) induce cell proliferation and migration in vitro and colonic mucosal proliferation in vivo. Our earlier NMR study defined the structure of Ggly and showed that ferric ions are essential to its biological activity, with the first binding to Glu7 and the second to Glu8 and Glu9 (Pannequin, J. et al. (2002) J. Biol. Chem. 277, 48602−48609). The aims of this study were to define the minimum biologically active fragment of Ggly and to determine whether ferric ions were also required for its activity. Cell-proliferation studies with Ggly fragments containing the five glutamate residues showed that the nonapeptide LE5AYG, the octapeptide LE5AY, and the heptapeptides E5AY and LE5A were fully active and that their activity was dependent on the presence of ferric ions. The activity of the hexapeptides LE5 and E5A and the pentapeptide E5 was reduced and independent of the presence of iron. The stoichiometry of ferric ion binding to LE5AYG, LE5AY, and E5AY, determined by absorption spectroscopy, was 2 mol/mol. NMR spectroscopy showed that the nonapeptide LE5AYG and shorter fragments had no defined structure and that the iron-binding sites differed from those in Ggly. We conclude that, in contrast to amidated gastrins where the C-terminal tetrapeptide is the minimum bioactive fragment, the shortest fully active fragments of Ggly are the heptapeptides LE5A and E5AY. These observations indicate that extensive proteolytic processing may not completely inactivate Ggly and that bioactive forms that are not detected by current radioimmunoassays may be present in tissues and/or plasma.