Retro-inverso forms of gastrin5-12 are as biologically active as glycine-extended gastrin in vitro but not in vivo.

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function.

Tyrosine modification increases the affinity of gastrin for ferric ions.

The peptide hormone gastrin17, which occurs naturally in both tyrosine sulphated and unsulphated forms, binds two ferric ions with pM affinities. The aim of this study was to investigate the hypothesis that sulphation or phosphorylation of gastrin17 altered ferric ion binding, and/or affinity for the CCK1 or CCK2 receptor. To investigate the effect of tyrosine modification on ferric ion binding, the changes in absorbance of gastrin17, gastrin17SO4 and gastrin17PO4 on addition of Fe(3+) ions were monitored. Binding of gastrin17, gastrin17SO4 and gastrin17PO4 to the human CCK1 and CCK2 receptors was assessed by competition with [(125)I]-Bolton and Hunter-labelled cholecystokinin8 in transiently transfected COS cells. Tyrosine sulphation or phosphorylation increased the affinity of gastrin17 for the first ferric ion bound from 267 to 83 pM and 14 pM, respectively, but had no effect on the stoichiometry of ferric ion binding. In contrast the affinity of gastrin17 for the second ferric ion bound was reduced from 94 pM to 7.32 µM and 671 nM, respectively. While sulphation of gastrin17 increased its affinity for the CCK2 receptor approximately 50 fold, phosphorylation had no effect on receptor binding. These results demonstrate that tyrosine modification may have profound effects on the interaction of gastrins with ferric ions and with the CCK2 receptor.

Tyrosine modification enhances metal-ion binding.

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.