Biologically Active Recombinant Human Progastrin6-80 Contains a Tightly Bound Calcium Ion

Abstract

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin6-80 and to investigate its structure and biological activities in vitro. Human progastrin6-80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin6-80 was purified by reverse phase high pressure liquid chromatography and characterized by radio-immunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6-80 at concentrations in the pM to nM range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin6-80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin6-80 suggested that progastrin6-80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin6-80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.