Ferric ions inhibit proteolytic processing of progastrin

Abstract

The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser(75) has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of progastrin after Arg(73)Arg(74). Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic progastrin-derived peptides. The affinity of synthetic progastrin(55-80) for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser(75). In contrast, in the presence of ferric ions the rate of cleavage of both progastrin(55-80) and phosphoSer(75)progastrin(55-80) by prohormone convertase 1 was significantly reduced. Hence iron binding to progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.

Fig. 1

Structure of progastrin and related peptides. The structures of progastrin_55-80, serine phosphoSer₇₅progastrin_55-80, Ggly (progastrin_55-72) and Gamide (amidated progastrin_55-71) are shown. A pyroglutamic acid residue (Z) protects the N-terminus of all four peptides.

Fig. 2

Binding of ferric ions by progastrin_55-80 and phosphoSer₇₅progastrin_55-80. Addition of aliquots of ferric chloride to 9.43 μM progastrin_55-80 (●) or 9.66 μM phosphoSer₇₅progastrin_55-80 (■) in 10 mM sodium acetate buffer (pH 4.0) containing 100 nM NaCl and 0.005% Tween 20 resulted in an increase in absorbance at 280 nm. Data were corrected for volume added during the titration, and expressed as a percentage of the absorbance of that peptide without ferric ions. Points are means of at least four separate experiments and bars represent the SEM. The lines represent the best fit to a two-site ordered model with the program BioEqs. The best fit values for the dissociation constants are given in the text.

Fig. 3

Effect of iron binding and serine phosphorylation on cleavage of progastrin by trypsin. The amount of residual progastrin_55-80 (●, ○) or phosphoSer₇₅progastrin_55-80 (■, □) after treatment with trypsin was determined in the absence (●, ■) or presence (○, □) of bound ferric ions by measurement of peak areas after HPLC separation of the cleavage products. Pre-incubation with ferric ions significantly reduced tryptic cleavage of progastrin_55-80 (*, p < 0.05). Ser₇₅ phosphorylation also significantly reduced tryptic cleavage of progastrin (#, p < 0.01). In contrast, pre-incubation with ferric ions did not inhibit tryptic cleavage of phosphoSer₇₅progastrin_55-80.

Fig. 4

Effect of iron binding and serine phosphorylation on cleavage of progastrin by PC1. The amount of residual progastrin_55-80 (●, ○) or phosphoSer₇₅progastrin_55-80 (■, □) after treatment with recombinant human PC1 was determined in the absence (●, ■) or presence (○, □) of bound ferric ions by measurement of peak areas after HPLC separation of the cleavage products. Pre-incubation with ferric ions significantly reduced cleavage of progastrin_55-80 (#, p 0.001) or phosphoSer₇₅progastrin_55-80 by PC1 (*, p < 0.05). In contrast Ser₇₅ phosphorylation did not affect cleavage of progastrin by PC1.