Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity

Abstract

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Fig. 1

MALDI-MS analysis of pTFF, WTFF, WTFF58, and WCys/ala in the presence and in the absence of twofold CuCl₂ molar excess, after 15 min of incubation. Peak A (1,969.52 Da), peak A* (2,033.65 Da), peak B* (4,001.52 Da), peak C (2,008.45 Da), peak C* (2,072.95 Da), peak D (4,015.1 Da), peak D* (4,078.85 Da), peak E (1,976.95 Da), peak F (1,776.83 Da), peak G (3,551.96 Da)

Fig. 2

Dimers formation of wild-type and mutant peptides. The increase of dimer formation after CuCl₂ incubation for 0, 3 and 36 h is shown for WTFF mutants and pTFF. (3 and 36 h vs 0 h; **p < 0.005)

Fig. 3

Stern–Volmer plots of peptides fluorescence quenched by copper. The slope change in a, b, c, d, f are diagnostic of a static component of the fluorescence quenching and a stable copper binding. F ₀/F = ratio of fluorescence intensities before (F ₀) and after (F) the addition of copper

Fig. 4

a The effect of metal ions on WTFF fluorescence. The quenching of fluorescence in the presence of Cu(II) and Ag(I) is diagnostic of a selective binding for both metals (***p < 0.0005). b pH dependence of the Cu (II) quenching of WTFF. The absence of quenching below pH 3.6 is indicative of Glu residue titration with a consequent lack of binding [Fluorescence intensities at different pHs were compared in the presence (+) and in the absence (−) of copper; *p < 0.05; **p < 0.005; ***p < 0.0005]

Fig. 5

Cu²⁺ quenching of WTFF fluorescence: glycine and histidine competition. a WTFF fluorescence in the presence of increasing concentration of Cu²⁺. Rescue of the tryptophan fluorescence after the addition of glycine (b) or histidine (c). ΔF = F − F ₀, ΔF _max = F _max − F ₀, where F ₀ is the fluorescence in the presence of 1 molar equivalent of Cu ²⁺

Fig. 6

Western blot analysis of secreted proteins in reducing (R) and non-reducing (NR) conditions; cells were cultured in copper free culture medium (Cu−) or 100 μM CuCl₂ culture medium (Cu+). a MCF-7cells (lane 1 recombinant TFF1 protein). b HT-29-E12 cells. Histograms of the densitometric analyses of MCF-7 (c) and HT-29-E12 (d) cell culture media (−Cu copper free, +Cu 100 μM CuCl₂) are representative of the mean signals obtained from western blot analyses of single samples from independent experiments carried out as biological triplicate (*p < 0.05, **p < 0.005)

Fig. 7

A western blot analysis of AGS-AC1 supernatants with anti-TFF1 antibody in reducing (a) and non-reducing (b) conditions: 1 20 μl of supernatant of non-induced cells; 2 20 μl of supernatant after induction with 0.1 mg ml⁻¹ doxycycline; 3 20 μl of supernatant after induction with 0.1 mg ml⁻¹ doxycycline + 100 μM CuCl₂. c Time-course of wound-healing assays. Comparison of the migration mean distances of induced or not induced AGS-AC1 cells, treated or not treated with BCS. Diamonds untreated control, squares induction with 0.1 mg ml⁻¹ doxycycline, circles treatment with 500 μM BCS, triangles induction with 0.1 mg ml⁻¹ doxycycline and treatment with 500 μM BCS. p < 0.05 (untreated + dox vs untreated − dox) is marked by asterisks