Screening and mechanistic study of chicken embryo egg-yolk peptide-Fe²⁺ chelates based on Caco-2 cells

Abstract

The iron-chelating peptides were screened from egg yolk, and the structure, and mechanism were investigated. By examining the incubation stages, day 12 had the highest iron chelation rate of 61.55 % ±1.69 %. On this basis, the peptide sequence PQETPPDTT (PT-9) was screened according to its molecular weight, amino acid composition, and molecular docking (binding energy < −4.2 kcal/mol). Compared with PT-9, the surface charge and average size of PT-9-Fe²⁺ changed from −17.07 mV and 1142 nm to −6.64 mV and 444 nm, respectively. Isothermal titration calorimetry (ITC) and Kinetic simulations revealed that the Gln2, Thr4, Asp7, and Thr9 in PT-9 formed a stable eight-coordination bond network spontaneously (ΔH = −1.689 kJ/mol) with Fe²⁺ via CO, N-H, and three water molecules, with an Fe^2 + release rate lower than 25 % in the gastrointestinal digestive tests. PT-9-Fe²⁺ enhanced Fe²⁺ uptake to 2.0-fold in Caco-2 cells compared with FeSO₄. Through a combination of methods, including RNA-seq and inhibitors, it was demonstrated that PT-9-Fe²⁺ up-regulated the expression of genes encoding Divalent Metal Transporter 1 (DMT1) and Transferrin Receptor (TFRC), and down-regulated the expression of genes encoding tight junction proteins and adhesion proteins. The iron uptake pathways mediated by PT-9-Fe²⁺ were predominantly paracellular transport, endocytosis and DMT1.