Collagen Peptides as a Hypoxia-Inducible Factor-2α-Stabilizing Prolyl Hydroxylase Inhibitor to Stimulate Intestinal Iron Absorption by Upregulating Iron Transport Proteins

Abstract

Iron intervention is not always safe and effective to correct iron deficiency. Host iron absorption stimulation is emerging as a promising adjunctive/alternative treatment. Here, porcine collagen hydrolysate (CH) and collagen-derived dipeptide prolyl-hydroxyproline, rather than collagen amino acids, namely, glycine, proline, and hydroxyproline, were found to increase cellular iron reduction, absorption, and transportation, to upregulate duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin (FPN), and hephaestin, and to nongenomically activate hypoxia-inducible factor-2α signaling in polarized Caco-2 cells. Prolyl-hydroxyproline showed both competitive and uncompetitive inhibition of recombinant human prolyl hydroxylase-3 activity with EC 50 and K i values of 10.62 and 6.73 μM, respectively. Docking simulations revealed collagen peptides as iron chelators and/or steric hindrances for prolyl hydroxylase-3. CH and prolyl-hydroxyproline acutely increased duodenal hypoxia-inducible factor-2α stability and Dcytb, DMT1, FPN, and hephaestin transcription in rats. Overall, collagen peptides act as a hypoxia-inducible factor-2α-stabilizing prolyl hydroxylase inhibitor to stimulate intestinal iron absorption.

Gum Arabic-Derived Hydroxyproline-Rich Peptides Stimulate Intestinal Nonheme Iron Absorption via HIF2α-Dependent Upregulation of Iron Transport Proteins

“… As shown in the two-dimensional (2D) and three-dimensional (3D) diagrams of docking simulations (Figure S8), Hyp–Hyp was predicted to fit well in the predominantly hydrophobic pocket at the active site of human PHD3 and was preferentially bound through coordination of its imino nitrogen and amino oxygen with the enzyme’s iron center, hydrogen bonds between its hydroxyproline residue and the enzyme’s Tyr125, Asp76, and His153 residues, and hydrophobic interactions between its hydrophobic group and the surrounding residues like Arg205, Val198, Ile149, Asp137, His196, Ala123, Ile78, Met121, Tyr132, and Phe188. The affinity energy was calculated to be −7.53 kcal/mol, which was close to those of Pro–Hyp, Ser–Hyp, and an oral PHD inhibitor drug of roxadustat as reported previously . Therefore, the protein fraction of GA might activate the enterocyte HIF2α signaling pathway in a similar way as collagen peptides through HCP-mediated PHD inhibition.…”

“…Collagen-derived HCPs have been recently identified as a PHD inhibitor to activate the HIF2α signaling pathway, which induces the expression of iron transporter genes in enterocytes. , In the present study, three GA-derived HCPs including Hyp–Hyp, Pro–Hyp, and Ser–Hyp were detected with high abundance by LC-MS/MS in the lysates of polarized Caco-2 cell monolayers (Figure a–d). In fact, Pro–Hyp and Ser–Hyp are among the most frequently detected collagen-derived HCPs, , and their PHD-inhibiting activities have been evaluated by using recombinant human PHD and docking simulations in our previous study . As the most abundant GA-derived HCPs, Hyp–Hyp seems to be typical for GA, owing to its absence in previously reported collagen-derived HCPs. , This was in line with the frequent motifs of Ser–Hyp 2–4 in the peptide backbones of GA (Table ).…”

“…In fact, Pro−Hyp and Ser−Hyp are among the most frequently detected collagenderived HCPs, 27,28 and their PHD-inhibiting activities have been evaluated by using recombinant human PHD and docking simulations in our previous study. 13 As the most abundant GA-derived HCPs, Hyp−Hyp seems to be typical for GA, owing to its absence in previously reported collagen-derived HCPs. 27,28 This was in line with the frequent motifs of Ser−Hyp 2−4 in the peptide backbones of GA (Table 2).…”

“…The affinity energy was calculated to be −7.53 kcal/mol, which was close to those of Pro−Hyp, Ser−Hyp, and an oral PHD inhibitor drug of roxadustat as reported previously. 13 Therefore, the protein fraction of GA might activate the enterocyte HIF2α signaling pathway in a similar way as collagen peptides through HCPmediated PHD inhibition.…”

“…Our recent study found that collagen-derived hydroxyproline-containing di/tripeptides (HCPs) could act as a PHD inhibitor to stimulate intestinal iron absorption by upregulating Dcytb, DMT1, FPN, and HEPH in vitro and in vivo. 13 Gum arabic (GA) is an Acacia exudate extensively used in the food industry as a bulking agent, carrier, emulsifier, glazing agent, stabilizer, and thickener. GA consists of a majority of arabinogalactan polysaccharides and a minority (1−5%) of hydroxyproline-rich peptides 14 and so could be a potential plant-based source of HCPs.…”