Lactoferrin is internalized by lactoferrin receptor via clathrin‐mediated endocytosis in Caco‐2 cells

Abstract

Lactoferrin (Lf) is a major iron‐binding protein in exocrine fluids such as breast milk and mucosal secretions. Mounting evidence has suggested that Lf plays important roles in immune competence, reproductive function, regulation of cellular proliferation and protection against cancer development. These functions are postulated to be mediated through Lf receptor (LfR) binding and internalization at cell surfaces and appear dependent upon the iron‐saturation of Lf. We previously cloned the intestinal LfR and in the present study, we investigated the role of LfR in Lf binding and internalization using Caco‐2 cells as a human enterocyte model. We detected LfR at the plasma membrane of Caco‐2 cells using a biotinylation assay. Both iron‐free Lf (apo‐Lf) and iron‐saturated Lf (holo‐Lf) uptake was significantly inhibited by pre‐incubation with LfR antibody or gene attenuation in cells transfected with siRNA. Lf uptake was significantly inhibited by hypertonic solution and suppression of clathrin by siRNA transfection significantly reduced Lf uptake. Additionally, the interaction of LfR with clathrin adaptor protein (AP2) was supported by LfR immunoprecipitation results. In conclusion, our results demonstrate that LfR regulates Lf internalization into enterocytes through a clathrin‐mediated endocytosis pathway and that the different functions of apo‐ and holo‐Lf are mediated by post‐endocytotic events.