Clathrin-Mediated Endocytosis of Soybean Ferritin by Caco-2 Cells.

Background: The mechanisms of intestinal absorption of non-heme and heme iron have been extensively studied. However ferritin, which has been shown to be a good source of bioavailable iron in the diet, could have a different mechanism.

Methods: Here we examined cellular mechanisms by which recombinant soybean ferritin (rSBFn) is absorbed in a cell model for intestinal epithelia. The internalization of mineralized (480 Fe) rSBFn, labeled with radioactive iodine, or the fluorescent probe Oregon Green®, was monitored in cultured, polarized Caco-2 cells. Additionally, internalization of rSBFn iron into the labile iron pool was determined by calcein fluorescence quenching.

Results: Caco-2 cells internalized ¹³¹ I-rSBFn from the apical border in a temperature, concentration and time dependent fashion. The apical membrane showed saturable binding sites with a K_D = 3.43 ×10⁻⁹ M. After internalization, rSBFn was degraded, increasing iron in the cytosolic, labile pool, analyzed as calcein quenching. Horse spleen ferritin also induced calcein quenching. The amount of iron incorporated from 5 nM rSBFn was similar to that contributed by micromolar amounts of heme and non-heme iron. rSBFn endocytosis was inhibited by treatment with short RNAi for the μ ₂ sub-unit of the assembly peptide 2 complex, by cytosol acidification and by hyperosmolarity, which all inhibit clathrin-mediated endocytosis. Filipin II, an inhibitor of caveolar endocytosis, had no effect.

Conclusion: These results suggests the presence of a third route of intestinal iron absorption in which ferritin from plant or animal sources is internalized from the apical medium of enterocytes by a clathrin-dependent endocytic process.