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Biochemical, Molecular, and Genetic Mechanisms

Caco-2 Intestinal Epithelial Cells Absorb Soybean Ferritin by µ₂ (AP2)-Dependent Endocytosis^1,2

³ Department of Biology, Faculty of Sciences, and Cell Dynamics and Biotechnology Institute and ⁴ Nutrition and Food Technology Institute, Universidad de Chile, Santiago, Chile; ⁵ Council for BioIron at Children's Hospital Oakland Research Institute, Oakland, CA 94609; and ⁶ Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720

^* To whom correspondence should be addressed. E-mail: mnunez{at}uchile.cl.

Iron deficiency, a condition currently affecting 3 billion people, persists in the 21st century despite half a millennium of medical treatment. Soybean ferritin (SBFn), a large, stable protein nanocage around a mineral with hundreds of iron and oxygen atoms, is a source of nutritional iron with an unknown mechanism for intestinal absorption. Iron absorption from SBFn is insensitive to phytate, suggesting an absorption mechanism different from for the ferrous transport. Here, we investigated the mechanism of iron absorption from mineralized SBFn using Caco-2 cells (polarized in bicameral inserts) as an intestinal cell mode and analyzed binding, internalization and degradation with labeled SBFn (¹³¹I or fluorescent labels), confocal microscopy, and immunoanalyses to show: 1) saturable binding to the apical cell surface; dissociation constant of 7.75 ± 0.88 nmol/L; 2) internalization of SBFn that was dependent on temperature, concentration, and time; 3) entrance of SBFn iron into the labile iron pool (calcein quenching); 4) degradation of the SBFn protein cage; and 5) assembly peptide 2 (AP2)-/clathrin-dependent endocytosis (sensitivity of SBFn uptake to hyperosmolarity, acidity, and RNA interference to the µ₂ subunit of AP2), and resistance to filipin, a caveolar endocytosis inhibitor. The results support a model of SBFn endocytosis through the apical cell membrane, followed by protein cage degradation, mineral reduction/dissolution, and iron entry to the cytosolic iron pool. The large number of iron atoms in SBFn makes iron transport across the cell membrane a much more efficient event for SBFn than for single iron atoms as heme or ferrous ions.