RESUMO
Ferritin, a food constituent of animal and vegetal origin, is a source of dietary iron. Its hollow central cavity has the capacity to store up to 4,500 atoms of iron, so its potential as an iron donor is advantageous to heme iron, present in animal meats and inorganic iron of mineral or vegetal origin. In intestinal cells, ferritin internalization by endocytosis results in the release of its iron into the cytosolic labile iron pool. The aim of this study was to characterize the endocytic pathway of exogenous ferritin absorbed from the apical membrane of intestinal epithelium Caco-2 cells, using both transmission electron microscopy and fluorescence confocal microscopy. Confocal microscopy revealed that endocytosis of exogenous AlexaFluor 488-labeled ferritin was initiated by its engulfment by clathrin-coated pits and internalization into early endosomes, as determined by codistribution with clathrin and early endosome antigen 1 (EEA1). AlexaFluor 488-labeled ferritin also codistributed with the autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and the lysosome marker lysosomal-associated membrane protein 2 (LAMP2). Transmission electron microscopy revealed that exogenously added ferritin was captured in plasmalemmal pits, double-membrane compartments, and multivesicular bodies considered as autophagosomes and lysosomes. Biochemical experiments revealed that the lysosome inhibitor chloroquine and the autophagosome inhibitor 3-methyladenine (3-MA) inhibited degradation of exogenously added (131)I-labeled ferritin. This evidence is consistent with a model in which exogenous ferritin is internalized from the apical membrane through clathrin-coated pits, and then follows a degradation pathway consisting of the passage through early endosomes, autophagosomes, and autolysosomes.
Assuntos
Ferritinas/metabolismo , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Autofagia , Células CACO-2 , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas de Transporte Vesicular/metabolismoRESUMO
Iron deficiency, a condition currently affecting approximately 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 ((131)I 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 mu(2) 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.