Caco-2 intestinal epithelial cells absorb soybean ferritin by μ2 (AP2)-dependent endocytosis
Caco-2 intestinal epithelial cells absorb soybean ferritin by μ2 (AP2)-dependent endocytosis
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DOI：
10.1093/jn/138.4.659
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发表时间：
2008-04-01
影响因子：
4.2
通讯作者：
Nunez, Marco T.
中科院分区：
文献类型：
--
作者：
Martin, Carol D. San;Garri, Carolina;Nunez, Marco T.
缺铁问题目前影响着大约30亿人，尽管经过了五千年的医疗，但在21世纪仍然存在。大豆铁蛋白（SBFn）是一种大而稳定的蛋白质纳米笼，包裹着一种含有数百个铁和氧原子的矿物质，是一种营养铁的来源，其肠道吸收机制尚不清楚。sfn对铁的吸收对植酸盐不敏感，表明其吸收机制不同于铁的运输。在这里，我们以Caco-2细胞（在双单元插入中极化）为肠道细胞模式，研究了矿化SBFn吸收铁的机制，并通过标记SBFn （I-131或荧光标记）、共聚焦显微镜和免疫分析分析了其结合、内化和降解，结果表明：1)与根尖细胞表面的饱和结合；解离常数为7.75 +/- 0.88 nmol/L；2) SBFn的内化与温度、浓度和时间有关；3) sfn铁进入不稳定铁池（钙黄素淬火）；4) SBFn蛋白笼的降解；5)组装肽2 (AP2)-/网格蛋白依赖的内吞作用（SBFn摄取对高渗透压、酸性和RNA干扰对AP2的mu(2)亚基的敏感性），以及对filipin（一种囊泡内吞抑制剂）的抗性。结果支持了一个SBFn通过顶细胞膜内吞的模型，随后是蛋白质笼降解，矿物质还原/溶解，铁进入细胞质铁池。SBFn中大量的铁原子使得铁在细胞膜上的运输比单个铁原子如血红素或亚铁离子更有效。
Iron deficiency, a condition currently affecting similar to 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 (I-131 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.