Interactions Between Ferroportin and Hephaestin in Rat Enterocytes Are Reduced After Iron Ingestion
Interactions Between Ferroportin and Hephaestin in Rat Enterocytes Are Reduced After Iron Ingestion
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DOI：
10.1053/j.gastro.2011.03.059
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发表时间：
2011-07-01
期刊：
影响因子：
29.4
通讯作者：
Glass, Jonathan
中科院分区：
文献类型：
--
作者：
Yeh, Kwo-Yih;Yeh, Mary;Glass, Jonathan
背景与目的：铁转运蛋白 (Fpn) 是一种与铁蛋白 (Heph) 配合将铁输出到体循环所需的多次跨膜蛋白。尽管 Fpn 在铁转运中很重要，但其与 Heph 相互作用时的拓扑结构和功能状态仍存在争议。方法：使用针对其不同表位的单特异性抗血清，在十二指肠用或未用去垢剂透化的细胞片中确定 Fpn 的拓扑结构。使用免疫沉淀和蓝色天然聚丙烯酰胺凝胶电泳，然后进行免疫印迹分析来确定 Fpn 和 Heph 之间的相互作用程度。针对细胞内、二价金属转运蛋白 (Dmt1) 和 Heph 的 C 末端的抗血清作为对照。结果：使用抗 Fpn 氨基酸 172-193 的抗血清（抗 Fpn 172）进行免疫荧光分析，仅在透化细胞中检测到 Fpn，而抗 Fpn 232（氨基酸 232-249）、抗 Fpn 370（氨基酸 370-420）和抗 Fpn C（C 末端）在透化细胞中检测到 Fpn。非透化细胞和透化细胞。免疫沉淀研究表明 Fpn 和 Heph 与抗 Fpn 或抗 Heph 共沉淀。蓝色天然聚丙烯酰胺凝胶电泳研究表明，Fpn 的一部分与 Heph 共迁移；摄入铁后，明显的相互作用减少。结论：针对Fpn不同表位的抗血清研究表明，Fpn的拓扑结构与11次跨膜模型一致，C端暴露于细胞表面。铁摄入后 Fpn 和 Heph 之间相互作用的减少表明，这是限制铁进一步吸收的调节机制。
BACKGROUND & AIMS: Ferroportin (Fpn) is a multiple transmembrane protein required for iron export into the systemic circulation, in cooperation with hephaestin (Heph). Despite the importance of Fpn in iron transport, there is controversy about its topology and functional state upon interaction with Heph. METHODS: The topology of Fpn was determined using monospecific antisera against its different epitopes, in sheets of cells from duodenum that were or were not permeabilized with detergent. Immunoprecipitation and blue native polyacrylamide gel electrophoresis, followed by immunoblot analysis, were used to determine the extent of interactions between Fpn and Heph. Antisera against the intracellular, C-termini of divalent metal transporter (Dmt1) and Heph served as controls. RESULTS: Immunofluorescence analysis with antisera against amino acids 172-193 of Fpn (anti-Fpn 172) detected Fpn only in permeabilized cells, whereas anti-Fpn 232 (amino acids 232-249), anti-Fpn 370 (amino acids 370-420), and anti-Fpn C (the C-terminus) detected Fpn in nonpermeabilized and permeabilized cells. Immunoprecipitation studies showed that Fpn and Heph coprecipitated with either anti-Fpn or anti-Heph. Blue native polyacrylamide gel electrophoresis studies revealed that a fraction of Fpn comigrates with Heph; the apparent interaction decreases after iron ingestion. CONCLUSIONS: Studies with antisera to different epitopes of Fpn indicate that the topology of Fpn is consistent with an 11-transmembrane model, with the C-terminus exposed on the cell surface. Reduced interactions between Fpn and Heph after iron ingestion indicate that this is a regulatory mechanism for limiting further iron absorption.