Cellular internalization of lactoferrin in intestinal epithelial cells
Cellular internalization of lactoferrin in intestinal epithelial cells
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DOI：
10.1023/b:biom.0000027710.13543.3f
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发表时间：
2004-06-01
期刊：
影响因子：
3.5
通讯作者：
Lönnerdal, B
中科院分区：
文献类型：
--
作者：
Ashida, K;Sasaki, H;Lönnerdal, B
为了探讨乳铁蛋白生物学作用的机制，我们研究了乳铁蛋白（Lf）在肠上皮细胞系Caco-2中的内化。RT-PCR和Western blotting分析显示，在还原条件下，分化的Caco-2细胞表达LfR mRNA及其蛋白，分子量为34 kD。生物素标记的Lf表现出特异性结合分化的Caco-2细胞的细胞膜的解离常数（Kd）为0.16 μ M。在Transwell插入物上单层生长的分化Caco-2细胞中研究了Lf的细胞内化，并与人转铁蛋白（Tf）进行了比较。用荧光染料标记后，将Lf或Tf从顶侧或基底侧加入Caco-2细胞。激光扫描共聚焦显微镜显示，标记的Lf只从顶侧内化，并定位于细胞核。另一方面，标记的Tf从基底外侧内化，而不是从顶侧，并定位于细胞质中。标记Lf的内化被过量的未标记Lf抑制，但不被Tf抑制。标记的Lf的内化，但不是标记的Tf，也被肝素抑制。这表明，肝素结合位点的N-末端区域的Lf可能是重要的Lf的内化。这些结果表明，摄入的Lf可能被肠上皮细胞内化的方式不同的Tf，并可能在细胞核中发挥作用。
We studied the cellular internalization of lactoferrin (Lf) in an intestinal epithelial cell line, Caco-2, to investigate the mechanism of biological actions of ingested Lf. RT-PCR and Western blotting analyses revealed that differentiated Caco-2 cells express LfR mRNA and its protein with a 34 kD molecular weight under reducing conditions. Biotin-labeled Lf showed specific binding to the cellular membrane of differentiated Caco-2 cells with a dissociation constant (K-d) of 0.16 muM. The cellular internalization of Lf was studied in differentiated Caco-2 cells grown as monolayers on Transwell inserts, and compared to that of human transferrin (Tf). After labeling with fluorescent dye, either Lf or Tf was added to Caco-2 cells from the apical side or the basolateral one. Laser scanning confocal microscopy showed that labeled Lf was internalized only from the apical side and localized to the nuclei. On the other hand, labeled Tf was internalized from the basolateral side, not from the apical side, and localized in the cytoplasm. The internalization of labeled Lf was inhibited by excess of unlabeled Lf, but not of Tf. The internalization of labeled Lf, but not of labeled Tf, was also suppressed by heparin. This indicates that a heparin-binding site in the N-terminal region of Lf could be important for the internalization of Lf. These findings suggest that ingested Lf might be internalized by the intestinal epithelium in a manner different from Tf and might function in the nucleus.