Institute of Physiology & Pathophysiology, ZBAF, University of Witten, Herdecke, Witten, Germany.
PLoS One. 2013 Aug 5;8(8):e71586. doi: 10.1371/journal.pone.0071586. Print 2013.
The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC₅₀ of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.
脂质运载蛋白 2//中性粒细胞明胶酶相关脂质运载蛋白/24p3 受体（NGAL-R/24p3-R）在啮齿动物远端肾单位表达，在那里它介导蛋白质内吞作用。蛋白质和肽在肠道中的顶膜内吞作用和转胞吞作用的机制知之甚少。在本研究中，通过免疫荧光和共聚焦激光扫描显微镜、免疫组织化学和免疫印迹研究了肠道各段的啮齿动物 24p3-R（r24p3-R）和人 NGAL-R（hNGAL-R）的表达和定位。r24p3-R/hNGAL-R 还在人 Caco-2BBE 细胞和瞬时转染 r24p3-R 的 CHO 细胞中通过免疫荧光显微镜、RT-PCR 和质膜富集囊泡（PM）的免疫印迹进行了研究。为了检测功能，通过测量荧光标记配体在塑料上生长或在 Transwell 插入物上作为单层生长的 Caco-2BBE 细胞中的内化，来检测假定配体植物螯合肽（PC₃）、金属硫蛋白（MT）和转铁蛋白（Tf）的内吞/转胞吞作用。通过微尺度热泳（MST）定量测定 Alexa 488-PC₃ 与结肠样 Caco-2BBE PM 的结合亲和力。r24p3-R/hNGAL-R 在所有肠道段均检测到顶端表达，但在回肠和结肠中表达最高。结肠样但不是十二指肠样 Caco-2BBE 细胞在其表面表达 hNGAL-R。结肠样 Caco-2BBE 细胞或转染 r24p3-R 的 CHO 细胞以亚微摩尔浓度的半最大饱和内化荧光标记的 PC₃ 或 MT。Caco-2BBE 细胞摄取 PC₃ 和 MT（0.7µM）的速度部分被 hNGAL（500pM）阻断，通过 MST 确定 Alexa 488-PC₃ 与 PM 囊泡结合的 EC₅₀为 18.6±12.2nM。Transwell 实验显示荧光 PC₃/MT/Tf（0.7µM）快速（0.5-2 小时）顶端摄取和基底外侧递呈。500pM hNGAL 显著阻断配体的顶端摄取。与 PC₃ 和 Tf 相比，MT 时 hNGAL-R 依赖性摄取更为明显，但转胞吞效率降低。因此，r24p3-R/hNGAL-R 可能代表一种高亲和力多配体受体，用于肠道下部对完整蛋白质/肽的顶端内化和转胞吞作用。
