Fate of E-cadherin in early RPE cultures: Transient accumulation of truncated peptides at nonjunctional sites
Fate of E-cadherin in early RPE cultures: Transient accumulation of truncated peptides at nonjunctional sites
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DOI：
10.1167/iovs.06-0104
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发表时间：
2006-08-01
影响因子：
4.4
通讯作者：
Hong, Jeehee
中科院分区：
文献类型：
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作者：
Burke, Janice M.;Hong, Jeehee
目的.已知E-钙粘蛋白在培养的人RPE细胞的连接处快速且缓慢地积累。本研究的目的是确定是什么限制了E-钙粘蛋白在RPE细胞中的蛋白积累，通过分析培养物在早期的postplating间隔时，连接的优势钙粘蛋白（N-钙粘蛋白）是第一次形成。在接种后48小时内，分析RPE细胞系hTERT-RPE 1和ARPE-19以及从人供体建立的RPE培养物的E-钙粘蛋白基因和蛋白表达（分别通过RT-PCR和Western印迹）以及蛋白分布（通过免疫荧光和免疫电子显微镜），包括与细胞器标记物的共分布。细胞表面定位分析的生物素化和胰蛋白酶切割细胞外钙粘蛋白结构域。E-钙粘蛋白基因组成型表达的RPE文化，但蛋白质没有积累大量的早期RPE文化。相反，少量的新合成的E-钙粘蛋白只能短暂检测到，在铺板后几小时内达到峰值，此时蛋白质以可变大小的肽的形式存在，而不是预测的120 kDa分子量。免疫反应性E-钙粘蛋白肽没有交通到细胞表面和本地化的连接。相反，它们与几种细胞器共分布，包括内质网（ER;但不是高尔基体），蛋白质降解位点（蛋白酶体，溶酶体和自噬体）和不寻常的隔室（中心体和与线粒体网络的亚结构域并列）。结果表明，在RPE细胞中，转录后机制涉及改变蛋白质加工和快速周转存在限制E-钙粘蛋白的积累。其结果可能是限制了RPE中E-钙粘蛋白特异性的诱导特性，RPE是一种细胞类型，其中N-钙粘蛋白是正常的显性钙粘蛋白。
PURPOSE. E-cadherin is known to accumulate variably and slowly at junctions of cultured human RPE cells. The intent of this investigation was to determine what limits E-cadherin protein accumulation in RPE cells by analyzing cultures at early postplating intervals when junctions of the dominant cadherin (N-cadherin) are first forming.METHODS. RPE cell lines hTERT-RPE1 and ARPE-19 and RPE cultures established from human donors were analyzed within 48 hours after plating for E-cadherin gene and protein expression (by RT-PCR and Western blotting, respectively) and for protein distribution (by immunofluorescence and immunoelectron microscopy), including codistribution with markers for organelles. Cell surface localization was analyzed by biotinylation and trypsin cleavage of extracellular cadherin domains.RESULTS. The E-cadherin gene was constitutively expressed by RPE cultures, but the protein did not accumulate substantially in early RPE cultures. Instead small amounts of newly synthesized E-cadherin were detectable only transiently, peaking within a few hours after plating, at which time the protein was in the form of peptides of variable size rather the predicted 120-kDa molecular mass. Immunoreactive E-cadherin peptides did not traffic to the cell surface and localize to junctions. Rather they codistributed with several organelles including the endoplasmic reticulum (ER; but not the Golgi), sites of protein degradation (proteasomes, lysosomes, and autophagosomes) and unusual compartments (centrosomes and apposed to subdomains of the mitochondrial network).CONCLUSIONS. The results suggest that in RPE cells posttranscriptional mechanisms involving altered protein processing and rapid turnover exist to limit E-cadherin accumulation. The consequence may be to limit E-cadherin-specific inductive properties in the RPE, a cell type in which N-cadherin is the normal dominant cadherin.