Expression analysis of the transmembrane mucin MUC20 in human corneal and conjunctival epithelia.
Expression analysis of the transmembrane mucin MUC20 in human corneal and conjunctival epithelia.
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跨膜粘蛋白MUC20在人角膜和结膜上皮中的表达分析。
DOI：
10.1167/iovs.14-15269
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发表时间：
2014
影响因子：
4.4
通讯作者：
Argüeso,Pablo
中科院分区：
文献类型：
--
作者：
Woodward,AshleyM;Argüeso,Pablo
目的：细胞表面粘蛋白是一组高度O-糖基化的跨膜糖蛋白，负责保护粘膜表面上的上皮细胞。本研究的目的是研究粘蛋白20（MUC 20）在眼表的定位和调节。通过免疫荧光显微镜评估MUC 20在人角膜和结膜上皮中的定位。永生化角膜（HCLE）和结膜（HCjE）细胞系生长在不同的分化阶段，并进行定量PCR和蛋白质印迹分析。将顶端细胞膜上的细胞表面蛋白生物素化并通过中性亲和素色谱法分离。MUC 20在整个人眼表上皮中检测到，主要在中间细胞层内的细胞膜中。在结膜中，MUC 20也被观察到在复层鳞状上皮内的顶端细胞的细胞质中，而不是在杯状细胞中。定量PCR和免疫印迹证实HCLE和HCjE细胞中MUC 20的表达。用含血清培养基诱导分化导致MUC 20 mRNA和蛋白的上调。分层培养物表面的生物素标记揭示了顶端糖萼上的低水平MUC 20蛋白。此外，在细胞培养基或人泪液中未检测到MUC 20，表明MUC 20的胞外结构域不像先前对于其他细胞表面粘蛋白所述的那样从眼表面释放。我们的研究结果表明，MUC 20是一种新的跨膜粘蛋白表达的人角膜和结膜上皮细胞，并建议MUC 20在分化过程中的差异表达在维持眼表稳态的作用。
Purpose.: Cell surface mucins are a group of highly O-glycosylated transmembrane glycoproteins responsible for the protection of epithelial cells on mucosal surfaces. The aim of this study was to investigate the localization and regulation of mucin 20 (MUC20) at the ocular surface.Methods.: Localization of MUC20 in human corneal and conjunctival epithelia was evaluated by immunofluorescence microscopy. Immortalized corneal (HCLE) and conjunctival (HCjE) cell lines were grown at different stages of differentiation and subjected to quantitative PCR and Western blot analyses. Cell surface proteins on apical cell membranes were biotinylated and isolated by neutravidin chromatography.Results.: The MUC20 was detected throughout the entire human ocular surface epithelia, predominantly in cell membranes within intermediate cell layers. In conjunctiva, MUC20 also was observed in the cytoplasm of apical cells within the stratified squamous epithelium, but not in goblet cells. Quantitative PCR and immunoblotting demonstrated expression of MUC20 in HCLE and HCjE cells. Induction of differentiation with serum-containing medium resulted in upregulation of MUC20 mRNA and protein. Biotin labeling of the surface of stratified cultures revealed low levels of MUC20 protein on apical glycocalyces. Further, MUC20 was not detected in the cell culture media or in human tears, suggesting that the extracellular domain of MUC20 is not released from the ocular surface as described previously for other cell surface mucins.Conclusions.: Our results indicate that MUC20 is a novel transmembrane mucin expressed by the human corneal and conjunctival epithelia, and suggest that differential expression of MUC20 during differentiation has a role in maintaining ocular surface homeostasis.
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