Sumiyoshi Mika, Ricciuto Jessica, Tisdale Ann, Gipson Ilene K, Mantelli Flavio, Argüeso Pablo
Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):197-203. doi: 10.1167/iovs.07-1038.
Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells.
Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber.
Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface.
These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).
在眼表、口腔、生殖道和肠道中会出现相对的黏膜表面长时间接触的情况，这需要一种特殊的顶端细胞表面来防止黏附。本研究的目的是评估黏蛋白O-聚糖对人角膜缘上皮（HCLE）细胞抗黏附特性的贡献。
通过用苄基-α- N -乙酰半乳糖胺进行代谢干扰来破坏HCLE细胞中黏蛋白O-聚糖的生物合成。通过免疫荧光和蛋白质印迹检测细胞表面黏蛋白MUC16及其碳水化合物表位H185。通过场发射扫描电子显微镜评估HCLE细胞表面特征。在静态条件下和平行板层流室中进行细胞间黏附试验。
苄基-α- N -乙酰半乳糖胺破坏了O-聚糖的生物合成，而不影响脱辅基黏蛋白的生物合成或细胞表面形态。静态黏附试验表明，表达MUC16和H185的分化HCLE细胞的顶端表面比缺乏MUC16的未分化HCLE细胞更具抗黏附性。用苄基-α- N -乙酰半乳糖胺对分化培养物中的黏蛋白O-糖基化进行消除，导致所施加的角膜上皮细胞和角膜成纤维细胞的黏附增加。动态流动黏附试验中的荧光视频显微镜进一步证明了黏蛋白O-聚糖的抗黏附作用。细胞表面的阳离子化铁蛋白标记表明，阴离子排斥对顶端表面的抗黏附特性没有贡献。
这些结果表明上皮O-聚糖有助于角膜上皮细胞中细胞表面相关黏蛋白的抗黏附特性，并表明黏蛋白O-糖基化的改变参与了干燥性黏膜疾病（如干眼症）的病理过程。
