Blalock Timothy D, Spurr-Michaud Sandra J, Tisdale Ann S, Heimer Susan R, Gilmore Michael S, Ramesh Vijaya, Gipson Ilene K
Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4509-18. doi: 10.1167/iovs.07-0430.
The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins.
The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium.
MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression.
These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.
膜相关黏蛋白MUC16是一种高度O-糖基化的跨膜蛋白，由眼表上皮细胞表达并定位于表面微褶的尖端。尽管其在眼表糖萼中的功能尚不清楚，但人们认为MUC16为上皮细胞膜提供了一种抗黏附屏障。眼表上皮细胞表达的另外两种膜相关黏蛋白MUC1和MUC4具有多种功能，分别通过其细胞质尾巴和表皮生长因子样结构域具有信号传导能力。MUC16的细胞质尾巴尚未得到表征，但由于它包含一个多碱性氨基酸序列，它可能通过埃兹蛋白/根蛋白/膜突蛋白（ERM）肌动蛋白结合蛋白与肌动蛋白细胞骨架相互作用。
使用细胞质尾巴肽和ERM下拉实验研究MUC16通过ERM与肌动蛋白细胞骨架的相互作用。使用RNA干扰在永生化人角膜缘上皮（HCLE）细胞中确定MUC16的功能。使用扫描和免疫电子显微镜确定MUC16敲低对HCLE细胞中微褶结构的影响。将HCLE细胞与孟加拉玫瑰红染料孵育，以测量MUC16在眼表屏障功能中的作用。测量荧光标记的金黄色葡萄球菌与HCLE细胞的结合，以确定MUC16在保护眼表上皮细胞免受病原体黏附中的作用。
MUC16细胞质尾巴肽与ERM的N端结合，而MUC1和MUC4肽未检测到结合。MUC16抑制后，在HCLE细胞中未检测到对表面膜突起的影响；然而，用孟加拉玫瑰红孵育的HCLE细胞显示，MUC16抑制的细胞中染料的排除显著减少。此外，MUC16抑制后，金黄色葡萄球菌与HCLE细胞的结合显著增加。
这些结果表明MUC16是一种与肌动蛋白细胞骨架相关的多功能分子。MUC16在眼表糖萼中的表达有助于为上皮表面提供抗黏附保护屏障。
