Understanding the basic elements of the airway mucosal surfaces and how they form a functional barrier is essential in understanding disease initiation, progression, pathogenesis and ultimately treating chronic lung diseases. Using primary airway epithelial cell cultures, atomic force microscopy (AFM), multiangle light scattering and quartz crystal micro balance with dissipation monitoring techniques, here we report that the membrane bound mucins (MBMs) found in the periciliary layer (PCL) of the airway surface are densely decorated with keratan sulfate (KS). AFM and immunoblotting show that the KS sidechains can be removed enzymatically with keratanase II (KII) treatment, and the antibody accessibility for B2729 (MUC1), MUCH4 (MUC4) and OC125 (MUC16) was substantially enhanced. Light scattering analysis confirmed that KII treatment removed ~40% of the mass from the mucin fractions. Surface binding experiments indicated that MBMs were able to pack into a tighter conformation following KS removal, suggesting that negatively charged KS sidechains play a role in mucin–mucin repulsion and contribute to “space filling” in the PCL. We also observed that soluble filtrate from the common airway pathogen Pseudomonas aeruginosa is capable of stripping KS from MBMs. Altogether, our findings indicate that KS glycosylation of MBMs may play an important role in the integrity of the airway mucosal barrier and its compromise in disease.

中文翻译：

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气道粘膜表面的膜结合粘蛋白被硫酸角质素密集装饰：重新审视它们在肺先天防御中的作用

了解气道黏膜表面的基本元素以及它们如何形成功能性屏障对于了解疾病的发生、进展、发病机制和最终治疗慢性肺病至关重要。使用初级气道上皮细胞培养、原子力显微镜 (AFM)、多角度光散射和石英晶体微天平以及耗散监测技术，我们在此报告在气道表面的纤毛层 (PCL) 中发现的膜结合粘蛋白 (MBM)用硫酸角质素（KS）密集装饰。AFM 和免疫印迹显示 KS 侧链可以通过角化酶 II (KII) 处理酶促去除，并且 B2729 (MUC1)、MUCH4 (MUC4) 和 OC125 (MUC16) 的抗体可及性显着增强。光散射分析证实，KII 处理从粘蛋白部分中去除了约 40% 的质量。表面结合实验表明，在去除 KS 后，MBM 能够形成更紧密的构象，这表明带负电荷的 KS 侧链在粘蛋白-粘蛋白排斥中发挥作用，并有助于 PCL 中的“空间填充”。我们还观察到常见气道病原体的可溶性滤液铜绿假单胞菌能够从 MBM 中剥离 KS。总之，我们的研究结果表明 MBM 的 KS 糖基化可能在气道黏膜屏障的完整性及其对疾病的损害中起重要作用。