Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia.
St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia.
Commun Biol. 2021 Sep 6;4(1):1038. doi: 10.1038/s42003-021-02528-w.
Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the 'cap' region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the 'propeller' regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.
机械敏感通道是一种整合膜蛋白，可感知机械刺激。与大多数质膜离子通道蛋白一样，它们必须通过内质网中的生物合成质量控制，才能到达质膜上的目的地。在这里，我们表明，机械敏感 Piezo1 通道“帽”区中两个高度保守的天冬酰胺残基的 N-连接糖基化对于成熟蛋白到达质膜是必要的。这两个天冬酰胺的突变（N2294Q/N2331Q）和用水解 N-连接寡糖的酶（PNGaseF）处理都会消除完全糖基化的成熟 Piezo1 蛋白。“桨叶”区中的 N-糖基化是“帽”区中 N-糖基化的前提条件，“桨叶”区存在于机械转导所必需的环中。重要的是，与广义淋巴管发育不良和二叶主动脉瓣相关的运输缺陷 Piezo1 变体显示出减少的完全 N-糖基化的 Piezo1 蛋白。因此，体外的 N-连接糖基化状态与有效的膜运输相关，并将有助于确定未知意义的 Piezo1 变体的功能影响。
