Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

Jinyuan Vero Li,Diane Fatkin,Yang Guo,Chai-Ann Ng,Philip W Kuchel,Delfine Cheng,Yogambha Ramaswamy,Mingxi Yao,Michael P Feneley,Zijing Zhou,Lining Arnold Ju,Ze-Yan Yu,Charles D Cox

doi:10.1038/s42003-021-02528-w

Abstract

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.

Highlights

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli
PIEZO1 have been linked to disease, we explored whether human Piezo[1] undergoes N-linked glycosylation and whether this serves as a predictor of efficient membrane trafficking
We report on the molecular events that occur during the trafficking of Piezo[1] channels, and we showed that N-linked glycosylation was critical in the biosynthetic quality control of this channel

Summary

Introduction

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. We show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo[1] channels are necessary for the mature protein to reach the plasma membrane. Like many other integral membrane proteins, Piezo[1] channels undergo biosynthetic quality control during their biogenesis[13]. Almost all membrane proteins undergo folding and maturation in the ER, where N-linked glycosylation usually fulfils a critical role in biosynthetic quality control[13,14]. N-linked glycosylation is the process by which oligosaccharides are covalently attached to asparagine residues in proteins, at specific asparagine containing motifs This process begins with the co-translational addition of core-glycans in the ER and culminates in the processing, modification and potential elongation of the N-glycan in the Golgi prior to vesicular transit to the plasma membrane. The hERG K+ channel (Kv11.1)[18,19], cystic fibrosis transmembrane conductance regulator (CFTR)[20] and polycystic kidney disease proteins[21] have all been studied in this regard

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Conclusion