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

Abstract

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi 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 higher-order 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 protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

The anchor domain is critical for Piezo1 channel mechanosensitivity

“…We then looked at the expression of each of the F2114 mutations using western blotting and the same monoclonal anti-Piezo1 antibody used in Figure 2 . We saw that compared to the other four F2114 mutations (A, C, T, Y) the F2114G in particular, but also the F2114A, has a lower intensity upper band (the fully glycosylated version) consistent with previous studies ( Figure 3d ) [ 32 ]. We then compared the mechanosensitivity of all the variants at the 2114 position ( Figure 3e ).…”

“…In addition, many other regions including the local curvature and “footprint” [ 23 , 44 ] of the Piezo1 channel are essential for mechanosensing [ 45 ]. This could include the importance of cytoskeletal [ 46 , 47 ] or extracellular-matrix-based molecular tethers (possibly N-glycans [ 32 ]) that work in unison with bilayer forces [ 38 ]. In conclusion, our data suggest that the anchor domain, although not the only, is a critical force conveying structure within the MS channel Piezo1.…”

“…To understand why this mutation lacks function we used western blotting. We have previously shown that human Piezo1 undergoes significant N-linked glycosylation and that the fully glycosylated version of the protein represents the major pool of the surface expressed mature Piezo1 32 . So we expressed wild-type P2113 + 2 G, T2128 + 2 G and G2163 + 2 G in Piezo1 −/-HEK293T and probed their expression using a primary monoclonal Piezo1 antibody [32].…”

“…We have previously shown that human Piezo1 undergoes significant N-linked glycosylation and that the fully glycosylated version of the protein represents the major pool of the surface expressed mature Piezo1 32 . So we expressed wild-type P2113 + 2 G, T2128 + 2 G and G2163 + 2 G in Piezo1 −/-HEK293T and probed their expression using a primary monoclonal Piezo1 antibody [32]. A representative western blot clearly showed that WT, P2113 + 2 G and G2163 + 2 G all run as a doublet with the upper band representing the fully glycosylated (FG) protein.…”