ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

Tejeshwar C Rao,Reena R Beggs,Katherine E Ankenbauer,Jihye Hwang,Victor Pui-Yan Ma,Khalid Salaita,Susan L Bellis,Alexa L Mattheyses

doi:10.1016/j.jbc.2022.101726

Abstract

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Highlights

Mechanical forces are key regulators of cell structure and function, playing a crucial role in many processes including mitosis, apoptosis, adhesion, and migration (1,2)
We found no significant difference in epidermal growth factor receptor (EGFR) sialylation regulates cell mechanics total EGFR protein levels in overexpress ST6Gal-I (OE) and empty vector (EV) cells (Fig. 1B, D)
To verify that overexpression of ST6Gal-I led to a concomitant increase in cell surface sialylation, wild type (WT), EV and ST6Gal-I OE Cos-7 cells were labeled with SNA, with or without EGF

Summary

Introduction

Mechanical forces are key regulators of cell structure and function, playing a crucial role in many processes including mitosis, apoptosis, adhesion, and migration (1,2). Using high resolution total internal reflection fluorescence (TIRF) microscopy, we show that ST6Gal-I mediated sialylation of EGFR in Cos-7 cells enhances EGF driven mechanical changes including increased integrin tension, focal adhesion (FA) maturation, cell spreading, and migration. At 10 and 90 min following EGF stimulation ST6Gal-I OE cells had increased EGFR activation relative to EV or WT cells, in agreement with previous work (Fig. 1E) (29,30).

Results

Conclusion