Trefoil factors share a lectin activity that defines their role in mucus

Michael A Järvå,Ethan D Goddard-Borger,James P Lingford,Nichollas E Scott,Alan John,Niccolay Madiedo Soler

doi:10.1038/s41467-020-16223-7

Michael A Järvå, Ethan D Goddard-Borger + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-16223-7

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Abstract

The mucosal epithelium secretes a host of protective disulfide-rich peptides, including the trefoil factors (TFFs). The TFFs increase the viscoelasticity of the mucosa and promote cell migration, though the molecular mechanisms underlying these functions have remained poorly defined. Here, we demonstrate that all TFFs are divalent lectins that recognise the GlcNAc-α-1,4-Gal disaccharide, which terminates some mucin-like O-glycans. Degradation of this disaccharide by a glycoside hydrolase abrogates TFF binding to mucins. Structural, mutagenic and biophysical data provide insights into how the TFFs recognise this disaccharide and rationalise their ability to modulate the physical properties of mucus across different pH ranges. These data reveal that TFF activity is dependent on the glycosylation state of mucosal glycoproteins and alludes to a lectin function for trefoil domains in other human proteins.

Highlights

The mucosal epithelium secretes a host of protective disulfide-rich peptides, including the trefoil factors (TFFs)
ELISA performed using these reagents (Fig. 1c) detected robust binding of the TFF2bio control, mTFF1bio, and mTFF3bio to both porcine gastric mucin (pMucin) and pMucinred, while no significant binding was observed for any TFF to pMucinred+GH89
This established that all TFF–mucin interactions require αGlcNAc-terminated glycans and that these interactions are independent of mucin tertiary structure

Summary

Introduction

The mucosal epithelium secretes a host of protective disulfide-rich peptides, including the trefoil factors (TFFs). Structural, mutagenic and biophysical data provide insights into how the TFFs recognise this disaccharide and rationalise their ability to modulate the physical properties of mucus across different pH ranges These data reveal that TFF activity is dependent on the glycosylation state of mucosal glycoproteins and alludes to a lectin function for trefoil domains in other human proteins. The binding mode of this disaccharide is revealed using X-ray crystallography and this information used to inform mutagenesis studies to determine which residues are critical for lectin activity We demonstrate how these residues define the pH profile of lectin activity and how this correlates with the different biological roles of the TFFs. The lectin activity of the TFFs, and the presence of the GlcNAc-α-1,4-Gal disaccharide, is shown to be essential for cross-linking mucus glycoproteins, suggesting that the mucusthickening properties of the TFFs arise from their ability to reversibly and non-covalently cross-link these large glycoproteins.

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