Reorganisation of the Salivary Mucin Network by Dietary Components: Insights from Green Tea Polyphenols

Abstract

The salivary mucins that include MUC5B (gel-forming) and MUC7 (non-gel-forming) are major contributors to the protective mucus barrier in the oral cavity, and it is possible that dietary components may influence barrier properties. We show how one dietary compound, the green tea polyphenol epigallocatechin gallate (EGCG), can substantially alter the properties of both the polymeric MUC5B network and monomeric MUC7. Using rate-zonal centrifugation, MUC5B in human whole saliva and MUC5B purified from saliva sedimented faster in the presence of EGCG. The faster sedimentation by EGCG was shown to be greater with increasing MUC5B concentration. Particle tracking microrheology was employed to determine the viscosity of purified MUC5B solutions and showed that for MUC5B solutions of 200–1600 µg/mL, EGCG caused a significant increase in mucin viscosity, which was greater at higher MUC5B concentrations. Visualisation of the changes to the MUC5B network by EGCG was performed using atomic force microscopy, which demonstrated increased aggregation of MUC5B in a heterogeneous manner by EGCG. Using trypsin-resistant, high-molecular weight oligosaccharide-rich regions of MUC5B and recombinant N-terminal and C-terminal MUC5B proteins, we showed that EGCG causes aggregation at the protein domains of MUC5B, but not at the oligosaccharide-rich regions of the mucin. We also demonstrated that EGCG caused the majority of MUC7 in human whole saliva to aggregate. Furthermore, purified MUC7 also underwent a large increase in sedimentation rate in the presence of EGCG. In contrast, the green tea polyphenol epicatechin caused no change in the sedimentation rate of either MUC5B or MUC7 in human whole saliva. These findings have demonstrated how the properties of the mucin barrier can be influenced by dietary components. In the case of EGCG, these interactions may alter the function of MUC5B as a lubricant, contributing to the astringency (dry puckering sensation) of green tea.

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

“…2 The average MW of glycan-rich regions was found to be 546 kDa by size-exclusion chromatography coupled with multi-angle laser light scattering. 20 An N-terminal construct of MUC5B, consisting of D1-D2-D′-D3 domains (NT5B, residues 26–1304) and a C-terminal construct of MUC5B, consisting of D4-B-C-CK domains (CT5B, residues 4958–5765), were created, expressed, and purified.…”

“…Furthermore, gel-forming mucins in the GI tract also interact with dietary molecules, as has recently been shown for the salivary mucin MUC5B and green tea polyphenols, which may affect the absorption of nutrients and alter mucin network organization. 2 …”

Surface rearrangement of adsorbed EGCG–mucin complexes on hydrophilic surfaces

“…Since there are only a few PPR/RPP sequences found in mucin [46], the observed behaviour must be attributed to predominantly non-specific binding. The validity of this assumption is supported by conclusions from Davies et al [20] and Georgiades et al [23], where clear evidence for crosslinking of mucins by EGCG via a less specific range of amino acids is reported. Davies et al [20] also established that glycosylated domains do not aggregate with EGCG, and interaction occurs nearly exclusively within the C-and N-termini of the mucin molecule.…”

“…This suggests that binding of EGCG elicits only a small change in the overall conformation of mucin. This can be understood, because EGCG, according to Davis et al [20], does not interact with mucin's glycans, which constitute the largest portion of the mucin molecule in terms of mass as well as hydrodynamic size. It is plausible to assume that conformational changes at the C-and N-termini do occur, as (multidentate) polyphenol molecules bind to these largely non-glycosylated fragments of the mucin molecule.…”

“…The interaction of polyphenols with saliva in the oral cavity is thought to be dominated by prolinerich proteins (PRPs) [14][15][16][17][18][19], with salivary mucins most likely playing a secondary role [5,20,21]. A radically different picture of polyphenol behaviour emerges in the gastrointestinal tract, where mucins are the dominant component of mucus.…”