Mucins in the alimentary canal: their structure and interactions with polyphenols

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Abstract

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here.Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency.The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.

Layman's Abstract

Spit, that gooey, sticky substance, is a substance full of purpose. We make 1.5 L of spit every day and it helps us to eat, chew and talk by lubricating the mouth. Spit also traps bacteria and stops them from harming our teeth so we don't have to go to the dentist so often. Spit has these roles due to its sticky molecules called mucins. Mucins are like long sugar-coated ropes, but their size and shape is not fully known. Here, the shape of mucins was studied using new methods that have not been used for this before, called Raman spectroscopies. I found out that the ends of mucins have well organised shape, but the middle part of mucins does not. The middle part of mucins is coated in sugars and so the sugars stop the mucin from forming an organised shape. This makes mucins very floppy, which may be key to their stickiness.As well as looking at mucin shape, I studied how the shape of mucins in spit might change when we eat and drink. This could be important for how we decide what food and drinks we like and dislike. I studied green tea, which is thought to be good for human health. I found that green tea makes huge changes to the shape of mucins in spit. After this, I showed that green tea does this by changing the shape of the ends of the mucins, but not the middle part of the mucin. Green tea then clumps mucins together. This might lead to the dry feeling in the mouth after drinking green tea, called astringency. This is the first time a food/drink item has been shown to change mucins in saliva.Mucins are not only found in spit, but also in snot and all mucus layers that line the stomach and intestine. I saw that mucins in the stomach are also clumped together by green tea. It is possible that these changes to mucins in spit and stomach mucus might keep green tea around in the mouth and stomach and boost its health benefits. In summary, this thesis has revealed the shape of mucins and how this is changed by food/drink.

Keyword(s)

EGCG; Epigallocatechin-3-gallate; Green tea polyphenols; MUC5B; MUC7; Muc5ac; Muc6; Mucin; Mucus; Raman optical activity; Raman spectroscopy; Saliva; Stomach; Tip-enhanced Raman spectroscopy

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