Mucin gel assembly is controlled by a collective action of non-mucin proteins, disulfide bridges, Ca2+-mediated links, and hydrogen bonding

Abstract

Mucus is characterized by multiple levels of assembly at different length scales which result in a unique set of rheological (flow) and mechanical properties. These physical properties determine its biological function as a highly selective barrier for transport of water and nutrients, while blocking penetration of pathogens and foreign particles. Altered integrity of the mucus layer in the small intestine has been associated with a number of gastrointestinal tract pathologies such as Crohn’s disease and cystic fibrosis. In this work, we uncover an intricate hierarchy of intestinal mucin (Muc2) assembly and show how complex rheological properties emerge from synergistic interactions between mucin glycoproteins, non-mucin proteins, and Ca2+. Using a novel method of mucus purification, we demonstrate the mechanism of assembly of Muc2 oligomers into viscoelastic microscale domains formed via hydrogen bonding and Ca2+-mediated links, which require the joint presence of Ca2+ ions and non-mucin proteins. These microscale domains aggregate to form a heterogeneous yield stress gel-like fluid, the macroscopic rheological properties of which are virtually identical to that of native intestinal mucus. Through proteomic analysis, we short-list potential protein candidates implicated in mucin assembly, thus paving the way for identifying the molecules responsible for the physiologically critical biophysical properties of mucus.

Formation of Protein Networks between Mucins: Molecular Dynamics Study Based on the Interaction Energy of the System

“…Similar behavior showing a threshold was noticed also in a recent experimental study, which analyzed more complex mucin solution (involving mucin (MUC2) oligomers, additional non-mucin proteins, and ions Ca 2+ ); see Figure 2A in [51]. In their system, they noticed a threshold at a much lower concentration, between 5 and 10 g/L, when the system started to behave subdiffusively, showing in this way gel formation.…”

“…In their system, they noticed a threshold at a much lower concentration, between 5 and 10 g/L, when the system started to behave subdiffusively, showing in this way gel formation. The discrepancy was most probably caused by the differences in the system, mainly by the length of the mucin molecule: it is much longer, branched, and unfolded at low concentrations (see Figure 2C in [51]). Despite this, the vast similarity noticed indicated the usefulness of the molecular simulation technique in the studies within the polymer sciences area.…”

Salivary Metabolomics: From Diagnostic Biomarker Discovery to Investigating Biological Function

“…Whether there is a causative role for citrate in contributing to the extensional rheology of saliva is at present unclear, as is any precise mechanism. Other salivary analytes associated with salivary rheology include calcium and bicarbonate [138], and it is known that calcium chelation is an important step for the unpacking of salivary glycoproteins (viscoelastic macromolecules) following secretion [139,140]. It is possible that the interactions between calcium, citrate, bicarbonate, and glycoproteins at the point of salivary secretion ultimately determine the net physical properties of the resulting fluid.…”

Towards bioinspiredin vitromodels of intestinal mucus

“…8B). 63 Below this threshold, the mono-component models show a tan d slightly higher than one, suggesting a viscous contribution to the rheological behaviour higher than the elastic one. Under constant strain rate tests, this is reected in a lower steady state viscosity at any strain rate for monocomponent mucus models with respect to the physiological mucus, even if the shear thinning behaviour is preserved (Fig.…”

“…8C). 40,63 Like physiological mucus, mono-component mucus models based on mucins from different portions of the gastrointestinal tract display different viscoelastic properties even if at the same concentration. For instance, the mono-component mucus model composed of 4% (w/v) duodenal mucins shows a storage modulus that is ten times higher than the one of 4% (w/v) jejunal mucins.…”