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.
Highlights
Mucus is a complex viscoelastic fluid produced by specialized secretory cells in the linings of the respiratory, gastrointestinal and urogenital tracts
The assembly mechanism stems from a common blueprint of their domain structure, which comprises a largely unstructured glycosylated (>50%) central domain flanked by the amino (N-) and carboxyl (C-) termini
We investigate the effect of divalent ions (Ca2+), chelating (EDTA), chaotropic (GuHCl), and reducing (DTT) agents, as well as pH on the viscoelastic properties of both native and extensively purified mucins to probe the influence of different components and interactions on the structure and flow properties of mucus
Summary
Mucus is a complex viscoelastic fluid produced by specialized secretory cells in the linings of the respiratory, gastrointestinal and urogenital tracts. Hyper-viscous mucus is a central pathogenic feature of cystic fibrosis, where mucus accumulation and stagnation results in airway and intestinal obstruction and chronic bacterial colonization[2,3] It is established the mucus network is formed via supramolecular assembly, the details of interactions remain poorly understood, which limits our ability to translate physical insights into tangible therapeutic interventions[4]. The central domain includes a tandem repeat region that due to genetic polymorphism comprises anywhere between 51 to 115 repeat fragments (23 amino acids each)[13] This polymorphism together with the broad distribution of glycosylation patterns confer MUC2 a wide distribution of molecular weights, with values reported in the literature varying from ∼2.7 MDa14 up to ∼7 MDa15. Mucus behaves as a solution of entangled polymers that may or may not display significant elasticity (G′ ≈ G′′) depending on the mucin genetic type and glycosylation pattern[22]
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