Mucus.

The biosynthesis of a mucin polymer is carried out in specialized secretory cells that must tackle a variety of challenges such as assembling the polymer, controlling glycosylation, and managing molecules whose fully hydrated dimensions surpass those of some of the intracellular structures in which they are trafficked and stored. Although the general stages and intracellular locations of the polymeric mucin biosynthetic pathway have been described — based mainly on studies of MUC2, MUC5AC, MUC5B, and pig submaxillary mucin (MUC19) — full mechanistic details have yet to be elucidated for any mucin. Mucins are translated in the rough endoplasmic reticulum (rER) of secretory cells, where membrane-bound ribosomes synthesize peptides that are co-translationally transported into the rER lumen. The rER harbors an abundance of modifying enzymes involved in folding, N -glycosylation, C -mannosylation, and disulfide-bond formation along the nascent protein. Next, individual mucin proteins dimerize through non-covalent twisting of domains downstream of the PTS, followed by formation of disulfide bonds between ‘cysteine knot’ domains at the distal carboxy termini. Finally, the newly formed dimers are exported to the Golgi apparatus. O -glycosylation occurs in the Golgi. Here, mucins obtain the bottlebrush-like structure that renders them slimy upon hydration. Creation of the glycan chains is a step-wise process where various glycosyltransferases coordinate their initiation, extension, and termination. Initiation of the glycan chains occurs via the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues by polypeptide GalNAc (ppGalNAc) transferases. As the O -glycosylated mucin dimer moves from the cis-Golgi to later compartments, glycan chains are extended via addition of galactose and N-acetylglucosamine (GlcNAc) residues and then terminated via addition of additional sugars, such as fucose or highly charged sialic acid groups. Terminal sialic acid residues confer a net negative charge, which is important for the rod-like structure and hydration capacity of mucins, among other signature properties. Importantly, these glycosylation steps happen rapidly — mucin dimers contain 4,000 potential glycosylation sites, but they pass through the Golgi in just 2–4 hours ( Figure 2A ). With most types of protein glycosylation, initiation is controlled by just 1–2 gene products. However, in the case of mucin-type O -glycosylation, initiation can be accomplished by an entire family of at least 15 homologous ppGalNAc transferases. After initiation, the O -GalNAc residues can be lengthened by 30 additional glycosyltransferases, thus extending the glycan structure ( Figure 3 ). Given that these glycosyltransferases have unique recognition motifs and distinct donor and acceptor sugars, differential expression of these enzymes in various cells and tissues means that the same mucin gene product can possess site-specific glycosylation patterns. Beyond being helpful for generating diversity in glycan composition, such a large number of glycosyltransferases may hint at the important biological role of O -glycans within the body. What appears as redundancy may actually provide protection against deleterious mutations that compromise this important protein modification. Although O -glycosylation is not exclusive to mucins, the sheer number of O -glycans present on a single mucin gene product suggests that this process is particularly demanding in mucus-producing cells. It has yet to be explored how the glycosylation machinery in mucin-secreting cells differs from that in other cell types, how glycosyltransferases interact with the mucin backbone, and whether distinct enzymes act together in complexes. Further, it is unclear whether polymer size is controlled stochastically or whether it is limited by the spatial constraints of intracellular structures involved in storage and trafficking and/or by temporal constraints related to continuous exocytosis versus stimulated secretory bursts. What is clear is that mucin biosynthesis, particularly O -glycosylation, requires a huge expenditure of energy and resources, including entire families of glycosyltransferases and countless sugars that are used to make thousands of glycan chains per mucin. Thus, the complexity and diversity of glycan structures must be functionally important.

Together, our emerging insights into the diverse properties, structures, and functions of mucus underscore this hydrogel’s multipronged roles in promoting health and preventing disease. Changes in the permeability, glycosylation profiles, and charge distributions of mucus barriers have been linked to altered physiology, resulting in devastating pulmonary, gastrointestinal, and urogenital diseases, among other ailments ( Figure 5 ). In several diseases, compromised selectivity of the mucosal layer results in serious physiological consequences. The cervical mucus of women at high risk for preterm birth is more permeable to peptide probes than samples from low-risk pregnant counterparts. This finding presents a possible connection between increased rates of intra-amniotic infection observed in cases of preterm birth and increased permeability of the cervical mucus plug to microbes. In general, healthy mucus layers serve as protective barriers against harmful microbes. However, certain pathogens are able to degrade or alter the rheological properties of the mucus barrier to facilitate penetration. Helicobacter pylori , a causative agent of peptic ulcers and gastric cancer, modifies the properties of mucus in the stomach by locally increasing pH, decreasing the viscoelastic properties of mucus and enabling the pathogen to swim more easily through it. On the other hand, mucus barriers that are too impermeable present challenges as well. In addition to causing poorly cleared mucus to act as an incubator for colonized microbes, dense mucus has lower permeability, which impairs drug delivery to the lung in cystic fibrosis. Important causes of these defects include hyper-concentration of mucus components, increased mucin cross-linking, and calcium-mediated compaction due to impaired bicarbonate secretion. Patients with chronic obstructive pulmonary disease also hypersecrete mucus, causing a decline in lung performance due to decreased airflow, chronic infection, and poor gas exchange. Other diseases are linked to mucus with altered glycosylation profiles. For example, Sjögren’s syndrome is an autoimmune disorder leading to chronic dry mouth and eyes that is associated with problems swallowing and tasting, as well as increased rates of tooth decay and yeast infections. Patients with this disorder have decreased residual saliva production and flow rate. Although the overall salivary concentrations of MUC5B and MUC7 (a non-polymeric secreted mucin) did not differ between healthy controls and Sjögren’s patients, MUC7 O -glycans in Sjögren’s patients have a significant reduction in hydrophilic sialic acid groups, resulting in dryness. Over the past 20 years, research has revealed that changes in secreted and membrane-bound mucins are linked to cancer progression and carcinogenesis. Compromised MUC2 barriers in the intestine, as observed with ulcerative colitis, are associated with genotoxic stress and chronic-inflammation-induced cancer progression. Further, MUC1, a transmembrane mucin, evolved as an anti-inflammatory protective mechanism for epithelial cells; however, its role in promoting the proliferation and survival of epithelial cells is exploited in cancer. Mucins are typically only present on the apical surface, but when epithelial cells lose polarity during carcinogenesis, mucins can become expressed all over the cell surface, meaning that they become much more available to interact with various growth factor receptors involved in cancer signaling. Consequently, 65% of tumors diagnosed in the United States each year aberrantly express MUC1. Another membrane-bound mucin that is upregulated in certain cancers, MUC4, is correlated with resistance to antibody treatment and evasion of the immune system, and also appears to contribute to growth, motility, and invasiveness of carcinoma cells. Further, ovarian, lung, breast, and pancreatic cancers are associated with aberrant expression of the membrane-bound mucin MUC16. Together, these examples point to mucins as promising anticancer drug targets. Mucosal defects and aberrant expression of mucin genes (both membrane-bound and secreted mucins) have been linked to many other ailments, including but not limited to infertility, endometriosis, kidney disease, and Chrohn’s disease. This wide variety of conditions underscores the potential of non-invasive, mucin-based diagnostics for disease. It also suggests that beyond harboring biomarkers of disease states, mucus and its component mucins can be directly involved in disease pathology.

After their secretion, hydrated mucins interact with each other and scores of other proteins and macromolecules. The resulting supramolecular organization is still not completely understood. One key technical limitation is that it is hard to ‘visualize’ native mucus without perturbing its structure, but here are a few high-level insights that we currently understand. In aqueous solution, high molecular-weight mucin molecules form a network via a complex series of reversible bonds that are sensitive to variations in pH as well as to concentrations of ions and small molecules. Mucus is a viscoelastic material, meaning that its mechanical response to an imposed deformation lies somewhere between that of a pure solid (Hooke’s law of elasticity) and that of a pure liquid (Newton’s law of viscosity). The spinnability of mucus, or its propensity to form filaments when stretched, is an important determinant of physiological functions such as mucociliary clearance, food bolus formation, and swallowing. The extent of cross-linking in the mucin network, the length of the mucin molecules, mucin concentration, and the composition of mucin glycans all impact spinnability. Crucially, mucus layers possess distinct mechanical and biochemical properties that depend on their location and intended physiological function. Consider that the eye has a thin, watery mucin solution on its surface for hydration and lubrication, while the mucus lining the stomach is a stiffer gel that protects the epithelium against acidic (pH ≈ 1–2) gastric juices. Mechanical and biochemical properties can be altered by changing the degree of physical and chemical cross-linking in the mucin network, modifying mucin conformations through variations in pH and salt, manipulating the types and densities of the mucin glycans to impact gel swelling, and changing mucin concentration. Mucus stiffness, or its ability to resist deformation, varies throughout the body; mucus can rapidly self-heal to recover its initial stiffness after mechanical deformations associated with processes such as coughing.

Mucus secretion is an explosive event — upon hydration, mucins expand several hundred-fold in volume almost instantaneously, as if they are being released from a pressurized container. It is clear that secretory cells must expertly handle dehydrated mucins on the pathway to secretion. This goal is achieved via a process of regulated exocytosis. Dehydrated mucins are compacted by shielding the negative charges on the glycans with calcium and by establishing calcium- and pH-dependent, reversible, non-covalent interactions between the cysteine-rich domains of the mucin polymer. After compaction, mucin polymer-containing vesicles are trafficked along the secretory pathway in vesicles decorated with Rab proteins. At the plasma membrane, Rabs interact with tethering proteins on the inner leaflet of the plasma membrane, where they mediate granule docking. In response to specific biological cues, docked granules associate more tightly to and fuse with the plasma membrane, releasing mucins to the epithelial surface, where they instantly become hydrated and balloon in size. This fusion process uses highly conserved molecular machinery comprising a four-helix bundle (the SNARE complex), a Munc18 scaffolding protein, and other regulatory proteins that respond to diverse secondary messengers ( Figure 2B ). To protect and prevent the drying of epithelial surfaces, mucins are constantly secreted at low rates. In the lungs, low levels of mucin exocytosis occur in equilibrium with mucin biosynthesis in order to maintain mucociliary clearance and innate defense. In tissues where mucin biosynthesis is robust, exocytosis results in an accumulation of mucus on epithelial surfaces. In the stomach, colon, or endocervix, this accumulation may be protective. In contrast, in the lungs, excessive mucin release can obstruct airflow and negatively impact defensive mucus clearance. Beyond a baseline level of secretion, mucin exocytosis can be triggered in a controlled manner by various signals, including increased levels of Ca 2+ , diacylglycerol, ATP, histamine, and acetylcholine. For instance, when stomach epithelial cells experience disruption of their plasma membrane, Ca 2+ is released rapidly into the local environment. This signal triggers the rapid release of mucus by stomach mucous cells, which is thought to promote the survival of cells with damaged plasma membranes and confer extra protection and lubrication to sites that endure high amounts of mechanical stress. The triggers and machinery that regulate mucin exocytosis appear to be similar across tissues, but variations in exocytic machinery isoforms may help tailor secretion rates and magnitudes to local needs. In the lung, for example, some components are shared between the low baseline and the rapidly stimulated secretory pathways (such as SNAP23 and Munc13-2). However, others are distinct (for example, Munc18-1 versus 18-2). Whether these exocytic components are linked selectively with secretion of specific mucin isoforms is still an open question. Overall, the presence of distinct machineries in the airway epithelium enables strategies for selective manipulation of the stimulated secretory machinery to reduce obstruction in diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease, while preserving the baseline secretory machinery to maintain particle clearance.

The ability of mucus to achieve such a wide variety of functions is due to the complexity that underpins mucin structure and the abilities of mucus gels to adapt their properties to diverse environments and needs. The more we learn about the importance of mucus, the more new and exciting areas of research emerge. For example, the identity and arrangement of mucin glycans have been shown to be essential for activity, but it is unknown how these properties are regulated. Similarly, the ability of mucin to signal to pathogens and alter physiology suggests that there must be other relationships between mucin and mucus-resident cells, such as immune cells and commensal bacteria, yet to be discovered. Expanding our understanding of how mucin functions in health will enable us to better understand and treat disease states associated with mucus dysfunction. Moreover, research on mucin structure will uncover its properties with potential applications in biomedical engineering, such as wound healing and infection prevention. These exciting discoveries will shift the textbook model of mucus from one in which mucus serves solely as a barrier to one that encompasses its additional, multifaceted and vital biochemical functions.

Although many mucins seem to have related roles in the body, their production is differentially regulated and their glycosylation signatures are distinct. This points to important functional differences. Unique glycosylation patterns on mucus gels likely mediate the interplay between epithelial cells, the immune system, and microbes (both commensal and pathogenic) in a manner specific to the challenges faced by each mucosal surface. Accordingly, mucin production and secretion are tightly controlled both spatially and temporally. For example, MUC2 is expressed by surface goblet cells throughout the intestines. In the stomach, MUC5AC is widely expressed by surface and glandular mucous cells, whereas MUC6 is restricted to pyloric gland mucous cells. In nasal and tracheobronchial airways, MUC5B is abundant in submucosal glands but largely absent from surface goblet cells. In healthy airways, MUC5AC is predominantly expressed by surface mucosal cells, and its expression can increase > 50-fold during inflammation. Epigenetic control has been proposed as one way of regulating the location of mucin expression, presumably by restricting transcription factor accessibility. Emerging evidence in the developing mouse respiratory tract shows that dedicated signaling pathways, namely Notch- and bHLH-dependent signals, coordinate secretory cell fate determination, affecting homeostatic mucus defenses by controlling the production of Muc5b, the murine counterpart of human MUC5B. In health, there is very little deviance from these distribution patterns. However, the rapid and sometimes dramatic changes in mucin expression and localization in disease have motivated biomedical investigations into mechanisms regulating homeostatic and inducible mucin expression. After injury or inflammation, mucosal surfaces may undergo profound remodeling that is characterized by induction of mucin expression within existing mucous cells or by differentiation of additional epithelial precursors into mucous cells. In acute and chronic airway disease, increased expression of MUC5B and the induction of MUC5AC are driven by these differentiation cues along with inflammatory mediator signals, such as NFκB and STAT6, and growth-factor-response signals such as EGFR, FOXA2/3, and SPEDF. As mentioned above, the spatial distribution of mucins and patterns of mucin glycosylation are also regulated by the immune system. The recent discovery that infection at one mucosal barrier site — the gut — initiated mucin-mediated host protection to pathogen challenge at a distal mucosal site — the lung — reveals an important innate defense mechanism in which mucins play a key role in protecting against potential infectious challenges encountered at multiple mucosal surfaces. These effects reinforce the need to understand how mucous cell differentiation and mucin biosynthesis are driven by the coordinated efforts of genetic, environmental, and immunological cues. The heterogeneity of these signaling mechanisms highlights the importance of adapting — and integrating — the responses of mucin-expressing cells to diverse stimuli, as well as the potential for things to go awry and lead to disease. Accordingly, these variable yet tightly controlled expression patterns are also coordinated with expression of genes involved in polymer assembly, glycosylation, and protein homeostasis.