The Biology of Mucus: Composition, Synthesis and Organization☆

Home , Glycoprotein, Intrinsic factor, Mucin, Mucus, Phlegm, Proteoglycan

Advanced Drug Delivery Reviews 124 (2018) 3–15

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Advanced Drug Delivery Reviews

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The biology of mucus: Composition, synthesis and organization☆

Rama Bansil a,⁎, Bradley S. Turner b,1 a Dept. of Physics, Boston University, USA b Dept. of Biological Engineering, MIT, USA article info abstract

Article history: In this review we discuss mucus, the viscoelastic secretion from goblet or mucous producing cells that lines the Received 15 July 2017 epithelial surfaces of all organs exposed to the external world. Mucus is a complex aqueous ﬂuid that owes its Received in revised form 24 September 2017 viscoelastic, lubricating and hydration properties to the glycoprotein mucin combined with electrolytes, lipids Accepted 27 September 2017 and other smaller proteins. Electron microscopy of mucosal surfaces reveals a highly convoluted surface with a Available online 29 September 2017 network of ﬁbers and pores of varying sizes. The major structural and functional component, mucin is a complex

Keywords: glycoprotein coded by about 20 mucin genes which produce a protein backbone having multiple tandem repeats Mucus of Serine, Threonine (ST repeats) where oligosaccharides are covalently O-linked. The N- and C-terminals of this Mucin apoprotein contain other domains with little or no glycosylation but rich in cysteines leading to dimerization and Secretory granule further multimerization via S\\S bonds. The synthesis of this complex protein starts in the endoplasmic reticulum Exocytosis with the formation of the apoprotein and is further modiﬁed via glycosylation in the cis and medial Golgi and Rheology packaged into mucin granules via Ca2+ bridging of the negative charges on the oligosaccharide brush in the trans Golgi. The mucin granules fuse with the plasma membrane of the secretory cells and following activation by signaling molecules release Ca2+ and undergo a dramatic change in volume due to hydration of the highly negatively charged polymer brush leading to exocytosis from the cells and forming the mucus layer. The rheological properties of mucus and its active component mucin and its mucoadhesivity are brieﬂy discussed in light of their importance to mucosal drug delivery. © 2017 Elsevier B.V. All rights reserved.

Contents

1. Introduction...... 5 2. Mucusstructuralorganization...... 5 2.1. Goblet/mucouscells...... 5 2.2. Mucusstructure...... 5 2.3. Mucusthickness...... 5 3. Mucuscomposition...... 6 3.1. Mucuselectrolytes...... 6 3.2. Mucuslipids...... 6 3.3. Mucusproteins...... 6 3.4. Mucins...... 6 3.5. Glycosylation...... 6 3.6. Mucinproteincore,tandemrepeatdomains,non-repeatdomainsandcysteinerichdomains...... 8 4. Mucinsynthesisandsecretion...... 8 4.1. Synthesispathways...... 8 4.2. Mucinsynthesis...... 8 4.3. N-glycosylation...... 8 4.4. C-mannosylation...... 9 4.5. ERtoGolgitransport...... 9

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Technological strategies to overcome the mucus barrier in mucosal drug delivery” ⁎ Corresponding author. E-mail addresses: [email protected] (R. Bansil), [email protected] (B.S. Turner). 1 This article is adapted in part from the Introductory Chapter of the Ph.D. dissertation of Bradley S. Turner, Boston University (2012).

4.6. O-glycosylation...... 9 4.7. Granulecondensation...... 9 4.8. Granulepackaging...... 10 4.9. Secretionandexocytosis...... 10 4.10. Mucingranules...... 10 4.11. Exocytosis...... 11 4.12. Mucusandmucinrheologyandmucoadhesion...... 12 5. Futureoutlookandconclusions...... 12 NoteAddedinProof...... 12 Acknowledgements...... 12 References...... 12

Fig. 1. Mucus producing epithelial goblet cells. A. Histological section of colonic mucosa showing the epithelial cell layer containing mucus producing cells. Goblet cells appear as cells containing a large dilated rounded apical region containing mucus secretory granules. The goblet cells are interspersed among simple columnar colonocytes (Condon [36]: website for Basic Histology D502, Indiana University Purdue University Indianapolis). B. Transmission electron micrograph of a single typical intestinal goblet cell. Morphological features include a basal nucleus (N) and rough endoplasmic reticulum (RER), a central supranuclear Golgi apparatus (G), and the characteristic large dilated apical region containing translucent mucus droplets (MD) secretory granules (Moran and Rowley [114] Visual Histology Atlas website). C. A schematic representation of a typical goblet/mucous cell showing basal nucleus and mitochondria, a supranuclear Golgi body, the large dilated apical region containing the mucus granules and apical microvilli [113]. Fig. 1A is reproduced from Condon [36] http:// www.iupui.edu/~anatd502/Labs.f04/epithelia%20lab/s9940x1.jpg by permission from K. Condon. Fig. 1B is reproduced from Moran and Rowley [114] by permission from http://www. visualhistology.com/ Fig. 1C Reproduced from Molinoff and Atkinson [113] by permission of the author, P. Molinoff. R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 5

1. Introduction reticulum and Golgi apparatus, a basal nucleus, and sub-perinuclear rough endoplasmic reticulum and mitochondria (Fig. 1BandC).These Mucus is a complex viscoelastic adherent secretion that is synthe- cells are surrounded by other columnar or squamocolumnar epithelial sized and secreted by specialized goblet and mucous cells in the colum- cells with specialized functions such as ciliated, absorptive, secretory nar epithelia that line the lumen of all of the organs and glands that are or proliferative cells, and are attached to their neighboring cells by exposed to and communicate with the external environment [3,38] (Fig. typical tight junction structures. The goblet mucous cell types are distin- 1). This includes the inner linings of organs of the respiratory tract, the guished based on gross morphology. In general goblet cells are tapered, gastrointestinal tract, the reproductive tract and the ocular surface. His- decreasing in circumference (e.g., get thinner) from apex to base, torically, mucus was certainly known since ancient times. For example whereas mucous cells have near constant diameter from apex to base. Phlegm has origins in 3rd century Greek φληγμα (phlegma) and Hip- Both types of cells have large stores of apical mucus secretory granules, pocrates (460–370 BCE), extended the humoral theories first stated by occupying 75% of the cell volume [53]. Goblet cells regulate the produc- Empedocles (504–433 BCE) postulating the existence of 4 humors, tion of mucins in response to external factors. For example, the response black bile, yellow bile, blood, and phlegm. A balance of these humors of goblet cells to enteric infections is discussed in [89]. Goblet cells, wasassociatedwithhealthandanexcessordeficit of any one was asso- mucus and mucins also interact with the immune system as discussed ciated with disease. The Chinese pharmacopeia Book of Herbs (Pen in [121]. Ts'ao) dating to 2500 BCE mentions the use of ‘ma huang’ which contains ephedrine and pseudoephedrine, found in current “Sudafed” cold 2.2. Mucus structure remedies which are used to alleviate symptoms associated with mucus hypersecretion in upper respiratory infections [182]. Scanning electron microscopy (SEM) of gastric mucosal surfaces [58, Mucus has been described in organisms from all kingdoms [97]. Vis- 119] show the highly folded structure of stomach epithelium with gas- cous, gel forming mucilages and structural glyco-substances are found tric glands and fibrous mucus. Electron microscopic studies of mucus in all forms of life, including viruses, bacteria, fungi, plants, insects, fish- gels are strongly influenced by dehydration during sample preparation es, etc. but this review concentrates specifically on mammalian mucus (see for e.g. [41,110]). Familiari et al. [55] developed a multi-step pro- and its mucin glycoproteins. Vertebrates contain mucus layers in their cess to preserve the real microarchitecture of mucus covering the jeju- corresponding organ systems. Additionally, most aquatic organisms num surface and the zona pellucida surrounding the mature oocyte also possess an external mucus layer on their skin [128].Mucushas and show the network of the dried mucosal fibers. Cryo-SEM [91] of pul- also been studied from invertebrates (worms, snails, slugs, insects, monary mucus preserves the structure of better showing a highly het- anemones). For example the hagfish is able to explosively release liters erogeneous mesh with both large and small pores and a thick polymer of mucus from its skin which is used as a prey escape mechanism [18,60, scaffold. We examined the structure of hydrated mucus on the sub-mi- 151]. Mucus layers have also been described in various pathogenic pro- cron length scale in vitro by atomic force microscopy (AFM) in a liquid tozoa [161]. Zebra fish provides an excellent model system currently cell under appropriate buffers. A tapping mode AFM measurement of much in vogue to study the effects of mucus in vivo [24,78]. a wet sample of human mucus taken from the discarded material Mucus serves many protective functions for the underlying epithe- obtained in the lavage following a stomach biopsy reveals a swollen lia, such as lubrication for material transport and hydration over the ep- network similar to that formed by the glycoprotein mucin [74] with ithelium particularly in the respiratory tracts, eyes and mouth that are aqueous pores of about 200–300 nm in diameter. directly exposed to the drying evaporative effects of air and providing a barrier to noxious agents and pathogen exposure by trapping them and hindering their access to the epithelium [37]. It serves a cleansing 2.3. Mucus thickness transport function where external particles trapped in the mucus layer can be eliminated from organ cavities by cilia facilitated expulsion of The thickness of the mucus layer varies considerably both within the mucus layer [146], and provides a selectively permeable gel layer and between different organ systems. For example, in the respiratory for the diffusion, exchange and absorption of gases (eye and lung) and tract mucus layer thickness is about 10 μm in the trachea [112] while nutrients (gastrointestinal tract) with the underlying epithelium [115]. in the gastrointestinal tract, the mucus layer increases in thickness from the upper to the lower GI tract varying from 200 μmto800μmre- 2. Mucus structural organization spectively [5]. Table 1 gives thicknesses of the mucus layer in the GI tract. In the eye the mucus layer that covers the corneal and conjunctival 2.1. Goblet/mucous cells surfaces has a thickness of 1–7 μm [64,73,82], and in the female reproductive tract the thickness and viscosity of the mucus varies with the Mucus is secreted by goblet/mucous cells (for a recent review see ovulatory cycle and regulates sperm penetration [86] and uteral implan- [17]) which are interspersed with many other types of specialized tation of the fertilized egg [28]. These differences in mucus thickness epithelial cells (Fig. 1A), and have a similar typical highly polarized mor- may reflect the various protective functions and challenges of mucus phology with a broad apical region and narrower basal region, giving within its specific organs. them their characteristic ‘wine glass goblet’ shapes (Fig. 1B and C). Mucus in the respiratory, ocular and gastrointestinal tracts has been The apical region contains large, supranuclear, mucus containing shown to exist in two layers with distinct rheological properties [155]. storage and secretory granules, a central supranuclear endoplasmic For example, in the gastrointestinal tract the first 100–150 μmthick layer which is more viscoelastic, is in intimate contact with the epithelial cell layer, and a second, less viscous layer of thickness 100–700 μm Table 1 lies on top of it extending to the luminal surface. It has also been Mucus thickness in the gastrointestinal tract. fi Average values reported by Atuma et al. [5]. Note: we have rounded the numbers to 5 μm, shown that bacteria have differing af nities for these two layers [80]. well within the range of values reported. In contrast, in the respiratory tract, the lower viscosity perciliary mucus layer consisting of hydrated membrane mucins and mucopoly- Thickness (μm) Location in gastric mucosa saccharides, is in contact with the airway epithelium and supporting Corpus Antrum Duodenum Jejunum Ileum Colon the higher viscosity mucus on top of it, which is exposed to the air. Total 190 275 170 125 480 830 This arrangement is thought to facilitate mucus expulsion and clearance Firmly adherent 80 155 15 15 30 115 from the lung and airway passages by the action of cilia via mucociliary Loosely adherent 110 120 155 110 450 715 transport [25,146]. 6 R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15

3. Mucus composition These include the alpha and beta defensins, which are antimicrobial peptides [14,105] and lysozyme (muramidase), an enzyme that digests Mucus is a complex dilute aqueous viscoelastic secretion consisting muramic acid mucopolysaccharide bacterial cell wall components. The of many components: water, electrolytes, lipids and various proteins. positive charge of lysozyme enables binding to the negatively charged Creeth [40] provides an excellent review describing the constituents mucus; its bactericidal properties were discovered by Alexander of mucus and methods to separate them. Water comprises approxi- Flemming [57]. Lactoferrin, in all mucosae, is an intrinsic factor (in the mately 90–95% of mucus and serves as the solvent and diffusion medi- stomach) that chelates iron which limits its availability for bacterial um for all the other mucus components. growth. Statherins, proline rich peptides and histatin defensive proteins have also been identified in mucus [76]. 3.1. Mucus electrolytes Immunoglobulins, secretory IgA and IgM are present in all mucus layers and are active against a variety of pathogens. Immunoglobulins Electrolytes are an important component of mucus and their compo- have also been reported to increase the viscosity of mucus in airways sition can be different in mucus from various tissues, and are deter- [65] and IgG and IgA have been shown to bind differentially to cervical mined by the underlying secretory epithelium. These differences may mucus [54]. Secretory IgA has been shown to be required to bind to be important to the specific environmental challenges found in each the mucus layer for effective antibacterial defense [138,158]. organ. Common to most mucus secretions are sodium and potassium The major growth factors present on almost all mucus layers are chloride and sodium bicarbonate, phosphate and magnesium and calci- transforming growth factor beta, epidermal growth factor and hepatic um, approximately isotonic with serum at approximately 1% (w/v)total nuclear factor [35]. One or more of these growth factors are present in concentration. Some organs modify the ionic composition of mucus mucus layers of all organs and may serve growth, maintenance, repair during their specialized functions. The stomach during active digestion, and regulatory functions to the underlying epithelium. Additionally, secretes large amounts of hydrochloric acid (HCl) with a concomitant there are various cytokines present that are released from epithelial or reduction in the potassium chloride concentration [106]. Changes in immune cells in response to various stimuli, pathogens or disease states. the electrolyte composition have a role in controlling mucus hydration Structural proteins include protease inhibitors such as secretory leu- and rheology e.g. increased concentration of divalent cations, Mg or kocyte proteinase inhibitor and pancreatic secretory trypsin inhibitor. Ca, can cause collapse of the mucus gel [165,167,168] whereas an in- These proteins protect the mucus proteins from proteolysis, depolymer- crease in concentration of monovalent cations, Na or K can cause a re- ization and loss of viscosity by enzymes of both host and pathogen ori- duction in mucus viscosity [149]. Bicarbonate, which is secreted by gins. Also present are the trefoil (TFF) peptides. These are small cysteine secretory cells and released by the submucosal vasculature, helps to rich peptides with a characteristic disulfide linked three loop clover leaf buffer the mucus layer adjacent to the epithelium to pH 7. This is partic- conformations [139,174]. They have been reported as agents associated ularly important in the stomach where bicarbonate establishes a protec- both with mucus interactions, increasing its viscosity [157], protection tive pH gradient through the mucus gel layer against HCl acid secretion from proteolysis and as epithelial repair growth factors. [2]. The pH also affects the viscosity of gastric mucus [15] leading to gelation below pH 4 [8,30]. 3.4. Mucins

3.2. Mucus lipids The major functional components of mucus are the mucin glycoproteins present at a concentration of 1–5%. They are responsible for the es- Lipids constitute 1–2% of mucus. Many lipids have been described to sential and predominant viscoelastic properties of the mucus layer. be both covalently and non-covalently associated with mucus layers Details of mucin genes, glycoprotein structure and biophysical proper- [71,102]. These include phospholipids, primarily phosphatidyl choline ties have been presented in considerable detail in previous reviews and phosphatidyl glycerol and small amounts of lysophosphatidyl cho- [6–8,38,45,72,123,134,137,158]. Mucins are broadly classified as two line generated by phospholipase A2 in some pathologic conditions, cho- types, secreted mucins and membrane bound mucins. Both membranes lesterol, fatty acids both free and acylated to proteins. The lipids interact and secreted mucins share many common characteristic compositional with mucus glycoproteins and affect the wettability (charged lipids) and structural features based on the fact that their underlying protein and hydrophobicity (neutral lipids) and barrier functions of the mucus sequence is governed by the same family of genes. Based on sequence layer [102]. Lipids may also contribute to the lubricating and surface and domain comparisons and similarities, and cellular origins, 22 tension properties of the mucus layer. The rheological properties of mucin genes have been identified and can be grouped into two broad mucus are also strongly affected by lipids [65,172].Lipidsalsoserveto classes representing membrane-bound mucins, anchored by insertion prevent evaporation of the aqueous phase, for example in the tear through the plasma membrane and secreted mucins, that are packaged mucus layer covering the eye [39,82]. in and secreted extracellularly from secretory granules. The membrane mucins include MUC 1, 3A, 3B, 4, 11, 12, 13, 15, 16, 17, 20 and 21. The 3.3. Mucus proteins secretory mucins include MUC 2, 5AC, 5B, 6, 7, 8, 9, and 19 [3,134]. The secretory mucins can be further subclassified as 1) large gel forming Mucin glycoproteins constitute the major functional component of mucins MUC 2, 5AC, 5B, 6, 19 and 2) small soluble mucins MUC 7, 8, 9. mucus, however ancillary non-mucus proteins play an important role Immunohistochemistry has been used to identify expression of differ- in the structural and protective functions of mucus. Recently many ent mucins in many organs in healthy as well as diseased states with a mucus proteins have been identified by high-throughput, proteomic significant focus on cancer (see for e.g. [10,16,34,81,99,100,104,129]). mass spectrometric, chromatographic and 2D-PAGE methods. One hun- dred eighty six different proteins have been identified in airway, 147 or 3.5. Glycosylation 685 in cervical, 111 in nasal and 145 in gastrointestinal tracts and are found in the mucus layers of all organs [29,143,147,154]. Many of the Both membrane and secreted mucins are highly glycosylated, proteins present are serum proteins, presumed to arise from epithelial consisting of approximately 50–80% (w/w) carbohydrates, primarily serum transudation. Most important proteins found can be broadly clas- composed of GalNAc, GlcNAc, Fuc, Gal and sialic acids or NANA or sified into functional groups including defensive proteins, growth fac- NeuNAc (N-acetyl neuraminic acid) and relatively small amounts of tors, structural proteins and glycoproteins. mannose. The oligosaccharide chains possess approximately 2–20 The major defensive proteins are involved in contributing to the monosaccharides in both linear and moderately branched structures mucus protective abilities against pathogens and particulate matter. (see for e.g. [21,104]). The oligosaccharides are attached to the mucin R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 7

Fig. 2. Structure of mucin glycoproteins. The hierarchical structure of mucin depicted schematically. A. The various non-glycosylated and glycosylated domains of mucin as discussed in the text under mucin structure are indicated. B. The mucin monomer consists of a linear protein core with a serine/threonine rich tandem repeat region where oligosaccharides (red) are linked O-glycosidically. C. Mucin monomers dimerize via formation of C terminal S\\S bonds of cysteine groups as indicated by the gray arrowhead. D. These dimers further polymerize forming multimers. Occasional branching can occur as indicated by an N terminal (blue circle) polymer branch. (Reproduced from Bansil et al. [8] under Creative Commons Attributable License).

protein core by alpha-1-O-glycosidic bonds from GalNAc to the hydrox- and combinations of the glycosyltransferases and their substrate speci- yl side chains of serines (Ser) and threonines (Thr). In a recent paper, Jin ficities [13,63,137]. Additionally, the oligosaccharides may be modified et al. [79] present a structural map of human gastric glycosylation from by the addition of blood group (ABO, Lewis), secretor (H) epitopes healthy and diseased subjects showing a large structural diversity with [135], and terminal negatively charged sialic acids and sulfates. The over 200 O-glycan structures identified in the total sample of 10 individ- large amounts, distribution and density of glycosylation, steric exclu- uals. To date, no single consensus O-glycosylation amino acid sequence sion, and charge repulsion, impart a highly hydrated, extended, relative- motif has been identified, however O-glycosylation sites can be roughly ly stiff elongated linear “rod-like” conformation to the mucins [62,109]. predicted by statistical, and artificial intelligence analysis of databases of These extended mucin structures have been directly observed by both experimentally determined glycosylated proteins (NetOGlyc, electron and atomic force microscopy and exhibit molecular lengths of OGlycBase) [26,27,70,84]. A large proportion of the Ser/Thr residues 50–1000 nm up to 8 μm [74,109,144] for a variety of secreted mucins are predicted to be glycosylated. This results in a “bottle brush” config- from various sources. This extensive glycosylation also serves to protect uration of the oligosaccharides arranged around the mucin protein core the mucin protein core from proteolytic degradation. Keismer et al. [88] (see Fig. 2). Elongation and branching of the oligosaccharide side chains used electron and atomic force microscopy to show 190–1500 nm long are governed and performed by a large family of glycosyltransferases tethered mucin polymers, and a finely textured glycocalyx matrix filling [23,156,160]. It is believed that the vast diversity in mucin oligosaccha- interciliary spaces of airway mucus. AFM methods for measuring bind- ride structures is determined by the tissue specific expression patterns ing forces of glycans to lectin and other groups deposited on AFM 8 R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 cantilevers have also been applied to investigate the distribution of gly- [47,97,123]. The cysteines can be involved with both intra- and inter- cans from mucins such as sialic acid of ocular mucins [9] and gastroin- molecular disulfide bonds or in contributing to protein flexibility be- testinal mucins [69]. tween tandem repeat domains. These regions contain few Ser/Thr or Both membrane and secreted mucins also contain N-glycosylated O-glycosylation and have an overall amino acid composition and con- multiantennary complex and hybrid oligosaccharide high mannose formation characteristic of globular secreted proteins. Multiple Cys- structures, which are synthesized in the secretory pathway by the en rich von Willebrand Factor D (vWF-D) domains are found at the bloc transfer of preformed mannose oligosaccharides to the amide side amino end of secreted mucin molecules [85,123]. These domains have chain of Asparagine (Asn) residues located in well-defined N-X-T/S se- been determined to be involved in the multimerization of both mucins quence motifs at the ends of mucin proteins [34,150,183].Secretedmu- and von Willebrand factor (vWF) [108,122,124]. cins also contain a novel type of glycosylation, C-mannosylation, At the carboxy terminal end of the central tandem repeat domains of modifications that involve a carbon-carbon bond between the C1 of secreted mucins, are Cys rich regions similar to vWF D domains, vWF C mannose and the indole ring of specific tryptophans (W) in W-X-X-W domains and carboxy terminal cysteine knot (CTCK) domains which are motifs [125]. required for dimerization [12,181]. The mucin carboxy terminal vWF-D The combined extensive glycosylation gives mucins a higher buoy- domains have similar sequence, structure and dimerization functions as ant density in CsCl (1.45 g/cm3) than typical non-glycosylated proteins those located at the amino terminal. (1.2 g/cm3)orlipids(b1.0 g/cm3) yet less than nucleic acids (N1.6 g/cm3) [42,67]. This feature along with their large molecular 4. Mucin synthesis and secretion sizes (N2 MDa) results in facile methods for purifying and analyzing mucins by isopycnic, density gradient ultracentrifugation [67] and size In this section we consider in some detail mucin synthesis and secre- fractionation by size exclusion chromatography and agarose electro- tion leading to the formation of a hydrated mucus layer. This subject has phoresis [67,130], respectively. been recently reviewed [34,43]. We will consider how mucins are synthesized, co- and post translationally modified, transported packaged, 3.6. Mucin protein core, tandem repeat domains, non-repeat domains and stored and secreted. Synthesis of large amounts of high molecular cysteine rich domains weight, complex extensively glycosylated mucins represents a significant metabolic commitment by the cell. We consider mainly secreted The apomucin protein core (100–500 kDa) for both membrane and gel forming mucins (MUC 2, 5AC, 5B, 6, 19), but the general description secreted mucins, comprises approximately 20% (w/w) of the mucin also applies to membrane mucins. These pathways have been studied mass. It is organized into two broadly distinct regions, 1) a centrally lo- for a wide variety of secretory products, including mucin in different cated tandem repeat region rich in Ser, Thr, and Pro (STP-tandem re- cell types, and organisms and have yielded basic mechanisms and com- peats) which contains O-glycosylation, and 2) the carboxy- and ponents that are relevant and applicable to all mucins. amino-terminal non-repeat regions, which may be rich in cysteine (Cys) residues with low amounts of Ser/Thr and relatively little O-glyco- 4.1. Synthesis pathways sylation, but possessing N-glycosylation. The amino acid composition, sequences and structures of these terminal Cys-rich regions are more Mucin synthesis and secretion proceeds by both constitutive path- typical of secreted globular proteins [38,123]. ways, characterized by continuous release of newly synthesized prod- The Ser/Thr repeat units can vary in length from 7 to 375 amino acids ucts and regulated pathways involving storage of mucin products that in MUC19 and MUC3A respectively. The number of repeats within a are released in response to various stimuli in a calcium dependent man- mucin molecule can also vary from 10 to 500 as inheritable Variable ner. In this description we consider primarily the regulated pathway, al- Number of Tandem Repeats (VNTR) polymorphisms [169]. The amino though the initial steps of synthesis and assembly through the trans acid sequences of the repeat units are very similar (70–90% identical) Golgi vesicular budding, also apply to the constitutive path. within a particular mucin gene within an individual of a single species, but can vary significantly between homologous mucin genes in different 4.2. Mucin synthesis species [137]. In secreted gel forming mucins, Cys-rich regions of the protein core The various steps in mucin synthesis are listed in Table 2. As with are located at both the amino and carboxy ends of the central tandem many other secreted glycoproteins, the synthesis of the protein core of repeat region. In some secreted mucins (MUC5AC an MUC5B) Cys-rich mucin (apomucin) begins on membrane bound ribosomes of the sequences (CysD) can also be found periodically interspersed within rough endoplasmic reticulum (RER). This involves ribosomal docking the Ser/Thr tandem repeats. The Cys-rich regions can contain up to to the translocon complex, a large (N125 Å) [111] membrane associated 10% Cys and relatively little Ser or Thr residues that are arranged in assembly of over 25 proteins including Sec61p [118] through which the highly conserved, specific patterns and can be used to classify mucins growing nascent apomucin is transported into the lumen of the endoplasmic reticulum (ER). A number of post translational modifications

Table 2 (PTM) of the apomucin protein take place co-translationally or very Intracellular synthesis and assembly of mucins. early in synthesis, namely N-glycosylation, C-mannosylation and A schematic of the various steps in the intracellular assembly of mucins as described in dimerization. [158].

Sub cellular Step. Process 4.3. N-glycosylation compartment No.

Endoplasmic 1 Translation of the peptide core, folding of cysteine rich N N-linked glycosylation is accomplished co-translationally by transfer reticulum and C terminal domains with formation of intracellular of preformed high mannose oligosaccharides from dolichoyl-pyrophos- disulﬁde bonds, and N-glycosylation in the ER phate-Glc3Man9GlcNAc2, to the side chain amide group of asparagines 2 Dimerization of mucin peptides in the ER (N) in consensus motif N-X-T/S (where X is any amino acid except pro- cis and medial 3 Initial O-glycosylation of serine and threonine by GalNAc Golgi in the cis Golgi line) sites located in the mucin Cys-rich regions catalyzed by the en- 4 Elongation of O-linked oligosaccharides in the medial Golgi zyme oligosaccharide transferase which is closely associated on the ER trans Golgi 5 Polymerization of mature mucins to form linear and luminal side of the translocon complex [32,150,178]. Immediately branched polymers. Packaging into secretory granules with after N-glycosylation, N-linked oligosaccharides are further modiﬁed Ca2+ in the ER by removal of two Glc residues by [177] α1,2- and α1,3 R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 9 glucosidase and one mannose residue by α 1,2 mannosidase I to yield STP-tandem repeat regions due to steric exclusion of the carbohydrates

Glc1Man9GlcNAc2-Asn [136]. This oligosaccharide structure serves as a [145]. ligand for the ER Ca2+ dependent lectins calnexin and calreticulin, All mucin O-linked oligosaccharide structures are built on a limited both present in the ER lumen which bind the N-linked oligosaccharides number of eight common “Core” structures, based on the addition of on the nascent peptide and serve as folding co-chaperones [163]. Bound monosaccharides Gal or GlcNAc directly to different positions on the ini- peptides undergo repeated cycles of binding and folding with formation tial GalNAc to form linear or branched Core structures. Many different of intramolecular and intermolecular disulfide bonds, leading to dimer Core structures can be present on a single mucin molecule [23,131, and trimer formation through the C-terminal cysteine knot domains. It 132]. Elongation of the basic core structures continues by stepwise se- has been demonstrated that elimination of N-glycosylation, by use of in- quential addition of monosaccharides from nucleotide precursors by al- hibitors, site directed mutation or expression of proteins in N-glycosyl- ternate action of various GlcNAc and Gal transferases. Alternating Gal- ation deficient cells, leads to decreased mucin secretion and GlcNAc disaccharides either beta 1-3 or beta 1-4 lactosamines are com- accumulation of mucin products within the ER [11]. mon. The elongated structures may be either linear or branched. The specific structures that are formed are determined by the cell or tissue 4.4. C-mannosylation specific expression patterns and substrate specificities of the glycosyltransferases. Thus it is possible for the same mucin peptide to have dif- C-mannosylation, an unusual novel PTM also occurs co- ferent glycosylation structures when expressed in different cells or translationally. It involves transfer by the enzyme C-mannosyl transfer- tissues. For example, for cervical mucin glycosylation, the specificcom- ase [49] of a single mannose residue from dolichoyl phosphate mannose bination of glycosyltransferases, and their levels are hormonally regu- to the indole C2 of the first tryptophan (W) in the consensus W-X-X-W lated during the menstrual cycle [4]. More than 100 different O-linked motifs [83]. These motifs are also usually found within the Cys-Rich do- oligosaccharide structures have been identified on a single colonic mains of mucins. Although the precise function of C-mannosylation is MUC2 protein [98]. unknown, mutation or absence of the mannosylation site or expression Termination of O-linked oligosaccharide chain growth occurs in the of mucins in C-mannosylation deficient cells, results in the accumula- medial- to trans-Golgi compartments by the competitive enzymatic tion of protein in the ER and decreased secretion, suggesting that C- addition of fucose, GalNAc or NeuNAc (Sialic Acid) mainly as the α- mannosylation may be required for export from the ER via cargo recep- anomers to the periphery of the growing oligosaccharide chains. These tor binding and transport vesicle packaging [125]. Thus both N-glycosyl- terminal structures can include blood group ABO, Lewis ABXY and H se- ation and C-mannosylation are required for mucin dimerization, folding cretor antigens [135]. Terminal sugars can also be modified with sul- and for export from ER to Golgi. fates. Of the eight core structures, only cores 1–4 and 6 are substrates Mucin products at this point are presumed to have a globular, ran- for the sulfotransferases [22]. It should be noted that the N-linked oligo- dom coil conformation with molecular weights of 250–500 kDa for disaccharides also undergo trimming, elongation and modification in the sulfide linked monomers and 0.5–1 MDa for dimers and multimers Golgitoobtainhighmannose,complexandhybridtypestructures. [158]. The addition of the terminal sialic acid (NeuNAc) and sulfates contribute large amounts of negative charges to the mucin oligosaccha- 4.5. ER to Golgi transport rides. This causes charge and steric repulsion between the oligosaccharide chains and an extension and stiffening of the mucin molecule to an Continued synthesis of correctly folded proteins proceeds by pack- extended rod-like configuration with a “bottle brush” arrangement of ing into COP II (coat protein complex) transport vesicles with the assis- oligosaccharides around the apomucin core, and with molecular tance of cargo receptors VIP36 (Vesicular Integral Protein 36 kDa) and weights from 2 to 10 MDa and occupation of a large hydrated volume ERGIC-53p (ER to Golgi Intermediate Compartment Protein 53 kDa) in solution. and transported to the cis-Golgi on microtubules [120,136,177]. 4.7. Granule condensation 4.6. O-glycosylation As oligosaccharide construction is completed, mucins in the trans- Once in the Golgi, mucin proteins acquire their most prominent and Golgi network (TGN) are packaged into vesicles that bud off by means characteristic salient feature, namely extensive O-glycosylation, which of the cargo receptor VIP36 (vesicular integral protein 36 kDa). Mucin can constitute 50–90% of the final mucin molecular mass. In contrast packaged in vesicles undergo maturation to secretory granules (SG) to N-glycosylation which transfers, en bloc, a large preformed oligosac- by condensation by the formation higher order mucin multimers by di- charide from a lipid precursor, O-glycosylation occurs by the sequential sulfide association of amino-terminal vWFD domains, to form mole- addition of single monosaccharides from nucleotide sugar precursors. cules that are 10–50 MDa extended rods with linear dimensions 1–10 O-glycosylation is accomplished in three principal steps, initiation, elon- μm [66,122], which are packaged into vesicles of b1 μm. Verdugo gation and termination [23]. [164] applied the ideas of a large volume gel phase transition [101, The first step of initiation, which occurs in the cis- and medial-Golgi 153] in charge polymer gels to mucins to both explain and demonstrate (reviewed in [137]), occurs by the addition of a single GalNAc from a how this packaging is possible (for a recent review see [166]). The many UDP-GalNAc precursor, from its reducing end C1 to the hydroxyl side repulsive negative charges (from sulfates and sialic acids) on the ex- chains of serine (Ser) and threonine (Thr) residues, by the action of en- tended mucin molecules can be neutralized, shielded and crosslinked/ zyme UDP-N-acetylgalactosamine:polypeptide galactosaminyl trans- complexed with positive counter ions such as H+,Na+,Ca2+,resulting ferases (GalNAc-Tase) to yield GalNAC-α1-O-Ser/Thr [159]. There are in charge shielding, allowing mucin gels to undergo a 100 to 200 fold re- 10–20 GalNAc-Tase type II membrane proteins [156] which are con- versible volume collapse phase transition. This was demonstrated with served from fungi to mammals. Some of these initiating GalNAc-Tases mucins by increasing the extracellular solution concentration of H+ and (GalNAc T1-T6 and T10-T14) transfer the initial GalNAc to Ca2+ to those concentrations found in secretory granules and observing unglycosylated protein substrates, whereas other GalNAc-Tases (T7- shrinkage of the mucin gels. T9) enzymes containing a carboxy-terminal rich in lectin (Gal/GalNAc It has been shown that progression through the secretory pathway binding) domain that is required for binding to substrate, complete results in luminal compartments with increasing H+ and Ca2+ concen- the dense O-glycosylation of clustered Ser/Thr residues [170]. After ad- trations [46] with ER at pH 6.8, cis Golgi at pH 6.7, medial-Golgi at dition of the initial GalNAc, mucin molecules have a molecular weight of pH 6.3, trans-Golgi at pH 6.0 and secretory granules at pH 5.5. This is ac- 0.5–2 MDa and would be slightly less flexible and more extended in the complished by an increasing density of a membrane bound vesicular 10 R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15

Fig. 3. Mucin secretory granule transport and exocytosis. The steps and components involved in mucin granule transport and exocytosis. Step 1 transport of mature mucin secretory granules along microtubules to the cell apex. Step 2 transfer of secretory granules to actin myosin mediated transport. Step 3 secretory granule docking to plasma membranes and disruption of the cortical actin network. Step 4 fusion of secretory granule with cell plasma membrane and release of granule contents. Components and their roles are discussed in the text [53]. Permission is automatically granted under the STM Permissions Guidelines, subject to proper acknowledgement, as Pharmacology & Therapeutics is also an Elsevier journal (http://www.sciencedirect.com/science/article/pii/S0163725808002015www.sciencedirect.com/science/article/pii/S0163725808002015).

H+-ATPase proton pump that transports H+ into the Golgi lumen and that allow movement of small molecules which is important for exocy- by a decrease in leakage of H+ from the lumen [46,175]. tosis [126]. Similarly, intragranular Ca2+concentrations (23 mmol/kg) are found to be higher than intracellular or other compartment Ca2+ con- 4.9. Secretion and exocytosis centrations (6 mmol/kg) [77,93,117]. Once mucin's charge repulsion has been neutralized by H+ and Ca2+, hydrophobic interactions be- After secretory granule budding from the TGN, condensation and tween mucin molecules and lipids can dominate causing mucin conden- maturation, mature secretory granules (MSG) move toward the periph- sation within the secretory granules. eral apical areas of the cell for storage and secretion. Four broad steps and many substeps and mediators are involved in the secretory process (see Fig. 3): 1) transport of MSG from the TGN to the apical inner cell 4.8. Granule packaging plasma membrane, 2) docking an tethering of MSG to the cell membrane, 3) pore formation by granule membrane to plasma membrane The intragranular condensed state of the mucin secretory granule fusion, and 4) exocytosis of MSG contents into the extracellular space. matrix has been elegantly investigated by Perez-Vilar using fluorescent More details can be found in reviews by Davis et al. [43], Evans et al. confocal microscopy and FRAP (fluorescence recovery after [53], Rogers [133], and Williams et al. [173]. photobleaching) of recombinant GFP-mucin domain constructs that are packaged into secretory granules. Results indicate that the diffusion 4.10. Mucin granules constant of GFP is greater in the ER or extracellularly (1.42 μm2/s) than in the secretory granule (0.014 μm2/s) demonstrating more restricted Mucin secretory cells showing granules have been imaged using movement and a more condensed state in the granules than ER. It also fluorescence microscopy [126] as well as with non-fluorescent confocal indicates the existence of pores or spaces within the granule matrix light absorption and scattering spectroscopy (CLASS) [56].They R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 11

A. B.

Fig. 4. Excocytosis of mucin from secretory granules. A. Image of gastric cell with granules. Gastric cell (AGS cell) imaged with a non-fluorescent optical technique, confocal light absorption and scattering spectroscopy (CLASS), showing mucin granules ranging in size from 0.5 to 2 μm. The scale bar is 2 μm. Note, the image is pseudo-colored for illustration purposes; the cells were not labeled with any chromophore or fluorescent dye. B. Exocytosis mechanism. Mucin exocytosis is depicted as (a) mucin is packaged in secretory vesicles in a condensed state by complexation with calcium Ca2+. The vesicles fuse with the plasma membrane via interaction with proteins, such as the Rab proteins and unidentified effectors. Activation of various receptors and proteins leads to generation of signaling molecules which activate release of Ca2+, destabilizing the binding of the negative charges on mucin. The diffusion of Ca2+ out of the fusion pore and increased repulsion due to negatively charged mucin, results in extrusion of a highly expanded mucin network. C. Expansion of mucin granules. Schematic summary of electron micrographs of isolated mucin granules in various stages of expansion [87]. (Image in panel A was provided by Dr. L. Qiu. Image in panel B was reproduced from Adler et al. [1]: Creative Commons Attribution License.) (Image in panel C was reproduced from Kesimer et al. [87] by obtaining license to reuse from the American Physiological Society via Rightslink.) Fig. 4C is reproduced from Kesimer et al. [87] by license provided by American Physiological Society and Copyright Clearance Center.

compared CLASS images to fluorescently labeled granules from the neutralize and crosslink mucin negative charges. When the MSG interi- same cell line and showed improved resolution from this label-free or is exposed to the extracellular milieu, which has lower Ca2+ and H+ technique. An image of a mucus producing gastric cell (AGS cell) obtain- concentrations, the MSG Ca2+ and H+ rapidly diffuse out of the MSG ed by CLASS showing mucin granules ranging in size from 0.5 to 2 μmis faster than the large mucin molecules. This results in de-shielding of shown in Fig. 4A. the mucin anions, yielding a net mucin negative charge and electrostatic repulsion of the mucin from its sialic acids and sulfates. This cationic dif- 4.11. Exocytosis fusion also results in the formation of a net positive charge in the area immediately surrounding the extracellular regions of the pore opening. MSG and plasma membrane fusion causes the formation of an open This diffusion driven charge separation defines a Donnan electric poten- pore having an “omega” structure ‘Ω’ [116]. The pore allows the interior tial with mucin serving as the semipermeable membrane containing of the MSG to be exposed to the extracellular environment. Verdugo ap- slowly diffusible negative charges [48,152]. The MSG mucin gel network plied the gel phase transition model to explain the mechanism by which undergoes a rapid volume expansion phase transition [167] driven by a mucins leave the secretory granule through the pore [165]. Adler et al. combination of Donnan potential, mutual electrostatic repulsion of the [1] describe the proteins involved in the fusion of vesicle with granules deshielded mucin anions and by osmotic hydration of mucin saccha- to the cell membrane and the signaling factors involved in activation of rides by water influx into the MSG. The mucin molecules expand and Ca2+ release leading to the secretion of mucin (Fig. 4B). As described spread away from the open fusion pore, covering the external apical above for mucin condensation, the MSG contains a condensed porous surface of the cell. gel network consisting of collapsed negatively charged mucins com- Experimental verification of this “jack-in-the-box” explosive exocy- plexed with high concentrations of H+ and Ca2+ counter ions which tosis process was demonstrated by the prevention of mucin expansion 12 R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews 124 (2018) 3–15 by increasing the extracellular H+ and Ca2+ concentrations to match mucosal surfaces. We refer the reader to two recent reviews on those of the MSG interior. This eliminates the MSG interior to extracel- mucoadhesivity [148,180] for this fascinating topic. Another topic of lular diffusion gradient and also eliminates development of the Donnan considerable current interest, that we have not discussed concerns the potential. Decreasing the external pH from 7.4 to 6.5 and increasing the transport of nanoparticles through mucus (see for e.g. [51,59,68,127, external Ca2+ from 1 to 4 mM causes a measurable increase in the 140,171]). mucin swelling times and decrease in the diffusion constants from −7 2 −8 2 2.25 × 10 cm /s to 1.11 × 10 cm /s [52]. The released mucin then 5. Future outlook and conclusions remains in an unexpanded, condensed state and does not undergo the rapid volume expansion phase transition “jack-in-the-box” exocytosis In summary, we have reviewed the basic structure, composition, [52,167,168]. Recently, this process has been experimentally observed synthesis and organization of mucus. Research on mucus and purified using electron microscopy of isolated mucin granules in various stages mucin is currently seeing a significant interest in the context of diseases, of exocytosis (Fig. 4C) [87]. particularly cancer and infectious diseases caused by agents that breach the mucosal barrier, as well as in the context of delivering drugs and nu- 4.12. Mucus and mucin rheology and mucoadhesion trients across the mucosal barrier. Understanding both the physical and chemical/biochemical interaction of this complex highly heterogeneous Although the main focus of this review is on mucus structure, soft material is crucial for progress in this area. One of the major obsta- composition and synthesis, we briefly discuss the rheological cles in the progress of this work has been the availability of well charac- properties of mucus and mucin, the molecule that gives rise to its terized and standardized preparation of mucus and purified mucin. An viscoelastic properties, as well as its ubiquitous mucoadhesive prop- alternative to using purified mucus/mucins for applications where erties. Rheology of mucus is a major factor in controlling the trans- large quantities of material are required is to use synthetic formulations, portoffood,drugs,particlesincluding nanoparticles and larger or even mucus from other sources such as hagfish. However, these will micron scale particles, and infectious organisms, across mucosal lin- not be identical to mucus, and thus results would have to be interpreted ings, as well as controlling its hydration and lubrication properties. with caution. Another area of research which is just beginning to devel- The rheology of mucus from many different animals/tissues/organs op is novel methods for in vitro and in vivo imaging and other tech- has been investigated for a long time (see for e.g., [44,90,95,142, niques to investigate mucus structure, rheology, adhesive properties 162]). The viscosity of purified mucin, particularly lung, salivary and transport of nutrients, drugs, nanoparticles, bacteria and viruses and gastric mucin has also been well studied using bulk, or macro- through mucus. At the biochemical and molecular biological end novel scale methods [30,75,94,158,176] and at the local, micro-scale immune-fluorescent and single molecule spectroscopy techniques are using microscopic particle tracking methods [8,61,103]. We refer pushing the frontier forward to investigating molecular details of muco- the reader to a recent review from our group [8] that provides a sim- sal surfaces and mucus secretions. Biochemical and molecular modifica- ple explanation of the basic concepts of rheology (i.e. elastic and vis- tions of the glycan coat may lead to development of new vaccines and cous properties) and describes the different techniques used. Briefly, drugs. mucus and mucin exhibit frequency dependent viscoelastic moduli and their viscosity decreases with increasing shear rate, i.e. they are Note Added in Proof shear thinning materials. Many of the gel forming mucins, such as fi MUC5AC from puri ed porcine gastric mucin exhibit elastic response The reference below confirms that hydrophobic interactions are in- as well [30].Georgiadisetal.[61] showed that pH induced gelation oc- volved in mucin gel formation and low pH viscoelasticity increase. The curs not only in porcine gastric MUC 5AC but also in porcine duodenal surfactant (very mild detergent) 1,2 hexandiol causes a reduction in mucin MUC2. The viscoelastic behavior of mucins depends on pH, mucin viscoelasticity. ionic strength, and degree of hydration and thus is particularly relevant Wagner CE, Turner BS, Rubinstein M, McKinley GH, Ribbeck K. A to the transport of particles through mucus [96]. For example, the vis- rheological study of the association and dynamics of MUC5AC gels. fl cosity of mucus or mucin has been shown to in uence binding to Biomacromolecules. 2017 Sep 14. (in press) [PubMed] https://www. small molecules such as nicotine [33] and other drugs [141], and the ncbi.nlm.nih.gov/pubmed/28903557. transport of sperm [50],bacteria[31,107],viruses[19,20] and nutrients [179] through purified mucin or mucus layers. Acknowledgements Finally we add a cautionary note concerning the effect on viscoelastic properties related to differences in the methods for purification of RB was supported by NSF PHY 1410798. BST was supported by mucin. The methods differ in many ways such as fractionation tech- MIT Center for Materials Science and Engineering, MRSEC NSF DMR niques, and treatment by digestive enzymes which can lead to degrada- 1419807. tion into subunits [92], or chaotropic agents like guanidium chloride GuHCl which remove associated proteins. 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