ADR-13193; No of Pages 13
Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews
journal homepage: www.elsevier.com/locate/addr
The biology of mucus: Composition, synthesis and organization☆
Rama Bansil a,⁎, Bradley S. Turner b,1
a
b
Dept. of Physics, Boston University, USA
Dept. of Biological Engineering, MIT, USA
a r t i c l e
i n f o
Article history:
Received 15 July 2017
Received in revised form 24 September 2017
Accepted 27 September 2017
Available online xxxx
Keywords:
Mucus
Mucin
Secretory granule
Exocytosis
Rheology
a b s t r a c t
In this review we discuss mucus, the viscoelastic secretion from goblet or mucous producing cells that lines the
epithelial surfaces of all organs exposed to the external world. Mucus is a complex aqueous fluid that owes its
viscoelastic, lubricating and hydration properties to the glycoprotein mucin combined with electrolytes, lipids
and other smaller proteins. Electron microscopy of mucosal surfaces reveals a highly convoluted surface with a
network of fibers and pores of varying sizes. The major structural and functional component, mucin is a complex
glycoprotein coded by about 20 mucin genes which produce a protein backbone having multiple tandem repeats
of Serine, Threonine (ST repeats) where oligosaccharides are covalently O-linked. The N- and C-terminals of this
apoprotein contain other domains with little or no glycosylation but rich in cysteines leading to dimerization and
further multimerization via S\\S bonds. The synthesis of this complex protein starts in the endoplasmic reticulum
with the formation of the apoprotein and is further modified via glycosylation in the cis and medial Golgi and
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.
1. Introduction
Mucus is a complex viscoelastic adherent secretion that is synthesized and secreted by specialized goblet and mucous cells in the columnar epithelia that line the lumen of all of the organs and glands that are
exposed to and communicate with the external environment [3,38] (Fig.
1). This includes the inner linings of organs of the respiratory tract, the
gastrointestinal tract, the reproductive tract and the ocular surface. Historically, mucus was certainly known since ancient times. For example
Phlegm has origins in 3rd century Greek φληγμα (phlegma) and Hippocrates (460–370 BCE), extended the humoral theories first stated by
Empedocles (504–433 BCE) postulating the existence of 4 humors,
black bile, yellow bile, blood, and phlegm. A balance of these humors
was associated with health and an excess or deficit of any one was associated with disease. The Chinese pharmacopeia Book of Herbs (Pen
Ts'ao) dating to 2500 BCE mentions the use of ‘ma huang’ which
☆ This article is part of the Advanced Drug Delivery Reviews theme issue on
"Technological strategies to overcome mucus barrier in mucosa".
⁎ Corresponding author.
E-mail addresses: rb@bu.edu (R. Bansil), bsturner@mit.edu (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).
contains ephedrine and pseudoephedrine, found in current “Sudafed”
cold remedies which are used to alleviate symptoms associated with
mucus hypersecretion in upper respiratory infections [182].
Mucus has been described in organisms from all kingdoms [97]. Viscous, gel forming mucilages and structural glyco-substances are found
in all forms of life, including viruses, bacteria, fungi, plants, insects, fishes, etc. but this review concentrates specifically on mammalian mucus
and its mucin glycoproteins. Vertebrates contain mucus layers in their
corresponding organ systems. Additionally, most aquatic organisms
also possess an external mucus layer on their skin [128]. Mucus has
also been studied from invertebrates (worms, snails, slugs, insects,
anemones). For example the hagfish is able to explosively release liters
of mucus from its skin which is used as a prey escape mechanism [18,60,
151]. Mucus layers have also been described in various pathogenic protozoa [161]. Zebra fish provides an excellent model system currently
much in vogue to study the effects of mucus in vivo [24,78].
Mucus serves many protective functions for the underlying epithelia, such as lubrication for material transport and hydration over the epithelium particularly in the respiratory tracts, eyes and mouth that are
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
transport function where external particles trapped in the mucus layer
https://doi.org/10.1016/j.addr.2017.09.023
0169-409X/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
2
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
can be eliminated from organ cavities by cilia facilitated expulsion of the
mucus layer [146], and provides a selectively permeable gel layer for the
diffusion, exchange and absorption of gases (eye and lung) and nutrients (gastrointestinal tract) with the underlying epithelium [115].
2. Mucus structural organization
2.1. Goblet/mucous cells
Mucus is secreted by goblet/mucous cells (for a recent review see
[17]) which are interspersed with many other types of specialized
epithelial cells (Fig. 1A), and have a similar typical highly polarized morphology with a broad apical region and narrower basal region, giving
them their characteristic ‘wine glass goblet’ shapes (Fig. 1B and C).
The apical region contains large, supranuclear, mucus containing
storage and secretory granules, a central supranuclear endoplasmic reticulum and Golgi apparatus, a basal nucleus, and sub-perinuclear
rough endoplasmic reticulum and mitochondria (Fig. 1B and C). These
cells are surrounded by other columnar or squamocolumnar epithelial
cells with specialized functions such as ciliated, absorptive, secretory
or proliferative cells, and are attached to their neighboring cells by
typical tight junction structures. The goblet mucous cell types are distinguished based on gross morphology. In general goblet cells are tapered,
decreasing in circumference (e.g., get thinner) from apex to base,
whereas mucous cells have near constant diameter from apex to base.
Both types of cells have large stores of apical mucus secretory granules,
occupying 75% of the cell volume [53]. Goblet cells regulate the production of mucins in response to external factors. For example, the response
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.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
of goblet cells to enteric infections is discussed in [89]. Goblet cells,
mucus and mucins also interact with the immune system as discussed
in [121].
2.2. Mucus structure
Scanning electron microscopy (SEM) of gastric mucosal surfaces [58,
119] show the highly folded structure of stomach epithelium with gastric glands and fibrous mucus. Electron microscopic studies of mucus
gels are strongly influenced by dehydration during sample preparation
(see for e.g. [41,110]). Familiari et al. [55] developed a multi-step process to preserve the real microarchitecture of mucus covering the jejunum surface and the zona pellucida surrounding the mature oocyte
and show the network of the dried mucosal fibers. Cryo-SEM [91] of pulmonary mucus preserves the structure of better showing a highly heterogeneous mesh with both large and small pores and a thick polymer
scaffold. We examined the structure of hydrated mucus on the sub-micron length scale in vitro by atomic force microscopy (AFM) in a liquid
cell under appropriate buffers. A tapping mode AFM measurement of
a wet sample of human mucus taken from the discarded material
obtained in the lavage following a stomach biopsy reveals a swollen
network similar to that formed by the glycoprotein mucin [74] with
aqueous pores of about 200–300 nm in diameter.
2.3. Mucus thickness
The thickness of the mucus layer varies considerably both within
and between different organ systems. For example, in the respiratory
tract mucus layer thickness is about 10 μm in the trachea [112] while
in the gastrointestinal tract, the mucus layer increases in thickness
from the upper to the lower GI tract varying from 200 μm to 800 μm respectively [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
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
ovulatory cycle and regulates sperm penetration [86] and uteral implantation of the fertilized egg [28]. These differences in mucus thickness
may reflect the various protective functions and challenges of mucus
within its specific organs.
Mucus in the respiratory, ocular and gastrointestinal tracts has been
shown to exist in two layers with distinct rheological properties [155].
For example, in the gastrointestinal tract the first 100–150 μm thick
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
lies on top of it extending to the luminal surface. It has also been
shown that bacteria have differing affinities for these two layers [80].
In contrast, in the respiratory tract, the lower viscosity perciliary
mucus layer consisting of hydrated membrane mucins and mucopolysaccharides, is in contact with the airway epithelium and supporting
the higher viscosity mucus on top of it, which is exposed to the air.
This arrangement is thought to facilitate mucus expulsion and clearance
from the lung and airway passages by the action of cilia via mucociliary
transport [25,146].
Table 1
Mucus thickness in the gastrointestinal tract.
Average values reported by Atuma et al. [5]. Note: we have rounded the numbers to 5 μm,
well within the range of values reported.
Thickness (μm)
Total
Firmly adherent
Loosely adherent
Location in gastric mucosa
Corpus
Antrum
Duodenum
Jejunum
Ileum
Colon
190
80
110
275
155
120
170
15
155
125
15
110
480
30
450
830
115
715
3
3. Mucus composition
Mucus is a complex dilute aqueous viscoelastic secretion consisting
of many components: water, electrolytes, lipids and various proteins.
Creeth [40] provides an excellent review describing the constituents
of mucus and methods to separate them. Water comprises approximately 90–95% of mucus and serves as the solvent and diffusion medium for all the other mucus components.
3.1. Mucus electrolytes
Electrolytes are an important component of mucus and their composition can be different in mucus from various tissues, and are determined by the underlying secretory epithelium. These differences may
be important to the specific environmental challenges found in each
organ. Common to most mucus secretions are sodium and potassium
chloride and sodium bicarbonate, phosphate and magnesium and calcium, approximately isotonic with serum at approximately 1% (w/v) total
concentration. Some organs modify the ionic composition of mucus
during their specialized functions. The stomach during active digestion,
secretes large amounts of hydrochloric acid (HCl) with a concomitant
reduction in the potassium chloride concentration [106]. Changes in
the electrolyte composition have a role in controlling mucus hydration
and rheology e.g. increased concentration of divalent cations, Mg or
Ca, can cause collapse of the mucus gel [165,167,168] whereas an increase in concentration of monovalent cations, Na or K can cause a reduction in mucus viscosity [149]. Bicarbonate, which is secreted by
secretory cells and released by the submucosal vasculature, helps to
buffer the mucus layer adjacent to the epithelium to pH 7. This is particularly important in the stomach where bicarbonate establishes a protective pH gradient through the mucus gel layer against HCl acid secretion
[2]. The pH also affects the viscosity of gastric mucus [15] leading to gelation below pH 4 [8,30].
3.2. Mucus lipids
Lipids constitute 1–2% of mucus. Many lipids have been described to
be both covalently and non-covalently associated with mucus layers
[71,102]. These include phospholipids, primarily phosphatidyl choline
and phosphatidyl glycerol and small amounts of lysophosphatidyl choline generated by phospholipase A2 in some pathologic conditions, cholesterol, fatty acids both free and acylated to proteins. The lipids interact
with mucus glycoproteins and affect the wettability (charged lipids)
and hydrophobicity (neutral lipids) and barrier functions of the mucus
layer [102]. Lipids may also contribute to the lubricating and surface
tension properties of the mucus layer. The rheological properties of
mucus are also strongly affected by lipids [65,172]. Lipids also serve to
prevent evaporation of the aqueous phase, for example in the tear
mucus layer covering the eye [39,82].
3.3. Mucus proteins
Mucin glycoproteins constitute the major functional component of
mucus, however ancillary non-mucus proteins play an important role
in the structural and protective functions of mucus. Recently many
mucus proteins have been identified by high-throughput, proteomic
mass spectrometric, chromatographic and 2D-PAGE methods. One hundred eighty six different proteins have been identified in airway, 147 or
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
proteins present are serum proteins, presumed to arise from epithelial
serum transudation. Most important proteins found can be broadly classified into functional groups including defensive proteins, growth factors, structural proteins and glycoproteins.
The major defensive proteins are involved in contributing to the
mucus protective abilities against pathogens and particulate matter.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
4
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
These include the alpha and beta defensins, which are antimicrobial
peptides [14,105] and lysozyme (muramidase), an enzyme that digests
muramic acid mucopolysaccharide bacterial cell wall components. The
positive charge of lysozyme enables binding to the negatively charged
mucus; its bactericidal properties were discovered by Alexander
Flemming [57]. Lactoferrin, in all mucosae, is an intrinsic factor (in the
stomach) that chelates iron which limits its availability for bacterial
growth. Statherins, proline rich peptides and histatin defensive proteins
have also been identified in mucus [76].
Immunoglobulins, secretory IgA and IgM are present in all mucus
layers and are active against a variety of pathogens. Immunoglobulins
have also been reported to increase the viscosity of mucus in airways
[65] and IgG and IgA have been shown to bind differentially to cervical
mucus [54]. Secretory IgA has been shown to be required to bind to
the mucus layer for effective antibacterial defense [138,158].
The major growth factors present on almost all mucus layers are
transforming growth factor beta, epidermal growth factor and hepatic
nuclear factor [35]. One or more of these growth factors are present in
mucus layers of all organs and may serve growth, maintenance, repair
and regulatory functions to the underlying epithelium. Additionally,
there are various cytokines present that are released from epithelial or
immune cells in response to various stimuli, pathogens or disease states.
Structural proteins include protease inhibitors such as secretory leukocyte proteinase inhibitor and pancreatic secretory trypsin inhibitor.
These proteins protect the mucus proteins from proteolysis, depolymerization and loss of viscosity by enzymes of both host and pathogen origins. Also present are the trefoil (TFF) peptides. These are small cysteine
rich peptides with a characteristic disulfide linked three loop clover leaf
conformations [139,174]. They have been reported as agents associated
both with mucus interactions, increasing its viscosity [157], protection
from proteolysis and as epithelial repair growth factors.
3.4. Mucins
The major functional components of mucus are the mucin glycoproteins present at a concentration of 1–5%. They are responsible for the essential and predominant viscoelastic properties of the mucus layer.
Details of mucin genes, glycoprotein structure and biophysical properties have been presented in considerable detail in previous reviews
[6–8,38,45,72,123,134,137,158]. Mucins are broadly classified as two
types, secreted mucins and membrane bound mucins. Both membranes
and secreted mucins share many common characteristic compositional
and structural features based on the fact that their underlying protein
sequence is governed by the same family of genes. Based on sequence
and domain comparisons and similarities, and cellular origins, 22
mucin genes have been identified and can be grouped into two broad
classes representing membrane-bound mucins, anchored by insertion
through the plasma membrane and secreted mucins, that are packaged
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
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
mucins MUC 2, 5AC, 5B, 6, 19 and 2) small soluble mucins MUC 7, 8, 9.
Immunohistochemistry has been used to identify expression of different mucins in many organs in healthy as well as diseased states with a
significant focus on cancer (see for e.g. [10,16,34,81,99,100,104,129]).
protein core by alpha-1-O-glycosidic bonds from GalNAc to the hydroxyl side chains of serines (Ser) and threonines (Thr). In a recent paper, Jin
et al. [79] present a structural map of human gastric glycosylation from
healthy and diseased subjects showing a large structural diversity with
over 200 O-glycan structures identified in the total sample of 10 individuals. To date, no single consensus O-glycosylation amino acid sequence
motif has been identified, however O-glycosylation sites can be roughly
predicted by statistical, and artificial intelligence analysis of databases of
experimentally determined glycosylated proteins (NetOGlyc,
OGlycBase) [26,27,70,84]. A large proportion of the Ser/Thr residues
are predicted to be glycosylated. This results in a “bottle brush” configuration of the oligosaccharides arranged around the mucin protein core
(see Fig. 2). Elongation and branching of the oligosaccharide side chains
are governed and performed by a large family of glycosyltransferases
[23,156,160]. It is believed that the vast diversity in mucin oligosaccharide structures is determined by the tissue specific expression patterns
and combinations of the glycosyltransferases and their substrate specificities [13,63,137]. Additionally, the oligosaccharides may be modified
by the addition of blood group (ABO, Lewis), secretor (H) epitopes
[135], and terminal negatively charged sialic acids and sulfates. The
large amounts, distribution and density of glycosylation, steric exclusion, and charge repulsion, impart a highly hydrated, extended, relatively stiff elongated linear “rod-like” conformation to the mucins [62,109].
These extended mucin structures have been directly observed by both
electron and atomic force microscopy and exhibit molecular lengths of
50–1000 nm up to 8 μm [74,109,144] for a variety of secreted mucins
from various sources. This extensive glycosylation also serves to protect
the mucin protein core from proteolytic degradation. Keismer et al. [88]
used electron and atomic force microscopy to show 190–1500 nm long
tethered mucin polymers, and a finely textured glycocalyx matrix filling
interciliary spaces of airway mucus. AFM methods for measuring binding forces of glycans to lectin and other groups deposited on AFM cantilevers have also been applied to investigate the distribution of glycans
from mucins such as sialic acid of ocular mucins [9] and gastrointestinal
mucins [69].
Both membrane and secreted mucins also contain N-glycosylated
multiantennary complex and hybrid oligosaccharide high mannose
structures, which are synthesized in the secretory pathway by the en
bloc transfer of preformed mannose oligosaccharides to the amide side
chain of Asparagine (Asn) residues located in well-defined N-X-T/S sequence motifs at the ends of mucin proteins [34,150,183]. Secreted mucins also contain a novel type of glycosylation, C-mannosylation,
modifications that involve a carbon-carbon bond between the C1 of
mannose and the indole ring of specific tryptophans (W) in W-X-X-W
motifs [125].
The combined extensive glycosylation gives mucins a higher buoyant density in CsCl (1.45 g/cm3) than typical non-glycosylated proteins
(1.2 g/cm3) or lipids (b1.0 g/cm3) yet less than nucleic acids
(N1.6 g/cm3) [42,67]. This feature along with their large molecular
sizes (N 2 MDa) results in facile methods for purifying and analyzing
mucins by isopycnic, density gradient ultracentrifugation [67] and size
fractionation by size exclusion chromatography and agarose electrophoresis [67,130], respectively.
3.6. Mucin protein core, tandem repeat domains, non-repeat domains and
cysteine rich domains
3.5. Glycosylation
Both membrane and secreted mucins are highly glycosylated,
consisting of approximately 50–80% (w/w) carbohydrates, primarily
composed of GalNAc, GlcNAc, Fuc, Gal and sialic acids or NANA or
NeuNAc (N-acetyl neuraminic acid) and relatively small amounts of
mannose. The oligosaccharide chains possess approximately 2–20
monosaccharides in both linear and moderately branched structures
(see for e.g. [21,104]). The oligosaccharides are attached to the mucin
The apomucin protein core (100–500 kDa) for both membrane and
secreted mucins, comprises approximately 20% (w/w) of the mucin
mass. It is organized into two broadly distinct regions, 1) a centrally located tandem repeat region rich in Ser, Thr, and Pro (STP-tandem repeats) which contains O-glycosylation, and 2) the carboxy- and
amino-terminal non-repeat regions, which may be rich in cysteine
(Cys) residues with low amounts of Ser/Thr and relatively little O-glycosylation, but possessing N-glycosylation. The amino acid composition,
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
5
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. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
(Reproduced from Bansil et al. [8] under Creative Commons Attributable License).
sequences and structures of these terminal Cys-rich regions are more
typical of secreted globular proteins [38,123].
The Ser/Thr repeat units can vary in length from 7 to 375 amino acids
in MUC19 and MUC3A respectively. The number of repeats within a
mucin molecule can also vary from 10 to 500 as inheritable Variable
Number of Tandem Repeats (VNTR) polymorphisms [169]. The amino
acid sequences of the repeat units are very similar (70–90% identical)
within a particular mucin gene within an individual of a single species,
but can vary significantly between homologous mucin genes in different
species [137].
In secreted gel forming mucins, Cys-rich regions of the protein core
are located at both the amino and carboxy ends of the central tandem
repeat region. In some secreted mucins (MUC5AC an MUC5B) Cys-rich
sequences (CysD) can also be found periodically interspersed within
the Ser/Thr tandem repeats. The Cys-rich regions can contain up to
10% Cys and relatively little Ser or Thr residues that are arranged in
highly conserved, specific patterns and can be used to classify mucins
[47,97,123]. The cysteines can be involved with both intra- and intermolecular disulfide bonds or in contributing to protein flexibility between tandem repeat domains. These regions contain few Ser/Thr or
O-glycosylation and have an overall amino acid composition and conformation characteristic of globular secreted proteins. Multiple Cysrich von Willebrand Factor D (vWF-D) domains are found at the
amino end of secreted mucin molecules [85,123]. These domains have
been determined to be involved in the multimerization of both mucins
and von Willebrand factor (vWF) [108,122,124].
At the carboxy terminal end of the central tandem repeat domains of
secreted mucins, are Cys rich regions similar to vWF D domains, vWF C
domains and carboxy terminal cysteine knot (CTCK) domains which are
required for dimerization [12,181]. The mucin carboxy terminal vWF-D
domains have similar sequence, structure and dimerization functions as
those located at the amino terminal.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
6
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
4. Mucin synthesis and secretion
In this section we consider in some detail mucin synthesis and secretion leading to the formation of a hydrated mucus layer. This subject has
been recently reviewed [34,43]. We will consider how mucins are synthesized, co- and post translationally modified, transported packaged,
stored and secreted. Synthesis of large amounts of high molecular
weight, complex extensively glycosylated mucins represents a significant metabolic commitment by the cell. We consider mainly secreted
gel forming mucins (MUC 2, 5AC, 5B, 6, 19), but the general description
also applies to membrane mucins. These pathways have been studied
for a wide variety of secretory products, including mucin in different
cell types, and organisms and have yielded basic mechanisms and components that are relevant and applicable to all mucins.
4.1. Synthesis pathways
Mucin synthesis and secretion proceeds by both constitutive pathways, characterized by continuous release of newly synthesized products and regulated pathways involving storage of mucin products that
are released in response to various stimuli in a calcium dependent manner. In this description we consider primarily the regulated pathway, although the initial steps of synthesis and assembly through the trans
Golgi vesicular budding, also apply to the constitutive path.
4.2. Mucin synthesis
The various steps in mucin synthesis are listed in Table 2. As with
many other secreted glycoproteins, the synthesis of the protein core of
mucin (apomucin) begins on membrane bound ribosomes of the
rough endoplasmic reticulum (RER). This involves ribosomal docking
to the translocon complex, a large (N125 Å) [111] membrane associated
assembly of over 25 proteins including Sec61p [118] through which the
growing nascent apomucin is transported into the lumen of the endoplasmic reticulum (ER). A number of post translational modifications
(PTM) of the apomucin protein take place co-translationally or very
early in synthesis, namely N-glycosylation, C-mannosylation and
dimerization.
4.3. N-glycosylation
N-linked glycosylation is accomplished co-translationally by transfer
of preformed high mannose oligosaccharides from dolichoyl-pyrophosphate-Glc3Man9GlcNAc2, to the side chain amide group of asparagines
(N) in consensus motif N-X-T/S (where X is any amino acid except proline) sites located in the mucin Cys-rich regions catalyzed by the enzyme oligosaccharide transferase which is closely associated on the ER
luminal side of the translocon complex [32,150,178]. Immediately
after N-glycosylation, N-linked oligosaccharides are further modified
in the ER by removal of two Glc residues by [177] α1,2- and α1,3
Table 2
Intracellular synthesis and assembly of mucins.
A schematic of the various steps in the intracellular assembly of mucins as described in
[158].
Sub cellular
compartment
Step. Process
No.
Endoplasmic
reticulum
1
cis and medial
Golgi
trans Golgi
2
3
4
5
Translation of the peptide core, folding of cysteine rich N
and C terminal domains with formation of intracellular
disulfide bonds, and N-glycosylation in the ER
Dimerization of mucin peptides in the ER
Initial O-glycosylation of serine and threonine by GalNAc
in the cis Golgi
Elongation of O-linked oligosaccharides in the medial Golgi
Polymerization of mature mucins to form linear and
branched polymers. Packaging into secretory granules with
Ca2+
glucosidase and one mannose residue by α 1,2 mannosidase I to yield
Glc1Man9GlcNAc2-Asn [136]. This oligosaccharide structure serves as a
ligand for the ER Ca2 + dependent lectins calnexin and calreticulin,
both present in the ER lumen which bind the N-linked oligosaccharides
on the nascent peptide and serve as folding co-chaperones [163]. Bound
peptides undergo repeated cycles of binding and folding with formation
of intramolecular and intermolecular disulfide bonds, leading to dimer
and trimer formation through the C-terminal cysteine knot domains. It
has been demonstrated that elimination of N-glycosylation, by use of inhibitors, site directed mutation or expression of proteins in N-glycosylation deficient cells, leads to decreased mucin secretion and
accumulation of mucin products within the ER [11].
4.4. C-mannosylation
C-mannosylation, an unusual novel PTM also occurs cotranslationally. It involves transfer by the enzyme C-mannosyl transferase [49] of a single mannose residue from dolichoyl phosphate mannose
to the indole C2 of the first tryptophan (W) in the consensus W-X-X-W
motifs [83]. These motifs are also usually found within the Cys-Rich domains of mucins. Although the precise function of C-mannosylation is
unknown, mutation or absence of the mannosylation site or expression
of mucins in C-mannosylation deficient cells, results in the accumulation of protein in the ER and decreased secretion, suggesting that Cmannosylation may be required for export from the ER via cargo receptor binding and transport vesicle packaging [125]. Thus both N-glycosylation and C-mannosylation are required for mucin dimerization, folding
and for export from ER to Golgi.
Mucin products at this point are presumed to have a globular, random coil conformation with molecular weights of 250–500 kDa for disulfide linked monomers and 0.5–1 MDa for dimers and multimers
[158].
4.5. ER to Golgi transport
Continued synthesis of correctly folded proteins proceeds by packing into COP II (coat protein complex) transport vesicles with the assistance of cargo receptors VIP36 (Vesicular Integral Protein 36 kDa) and
ERGIC-53p (ER to Golgi Intermediate Compartment Protein 53 kDa)
and transported to the cis-Golgi on microtubules [120,136,177].
4.6. O-glycosylation
Once in the Golgi, mucin proteins acquire their most prominent and
characteristic salient feature, namely extensive O-glycosylation, which
can constitute 50–90% of the final mucin molecular mass. In contrast
to N-glycosylation which transfers, en bloc, a large preformed oligosaccharide from a lipid precursor, O-glycosylation occurs by the sequential
addition of single monosaccharides from nucleotide sugar precursors.
O-glycosylation is accomplished in three principal steps, initiation, elongation and termination [23].
The first step of initiation, which occurs in the cis- and medial-Golgi
(reviewed in [137]), occurs by the addition of a single GalNAc from a
UDP-GalNAc precursor, from its reducing end C1 to the hydroxyl side
chains of serine (Ser) and threonine (Thr) residues, by the action of enzyme UDP-N-acetylgalactosamine:polypeptide galactosaminyl transferases (GalNAc-Tase) to yield GalNAC-α1-O-Ser/Thr [159]. There are
10–20 GalNAc-Tase type II membrane proteins [156] which are conserved from fungi to mammals. Some of these initiating GalNAc-Tases
(GalNAc T1-T6 and T10-T14) transfer the initial GalNAc to
unglycosylated protein substrates, whereas other GalNAc-Tases (T7T9) enzymes containing a carboxy-terminal rich in lectin (Gal/GalNAc
binding) domain that is required for binding to substrate, complete
the dense O-glycosylation of clustered Ser/Thr residues [170]. After addition of the initial GalNAc, mucin molecules have a molecular weight of
0.5–2 MDa and would be slightly less flexible and more extended in the
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
STP-tandem repeat regions due to steric exclusion of the carbohydrates
[145].
All mucin O-linked oligosaccharide structures are built on a limited
number of eight common “Core” structures, based on the addition of
monosaccharides Gal or GlcNAc directly to different positions on the initial GalNAc to form linear or branched Core structures. Many different
Core structures can be present on a single mucin molecule [23,131,
132]. Elongation of the basic core structures continues by stepwise sequential addition of monosaccharides from nucleotide precursors by alternate action of various GlcNAc and Gal transferases. Alternating GalGlcNAc disaccharides either beta 1-3 or beta 1-4 lactosamines are common. The elongated structures may be either linear or branched. The
specific structures that are formed are determined by the cell or tissue
specific expression patterns and substrate specificities of the glycosyltransferases. Thus it is possible for the same mucin peptide to have different glycosylation structures when expressed in different cells or
tissues. For example, for cervical mucin glycosylation, the specific combination of glycosyltransferases, and their levels are hormonally regulated during the menstrual cycle [4]. More than 100 different O-linked
oligosaccharide structures have been identified on a single colonic
MUC2 protein [98].
Termination of O-linked oligosaccharide chain growth occurs in the
medial- to trans-Golgi compartments by the competitive enzymatic
addition of fucose, GalNAc or NeuNAc (Sialic Acid) mainly as the αanomers to the periphery of the growing oligosaccharide chains. These
terminal structures can include blood group ABO, Lewis ABXY and H secretor antigens [135]. Terminal sugars can also be modified with sulfates. Of the eight core structures, only cores 1–4 and 6 are substrates
for the sulfotransferases [22]. It should be noted that the N-linked oligosaccharides also undergo trimming, elongation and modification in the
Golgi to obtain high mannose, complex and hybrid type structures.
The addition of the terminal sialic acid (NeuNAc) and sulfates
contribute large amounts of negative charges to the mucin oligosaccharides. This causes charge and steric repulsion between the oligosaccharide chains and an extension and stiffening of the mucin molecule to an
extended rod-like configuration with a “bottle brush” arrangement of
oligosaccharides around the apomucin core, and with molecular
weights from 2 to 10 MDa and occupation of a large hydrated volume
in solution.
4.7. Granule condensation
As oligosaccharide construction is completed, mucins in the transGolgi network (TGN) are packaged into vesicles that bud off by means
of the cargo receptor VIP36 (vesicular integral protein 36 kDa). Mucin
packaged in vesicles undergo maturation to secretory granules (SG)
by condensation by the formation higher order mucin multimers by disulfide association of amino-terminal vWFD domains, to form molecules that are 10–50 MDa extended rods with linear dimensions 1–10
μm [66,122], which are packaged into vesicles of b 1 μm. Verdugo
[164] applied the ideas of a large volume gel phase transition [101,
153] in charge polymer gels to mucins to both explain and demonstrate
how this packaging is possible (for a recent review see [166]). The many
repulsive negative charges (from sulfates and sialic acids) on the extended mucin molecules can be neutralized, shielded and crosslinked/
complexed with positive counter ions such as H+, Na+, Ca2+, resulting
in charge shielding, allowing mucin gels to undergo a 100 to 200 fold reversible volume collapse phase transition. This was demonstrated with
mucins by increasing the extracellular solution concentration of H+ and
Ca2+ to those concentrations found in secretory granules and observing
shrinkage of the mucin gels.
It has been shown that progression through the secretory pathway
results in luminal compartments with increasing H+ and Ca2+ concentrations [46] with ER at pH 6.8, cis Golgi at pH 6.7, medial-Golgi at
pH 6.3, trans-Golgi at pH 6.0 and secretory granules at pH 5.5. This is accomplished by an increasing density of a membrane bound vesicular
7
H+-ATPase proton pump that transports H+ into the Golgi lumen and
by a decrease in leakage of H+ from the lumen [46,175].
Similarly, intragranular Ca2 + concentrations (23 mmol/kg) are
found to be higher than intracellular or other compartment Ca2+ concentrations (6 mmol/kg) [77,93,117]. Once mucin's charge repulsion
has been neutralized by H+ and Ca2 +, hydrophobic interactions between mucin molecules and lipids can dominate causing mucin condensation within the secretory granules.
4.8. Granule packaging
The intragranular condensed state of the mucin secretory granule
matrix has been elegantly investigated by Perez-Vilar using fluorescent
confocal microscopy and FRAP (fluorescence recovery after
photobleaching) of recombinant GFP-mucin domain constructs that
are packaged into secretory granules. Results indicate that the diffusion
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
movement and a more condensed state in the granules than ER. It also
indicates the existence of pores or spaces within the granule matrix
that allow movement of small molecules which is important for exocytosis [126].
4.9. Secretion and exocytosis
After secretory granule budding from the TGN, condensation and
maturation, mature secretory granules (MSG) move toward the peripheral 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
plasma membrane, 2) docking an tethering of MSG to the cell membrane, 3) pore formation by granule membrane to plasma membrane
fusion, and 4) exocytosis of MSG contents into the extracellular space.
More details can be found in reviews by Davis et al. [43], Evans et al.
[53], Rogers [133], and Williams et al. [173].
4.10. Mucin granules
Mucin secretory cells showing granules have been imaged using
fluorescence microscopy [126] as well as with non-fluorescent confocal
light absorption and scattering spectroscopy (CLASS) [56]. They compared CLASS images to fluorescently labeled granules from the same
cell line and showed improved resolution from this label-free technique. An image of a mucus producing gastric cell (AGS cell) obtained
by CLASS showing mucin granules ranging in size from 0.5 to 2 μm is
shown in Fig. 4A.
4.11. Exocytosis
MSG and plasma membrane fusion causes the formation of an open
pore having an “omega” structure ‘Ω’ [116]. The pore allows the interior
of the MSG to be exposed to the extracellular environment. Verdugo applied the gel phase transition model to explain the mechanism by which
mucins leave the secretory granule through the pore [165]. Adler et al.
[1] describe the proteins involved in the fusion of vesicle with granules
to the cell membrane and the signaling factors involved in activation of
Ca2+ release leading to the secretion of mucin (Fig. 4B). As described
above for mucin condensation, the MSG contains a condensed porous
gel network consisting of collapsed negatively charged mucins complexed with high concentrations of H+ and Ca2 + counter ions which
neutralize and crosslink mucin negative charges. When the MSG interior is exposed to the extracellular milieu, which has lower Ca2+ and H+
concentrations, the MSG Ca2+ and H+ rapidly diffuse out of the MSG
faster than the large mucin molecules. This results in de-shielding of
the mucin anions, yielding a net mucin negative charge and electrostatic
repulsion of the mucin from its sialic acids and sulfates. This cationic
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
8
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
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).
diffusion also results in the formation of a net positive charge in the area
immediately surrounding the extracellular regions of the pore opening.
This diffusion driven charge separation defines a Donnan electric potential with mucin serving as the semipermeable membrane containing
slowly diffusible negative charges [48,152]. The MSG mucin gel network
undergoes a rapid volume expansion phase transition [167] driven by a
combination of Donnan potential, mutual electrostatic repulsion of the
deshielded mucin anions and by osmotic hydration of mucin saccharides by water influx into the MSG. The mucin molecules expand and
spread away from the open fusion pore, covering the external apical
surface of the cell.
Experimental verification of this “jack-in-the-box” explosive exocytosis process was demonstrated by the prevention of mucin expansion
by increasing the extracellular H+ and Ca2+ concentrations to match
those of the MSG interior. This eliminates the MSG interior to extracellular diffusion gradient and also eliminates development of the Donnan
potential. Decreasing the external pH from 7.4 to 6.5 and increasing the
external Ca2 + from 1 to 4 mM causes a measurable increase in the
mucin swelling times and decrease in the diffusion constants from
2.25 × 10−7 cm2/s to 1.11 × 10−8 cm2/s [52]. The released mucin then
remains in an unexpanded, condensed state and does not undergo the
rapid volume expansion phase transition “jack-in-the-box” exocytosis
[52,167,168]. Recently, this process has been experimentally observed
using electron microscopy of isolated mucin granules in various stages
of exocytosis (Fig. 4C) [87].
4.12. Mucus and mucin rheology and mucoadhesion
Although the main focus of this review is on mucus structure,
composition and synthesis, we briefly discuss the rheological
properties of mucus and mucin, the molecule that gives rise to its
viscoelastic properties, as well as its ubiquitous mucoadhesive properties. Rheology of mucus is a major factor in controlling the transport of food, drugs, particles including nanoparticles and larger
micron scale particles, and infectious organisms, across mucosal linings, as well as controlling its hydration and lubrication properties.
The rheology of mucus from many different animals/tissues/organs
has been investigated for a long time (see for e.g., [44,90,95,142,
162]). The viscosity of purified mucin, particularly lung, salivary
and gastric mucin has also been well studied using bulk, or macroscale methods [30,75,94,158,176] and at the local, micro-scale
using microscopic particle tracking methods [8,61,103]. We refer
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
A.
9
B.
C.
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.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 4C is reproduced from Kesimer et
al. [87] by license provided by American Physiological Society and Copyright Clearance Center.
the reader to a recent review from our group [8] that provides a simple explanation of the basic concepts of rheology (i.e. elastic and viscous properties) and describes the different techniques used. Briefly,
mucus and mucin exhibit frequency dependent viscoelastic moduli
and their viscosity decreases with increasing shear rate, i.e. they are
shear thinning materials. Many of the gel forming mucins, such as
MUC5AC from purified porcine gastric mucin exhibit elastic response
as well [30]. Georgiadis et al. [61] showed that pH induced gelation occurs not only in porcine gastric MUC 5AC but also in porcine duodenal
mucin MUC2. The viscoelastic behavior of mucins depends on pH,
ionic strength, and degree of hydration and thus is particularly relevant
to the transport of particles through mucus [96]. For example, the viscosity of mucus or mucin has been shown to influence binding to
small molecules such as nicotine [33] and other drugs [141], and the
transport of sperm [50], bacteria [31,107], viruses [19,20] and nutrients
[179] through purified mucin or mucus layers.
Finally we add a cautionary note concerning the effect on viscoelastic properties related to differences in the methods for purification of
mucin. The methods differ in many ways such as fractionation
techniques, and treatment by digestive enzymes which can lead to degradation into subunits [92], or chaotropic agents like guanidium chloride GuHCl which remove associated proteins. These differences in
methodology can lead to products differing in the fraction of soluble
and insoluble mucins, varying in molecular weight and charge distribution, and the degree to which associated DNA and proteins are removed.
Similarly the use of different chromatographic techniques for separation
could also account for differences in the properties of the mucin. Recent
studies have shown that the use of different methods influences rheology and gelation of the purified mucin [61].
Mucus is highly adhesive to many different substrates owing to the
propensity of molecular interactions. Its glycan coat can bind to both
neutral and charged surfaces by non-covalent and electrostatic interactions. It also exhibits hydrophobic binding most likely related to the
non-glycosylated regions of the peptide. There is considerable interest
in controlling mucoadhesivity and coating with polymers, ionic or
other materials in designing drug delivery systems capable of
transporting through mucosal lining or for topical applications on
mucosal surfaces. We refer the reader to two recent reviews on
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
10
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
mucoadhesivity [148,180] for this fascinating topic. Another topic of
considerable current interest, that we have not discussed concerns the
transport of nanoparticles through mucus (see for e.g. [51,59,68,127,
140,171]).
5. Future outlook and conclusions
In summary, we have reviewed the basic structure, composition,
synthesis and organization of mucus. Research on mucus and purified
mucin is currently seeing a significant interest in the context of diseases,
particularly cancer and infectious diseases caused by agents that breach
the mucosal barrier, as well as in the context of delivering drugs and nutrients across the mucosal barrier. Understanding both the physical and
chemical/biochemical interaction of this complex highly heterogeneous
soft material is crucial for progress in this area. One of the major obstacles in the progress of this work has been the availability of well characterized and standardized preparation of mucus and purified mucin. An
alternative to using purified mucus/mucins for applications where
large quantities of material are required is to use synthetic formulations,
or even mucus from other sources such as hagfish. However, these will
not be identical to mucus, and thus results would have to be interpreted
with caution. Another area of research which is just beginning to develop is novel methods for in vitro and in vivo imaging and other techniques to investigate mucus structure, rheology, adhesive properties
and transport of nutrients, drugs, nanoparticles, bacteria and viruses
through mucus. At the biochemical and molecular biological end novel
immune-fluorescent and single molecule spectroscopy techniques are
pushing the frontier forward to investigating molecular details of mucosal surfaces and mucus secretions. Biochemical and molecular modifications of the glycan coat may lead to development of new vaccines and
drugs.
Note Added in Proof
The reference below confirms that hydrophobic interactions are involved in mucin gel formation and low pH viscoelasticity increase. The
surfactant (very mild detergent) 1,2 hexandiol causes a reduction in
mucin viscoelasticity.
Wagner CE, Turner BS, Rubinstein M, McKinley GH, Ribbeck K. A
rheological study of the association and dynamics of MUC5AC gels.
Biomacromolecules. 2017 Sep 14. (in press) [PubMed] https://www.
ncbi.nlm.nih.gov/pubmed/28903557.
Acknowledgements
RB was supported by NSF PHY 1410798. BST was supported by
MIT Center for Materials Science and Engineering, MRSEC NSF DMR
1419807.
References
[1] K. Adler, M. Tuvim, B. Dickey, Regulated mucin secretion from airway epithelial
cells, Front. Endocrinol. 4 (2013) 129, https://doi.org/10.3389/fendo.2013.00129.
[2] A. Allen, G. Flemstrom, Gastroduodenal mucus bicarbonate barrier: protection
against acid and pepsin, Am. J. Phys. Cell Phys. 288 (1) (2005) C1–19.
[3] M. Andrianifahanana, N. Moniaux, S. Batra, Regulation of mucin expression: mechanistic aspects and implications for cancer and inflammatory diseases, Biochim.
Biophys. Acta 1765 (2) (2006) 189–222.
[4] P. Argüeso, S. Spurr-Michaud, A. Tisdale, I.K. Gipson, Variation in the amount of T
antigen and N-acetyllactosamine oligosaccharides in human cervical mucus secretions with the menstrual cycle, J. Clin. Endocrinol. Metab. 87 (12) (2002)
5641–5648, https://doi.org/10.1210/jc.2002-020766.
[5] C. Atuma, V. Strugala, A. Allen, L. Holm, The adherent gastrointestinal mucus gel
layer: thickness and physical state in vivo, Am. J. Physiol. Gastrointest. Liver Physiol. 280 (5) (2001) G922–929.
[6] R. Bansil, B.S. Turner, Mucin structure, aggregation, physiological functions and
biomedical applications, Curr. Opin. Colloid Interface Sci. 11 (2006) 164–170.
[7] R. Bansil, H.E. Stanley, J.T. LaMont, Mucin biophysics, Annu. Rev. Physiol. 57 (1995)
635–657.
[8] R. Bansil, J. Celli, J. Hardcastle, B.S. Turner, The influence of mucus microstructure
and rheology in H. pylori infection, Front. Immunol. 4 (2013) 00310, https://doi.
org/10.3389/fimmu.2013.00310.
[9] S.C. Baos, D.B. Phillips, L. Wildling, T.J. McMaster, M. Berry, Distribution of sialic
acids on mucins and gels: a defense mechanism, Biophys. J. 102 (1) (2012)
176–184, https://doi.org/10.1016/j.bpj.2011.08.058.
[10] S.K. Behera, A.B. Praharaj, B. Dehury, S. Negi, Exploring the role and diversity of mucins in health and disease with special insight into non-communicable diseases,
Glycoconj. J. 32 (8) (2015) 575–613, https://doi.org/10.1007/s10719-015-9606-6.
[11] S.L. Bell, I.A. Khatri, G.Q. Xu, J.F. Forstner, Evidence that a peptide corresponding to
the rat Muc2 C-terminus undergoes disulphide-mediated dimerization, Eur. J.
Biochem. 253 (1) (1998) 123–131.
[12] S.L. Bell, G. Xu, J.F. Forstner, Role of the cystine-knot motif at the C-terminus of rat
mucin protein Muc2 in dimer formation and secretion, Biochem. J. 357 (Pt 1)
(2001) 203–209 (PubMed PMID: 11415450; PubMed Central PMCID:
PMC1221942).
[13] E.P. Bennett, U. Mandel, H. Clausen, T.A. Gerken, T.A. Fritz, L.A. Tabak, Control of
mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase
gene family, Glycobiology 22 (6) (2012) 736–756, https://doi.org/10.1093/glycob/
cwr182.
[14] C. Bevins, E. Martin-Porter, T. Ganz, Defensins and innate host defence of the gastrointestinal tract, Gut 45 (6) (1999) 911–915.
[15] K.R. Bhaskar, D. Gong, R. Bansil, S. Pajevic, J.A. Hamilton, B.S. Turner, J.T. LaMont,
Profound increase in viscosity and aggregation of pig gastric mucin at low pH,
Am. J. Phys. 261 (1991) G827–G832.
[16] A.-E. Biemer-Hüttmann, M.D. Walsh, M.A. McGuckin, Y. Ajioka, H. Watanabe, B.A.
Leggett, J.R. Jass, Immunohistochemical staining patterns of MUC1, MUC2, MUC4,
and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional
adenomas of the colorectum, J. Histochem. Cytochem. 47 (8) (1999) 1039–1048,
https://doi.org/10.1177/002215549904700808.
[17] G.M. Birchenough, M.E. Johansson, J.K. Gustafsson, J.H. Bergström, G.C.
Hansson, New developments in goblet cell mucus secretion and function, Mucosal Immunol. 8 (4) (2015) 712–719, https://doi.org/10.1038/mi.2015.32
(Epub 2015 Apr 15).
[18] L. Böni, P. Fischer, L. Böcker, S. Kuster, P.A. Rühs, Hagfish slime and mucin flow
properties and their implications for defense, Sci Rep 6 (2016) 30371.
[19] H. Boukari, B. Brichacek, P. Stratton, S.F. Mahoney, J.D. Lifson, L. Margolis, R. Nossal,
Movements of HIV-virions in human cervical mucus, Biomacromolecules 10 (9)
(2009) 2482–2488, https://doi.org/10.1021/bm900344q (PubMed PMID:
19711976; PubMed Central PMCID: PMC2768114).
[20] F. Boukari, S. Makrogiannis, R. Nossal, H. Boukari, Imaging and tracking HIV viruses
in human cervical mucus, J. Biomed. Opt. 21 (9) (2016) 96001, https://doi.org/10.
1117/1.JBO.21.9.096001 (PubMed PMID: 27598560; PubMed Central PMCID:
PMC5010625).
[21] I. Brockhausen, Pathways of O-glycan biosynthesis in cancer cells, Biochim.
Biophys. Acta 1473 (1) (1999) 67–95.
[22] I. Brockhausen, Sulphotransferases acting on mucin-type oligosaccharides,
Biochem. Soc. Trans. 31 (2) (2003) 318–325.
[23] I. Brockhausen, P. Stanley, O-GalNAc glycans, in: A. Varki, R.D. Cummings, J.D. Esko,
P. Stanley, G.W. Hart, M. Aebi, A.G. Darvill, T. Kinoshita, N.H. Packer, J.H. Prestegard,
R.L. Schnaar, P.H. Seeberger (Eds.), Essentials of Glycobiology [Internet], 3rd
editionCold Spring Harbor Laboratory Press, Cold Spring Harbor (NY) 2017,
pp. 2015–2017 (Chapter 10).
[24] S. Brugman, The zebrafish as a model to study intestinal inflammation, Dev. Comp.
Immunol. 64 (2016) 82–92, https://doi.org/10.1016/j.dci.2016.02.020.
[25] B. Button, L.H. Cai, C. Ehre, M. Kesimer, D.B. Hill, J.K. Sheehan, R.C. Boucher, M.
Rubinstein, A periciliary brush promotes the lung health by separating the
mucus layer from airway epithelia, Science 337 (6097) (2012) 937–941.
[26] J.J. Calvete, L. Sanz, Analysis of O-glycosylation, Methods Mol. Biol. 194 (2002) 89–100.
[27] J.J. Calvete, L. Sanz, Analysis of O-glycosylation, Methods Mol. Biol. 446 (2008)
281–292.
[28] D.D. Carson, M.M. DeSouza, E.G. Regisford, Mucin and proteoglycan functions in
embryo implantation, BioEssays 20 (7) (1998) 577–583.
[29] B. Casado, L.K. Pannell, P. Iadarola, J. Baraniuk, Identification of human nasal mucous proteins using proteomics, Proteomics 5 (11) (2005) 2949–2959.
[30] J.P. Celli, B.S. Turner, N.H. Afdhal, R.H. Ewoldt, G.H. McKinley, R. Bansil, S. Erramilli,
Rheology of gastric mucin exhibits a pH-dependent sol-gel transition,
Biomacromolecules 8 (2007) 1580–1586.
[31] J.P. Celli, B.S. Turner, N.H. Afdhal, S. Keates, I. Ghiran, C.P. Kelly, R.H. Ewoldt, G.H.
McKinley, P.T. So, S. Erramilli, R. Bansil, Helicobacter pylori moves through mucus
by reducing mucin viscoelasticity, Proc. Natl. Acad. Sci. U. S. A. 106 (34) (2009)
14321–14326, https://doi.org/10.1073/pnas.0903438106 (Epub 2009 Aug 11.
PubMed PMID: 19706518).
[32] M. Chavan, W. Lennarz, The molecular basis of coupling of translocation and N-glycosylation, Trends Biochem. Sci. 31 (1) (2006) 17–20 (Epub 2005 Dec 13. PubMed
PMID: 16356726).
[33] E.Y. Chen, A. Sun, C.-S. Chen, A.J. Mintz, W.-C. Chin, Nicotine alters mucin rheological properties, Am. J. Phys. Lung Cell. Mol. Phys. 307 (2) (2014) L149–L157,
https://doi.org/10.1152/ajplung.00396.2012.
[34] S. Chugh, V.S. Gnanapragassam, M. Jain, S. Rachagani, M.P. Ponnusamy, S.K. Batra,
Pathobiological implications of mucin glycans in cancer: sweet poison and novel
targets, Biochim. Biophys. Acta 1856 (2) (2015) 211–225.
[35] R.J. Coffey, M. Romano, J. Goldenring, Roles for transforming growth factor-alpha in
the stomach, J. Clin. Gastroenterol. 21 (Suppl. 1) (1995) S36–9.
[36] K. Condon, Basic Histology D502; Laboratory 2 Epitheliafrom website http://www.
iupui.edu/~anatd502/Labs.f04/epithelia%20lab/images/s9940x1_jpg.jpg
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
2008http://www.iupui.edu/~anatd502/Labs.f04/epithelia%20lab/Epithelial%20Lab.
html.
R.A. Cone, Barrier properties of mucus, Adv. Drug Deliv. Rev. 61 (2) (2009) 75–85.
A.P. Corfield, Mucins: a biologically relevant glycan barrier in mucosal protection,
Biochim. Biophys. Acta 1850 (1) (2015) 236–252.
J.P. Craig, A. Tomlinson, Importance of the lipid layer in human tear film stability
and evaporation, Optom. Vis. Sci. 74 (1) (1997) 8–13.
J.M. Creeth, Constituents of mucus and their separation, Br. Med. Bull. 34 (1)
(1978) 17–24.
R.S. Crowther, R.L. David, C.M. Hughes, Mucus glycoprotein gels: a scanning electron microscopy study of the effect of cations, Micron Microsc. Acta 15 (1)
(1984) 37–45, https://doi.org/10.1016/0739-6260(84)90029-7.
J.R. Davies, I. Carlstedt, Isolation of large gel-forming mucins, Methods Mol. Biol.
125 (2000) 3–13.
C.W. Davis, B.F. Dickey, Regulated airway goblet cell mucin secretion, Annu. Rev.
Physiol. 70 (2008) 487–512.
G.T. De Sanctis, B.K. Rubin, O. Ramirez, M. King, Ferret tracheal mucus rheology,
clearability and volume following administration of substance P or methacholine,
Eur. Respir. J. 6 (1993) 76–82.
J. Dekker, J.W. Rossen, H.A. Büller, A.W. Einerhand, The MUC family: an obituary,
Trends Biochem. Sci. 27 (3) (2002) 126–131.
N. Demaurex, S. Arnaudeau, M. Opas, Measurement of intracellular Ca2+ concentration, Methods Cell Biol. 70 (2002) 453–474.
J.L. Desseyn, Mucin CYS domains are ancient and highly conserved modules that
evolved in concert, Mol. Phylogenet. Evol. 52 (2) (2009) 284–292.
F.G. Donnan, Theory of ion distribution equilibria in a gel system with variable micelle distribution, Kolloid-Z. 51 (1930) 24–27.
M.A. Doucey, D. Hess, R. Cacan, J. Hofsteenge, Protein C-mannosylation is enzymecatalysed and uses dolichyl-phosphate-mannose as a precursor, Mol. Biol. Cell 9
(2) (1998) 291–300.
M. Dubois, P. Jouannet, P. Berge, G. David, Spermatozoa motility in human cervical
mucus, Nature 252 (5485) (1974) 711–713.
L.M. Ensign, R. Cone, J. Hanes, Oral drug delivery with polymeric nanoparticles: the
gastrointestinal mucus barriers, Adv. Drug Deliv. Rev. 64 (6) (2012) 557–570,
https://doi.org/10.1016/j.addr.2011.12.009 (PubMed PMID: 4474603).
M. Espinosa, G. Noe, C. Troncoso, S.B. Ho, M. Villalón, Acidic pH and increasing
[Ca(2+)] reduce the swelling of mucins in primary cultures of human cervical
cells, Hum. Reprod. 17 (8) (2002) 1964–1972.
C.M. Evans, J.S. Koo, Airway mucus: the good, the bad, the sticky, Pharmacol. Ther.
121 (3) (2009) 332–348.
K.M. Fahrbach, O. Malykhina, D.J. Stieh, T.J. Hope, Differential binding of IgG and
IgA to mucus of the female reproductive tract, PLoS One 8 (10) (Oct 2 2013),
e76176. https://doi.org/10.1371/journal.pone.0076176 (eCollection 2013).
G. Familiari, M. Relucenti, A. Familiari, E. Battaglione, G. Franchitto, R. Heyn, Visualization of the real microarchitecture of glycoprotein matrices with scanning electron microscopy, Modern Res. Educ. Topics Microsc. (2007) 224–228.
H. Fang, L. Qiu, E. Vitkin, M.M. Zaman, C. Andersson, S. Salahuddin, L.M. Kimerer,
P.B. Cipollini, M.D. Modell, B.S. Turner, S. Keates, I. Bigio, I. Itzkan, S.D. Freedman,
R. Bansil, E.B. Hanlon, L.T. Perelman, Confocal light absorption and scattering spectroscopic microscopy, Appl. Opt. 46 (10) (2009) 1760–1769.
A. Fleming, V.D. Allison, Observations on a bacteriolytic substance (“lysozyme”)
found in secretions and tissues on the development of strains of bacteria resistant
to lysozyme action and the relation of lysozyme action to intracellular digestion,
Br. J. Exp. Pathol. 3 (5) (1922) 252–260.
J.G. Forte, Gastric function, in: R. Greger, U. Windhorst (Eds.), Comprehensive
Human Physiology, Vol. 2, Springer Verlag, Berlin, Heidelberg, 1996.
E. Fröhlich, E. Roblegg, Mucus as barrier for drug delivery by nanoparticles, J.
Nanosci. Nanotechnol. 14 (1) (Jan 2014) 126–136.
D.S. Fudge, S. Schorno, S. Ferraro, Physiology, biomechanics and biomimetics of
hagfish slime, Annu. Rev. Biochem. 84 (2015) 947–967, https://doi.org/10.1146/
annurev-biochem-060614-034048 ((Volume publication date June 2015) First
published online as a Review in Advance on December 12, 2014).
P. Georgiadis, P.D. Pudney, D.J. Thornton, T.A. Waigh, Particle tracking
microrheology of purified gastrointestinal mucins, Biopolymers 101 (2014)
366–377.
T.A. Gerken, Biophysical approaches to salivary mucin structure, conformation and
dynamics, Crit. Rev. Oral Biol. Med. 4 (3–4) (1993) 261–270.
T.A. Gerken, O. Jamison, C.L. Perrine, J.C. Collette, H. Moinova, L. Ravi, S.D.
Markowitz, W. Shen, H. Patel, L.A. Tabak, Emerging paradigms for the initiation
of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases, J. Biol. Chem. 286 (16) (2011) 14493–14507, https://
doi.org/10.1074/jbc.M111.218701 (Epub 2011 Feb 24. PubMed PMID: 21349845;
PubMed Central PMCID: PMC3077648).
I.K. Gipson, T. Inatomi, Cellular origin of mucins of the ocular surface tear film, Adv.
Exp. Med. Biol. 438 (1998) 221–227.
S. Girod, J.-M. Zahm, C. Plotkowski, G. Beck, E. Puchelle, Role of the physiochemical
properties of mucus in the protection of the respiratory epithelium, Eur. Respir. J. 5
(4) (1992) 477–487.
K. Godl, M.E.V. Johansson, M.E. Lidell, M. Mörgelin, H. Karlsson, F.J. Olson, J.R. Gum,
Y.S. Kim, G.C. Hansson, The N terminus of the MUC2 mucin forms trimers that are
held together within a trypsin-resistant core fragment, J. Biol. Chem. 277 (49)
(2002) 47248–47256.
D. Gong, B. Turner, K.R. Bhaskar, J.T. LaMont, Lipid binding to gastric mucin: protective effect against oxygen radicals, Am. J. Phys. 259 (1990) G681–G686.
X. Gong, Q. Xu, X. Song, T. Yu, B. Schuster, J.-C. Yang, N.W. Lamb, J. Chang, J. Hanes,
Magnetic mucus-penetrating nanoparticles for drug delivery through human
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
11
cervicovaginal mucus, Nanomed.: Nanotechnol., Biol. Med. 12 (2) (2015) 521,
https://doi.org/10.1016/j.nano.2015.12.210.
A.P. Gunning, A.R. Kirby, C. Fuell, C. Pin, L.E. Tailford, N. Juge, Mining the
“glycocode”—exploring the spatial distribution of glycans in gastrointestinal
mucin using force spectroscopy, FASEB J. 27 (2013) 2342–2354, https://doi.org/
10.1096/fj.12-221416 (published ahead of print March 14, 2013).
R. Gupta, H. Birch, K. Rapacki, S. Brunak, J.E. Hansen, O-GLYCBASE version 4.0: a revised database of O-glycosylated proteins, Nucleic Acids Res. 27 (1) (1999)
370–372.
K. Gwozdzinski, A. Slomiany, H. Nishikawa, K. Okazaki, B.L. Slomiany, Gastric
mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction, Biochem. Int. 17 (5) (1988) 907–917 (PubMed PMID:
2473750).
C.L. Hattrup, S.J. Gendler, Structure and function of the cell surface (tethered) mucins, Annu. Rev. Physiol. 70 (2008) 431–457.
R.R. Hodges, D.A. Dartt, Tear film mucins: front line defenders of the ocular surface;
comparison with airway and gastrointestinal tract mucins, Exp. Eye Res. 117
(2013) 62–78.
Z. Hong, B. Chasan, R. Bansil, B.S. Turner, K.R. Bhaskar, N.H. Afdhal, Atomic force microscopy reveals aggregation of gastric mucin at low pH, Biomacromolecules 6
(2005) 3458–3466.
A. Horsley, K. Rousseau, C. Ridley, W. Flight, A. Jones, T.A. Waigh, D.J. Thornton, Reassessment of the importance of mucins in determining sputum properties in cystic fibrosis, J. Cyst. Fibros. 13 (3) (2014) 260–266, https://doi.org/10.1016/j.jcf.
2013.11.002 (Epub 2013 Dec 12. PubMed PMID: 24332705; PubMed Central
PMCID: PMC3994278).
I. Iontcheva, F.G. Oppenheim, G.D. Offner, R.F. Troxler, Molecular mapping of
statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B)
by the yeast two-hybrid system, J. Dent. Res. 79 (2) (2000) 732–739.
K.T. Izutsu, M.E. Cantino, D.E. Johnson, A review of electron probe X-ray microanalysis studies of salivary gland cells, Microsc. Res. Tech. 27 (1) (1994) 71–79.
I. Jevtov, T. Samuelsson, G. Yao, A. Amsterdam, K. Ribbeck, Zebrafish as a model to
study live mucus physiology, Sci Rep 4 (2014) 6653, https://doi.org/10.1038/
srep06653.
C. Jin, D.T. Kenny, E.C. Skoog, M. Padra, B. Adamczyk, V. Vitizeva, A. Thorell, V.
Venkatakrishnan, S.K. Lindén, N.G. Karlsson, Structural diversity of human gastric
mucin glycans, Mol. Cell. Proteomics 16 (5) (2017) 743–758, https://doi.org/10.
1074/mcp.M116.067983.
M.E. Johansson, M. Phillipson, J. Petersson, A. Velcich, L. Holm, G.C. Hansson, The
inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria, Proc. Natl. Acad. Sci. U. S. A. 105 (39) (2008) 15064–15069, https://doi.org/10.
1073/pnas.0803124105 (Epub 2008 Sep 19).
M.E. Johansson, H. Sjövall, G.C. Hansson, The gastrointestinal mucus system in
health and disease, Nat. Rev. Gastroenterol. Hepatol. 10 (6) (2013) 352–361.
M.E. Johnson, P.J. Murphy, Changes in the tear film and ocular surface from dry eye
syndrome, Prog. Retin. Eye Res. 23 (4) (2004) 449–474.
K. Julenius, NetCGlyc 1.0: prediction of mammalian C-mannosylation sites,
Glycobiology 17 (8) (2007) 868–876.
K. Julenius, A. Mølgaard, R. Gupta, S. Brunak, Prediction, conservation analysis, and
structural characterization of mammalian mucin-type O-glycosylation sites,
Glycobiology 15 (2) (2005) 153–164 (Epub 2004 Sep 22. PubMed PMID:
15385431).
W.M. Junker, S.K. Batra, The Mucin Domain Structures and Their Contributions,
Transworld Research Network, 2007.
D.F. Katz, D.A. Slade, S.T. Nakajima, Analysis of pre-ovulatory changes in cervical
mucus hydration and sperm penetrability, Adv. Contracept. 13 (2–3) (1997)
143–151.
M. Kesimer, A.M. Makhov, J.D. Griffith, P. Verdugo, J.K. Sheehan, Unpacking a gelforming mucin: a view of MUC5B organization after granular release, Am. J. Phys.
Lung Cell. Mol. Phys. 298 (1) (2010) L15–22.
M. Kesimer, C. Ehre, K.A. Burns, K.A. Davis, J.K. Sheehan, R.J. Pickles, Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal
surfaces of the airways, Mucosal Immunol. 6 (2012) 379–392, https://doi.org/10.
1038/mi.2012.81 (published online 29 August 2012).
J.J. Kim, W.I. Khan, Goblet cells and mucins: role in innate defense in enteric infections, Pathogens 2 (1) (2013) 55–70.
M. King, B.K. Rubin, Mucus rheology, relationship with transport, in: Sanae
Shimura, Tamotsu Takishima (Eds.), Airway Secretion: Physiological Bases for
the Control of Mucus Hypersecretion, Series: Lung Biology in Health and Disease,
Vol. 72, Dekker, New York, 1994 (Chapter 7).
J. Kirch, A. Schneider, B. Abou, A. Hopf, U.F. Schaefer, M. Schneider, C. Schall, C.
Wagner, C.M. Lehr, Optical tweezers reveal relationship between microstructure
and nanoparticle penetration of pulmonary mucus, Proc. Natl. Acad. Sci. U. S. A.
109 (45) (2012) 18355–18360, https://doi.org/10.1073/pnas.1214066109
(Epub 2012 Oct 22. PubMed PMID: 23091027; PubMed Central PMCID:
PMC3494950).
J. Kočevar-Nared, J. Kristl, J. Šmid-Korbar, Comparative rheological investigation of
crude gastric mucin and natural gastric mucus, Biomaterials 18 (9) (1997)
677–681.
R. Kuver, J.H. Klinkspoor, W.R. Osborne, S.P. Lee, Mucous granule exocytosis
and CFTR expression in gallbladder epithelium, Glycobiology 10 (2) (2000)
149–157.
G. Lafitte, O. Söderman, K. Thuresson, J. Davies, PFG-NMR diffusometry: a tool for
investigating the structure and dynamics of noncommercial purified pig gastric
mucin in a wide range of concentrations, Biopolymers 86 (2007) 165–175,
https://doi.org/10.1002/bip.20717.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
12
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
[95] S.K. Lai, Y.Y. Wang, J. Hanes, Micro- and macrorheology of mucus, Adv. Drug Deliv.
Rev. 61 (2) (2009) 86–100.
[96] S.K. Lai, Y.-Y. Wang, J. Hanes, Mucus-penetrating nanoparticles for drug and gene
delivery to mucosal tissues, Adv. Drug Deliv. Rev. 61 (2) (2009) 158–171,
https://doi.org/10.1016/j.addr.2008.11.002.
[97] T. Lang, S. Klasson, E. Larsson, M.E. Johansson, G.C. Hansson, T. Samuelsson,
Searching the evolutionary origin of epithelial mucus protein components-mucins
and FCGBP, Mol. Biol. Evol. 33 (8) (2016) 1921–1936.
[98] J.M. Larsson, H. Karlsson, H. Sjovall, G.C. Hansson, A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MS,
Glycobiology 19 (7) (2009) 756–766.
[99] S.K. Lau, L.M. Weiss, P.G. Chu, Differential expression of MUC1, MUC2, and
MUC5AC in carcinomas of various sites: an immunohistochemical study, Am. J.
Clin. Pathol. 122 (1) (2004) 61–69.
[100] D. Lazar, S. Taban, S. Ursoniu, Immunohistochemical profile of mucins in gastric
carcinoma, in: Prof. Mahmoud Lotfy (Ed.), Carcinoma — Molecular Aspects and
Current Advances, InTech Publisher June, 2011, p. 201 (Chapter 11).
[101] Y. Li, T. Tanaka, Phase transitions of gels, Annu. Rev. Mater. Sci. 22 (1) (1992)
243–277.
[102] L.M. Lichtenberger, The hydrophobic barrier properties of gastrointestinal mucus,
Annu. Rev. Physiol. 57 (1995) 565–583.
[103] O. Lieleg, I. Vladescu, K. Ribbeck, Characterization of particle translocation through
mucin hydrogels, Biophys. J. 98 (2010) 1782–1789.
[104] S.K. Linden, P. Suttonet, N.G. Karlsson, V. Korolik, M.A. McGuckin, Mucins in the
mucosal barrier to infection, Mucosal Immunol. 1 (2008) 183–197, https://doi.
org/10.1038/mi.2008.5 (published online 5 March 2008).
[105] Y.R. Mahida, R.N. Cunliffe, Defensins and mucosal protection, Novartis Found.
Symp. 263 (2004) 71–77 (discussion 77-84, 211-8).
[106] G.M. Makhlouf, J.P. McManus, W.I. Card, A quantitative statement of the twocomponent hypothesis of gastric secretion, Gastroenterology 51 (2) (1966)
149–171.
[107] L.E. Martinez, J.M. Hardcastle, J. Wang, Z. Pincus, J. Tsang, T. Hoover, R. Bansil, N.R.
Salama, Helicobacter pylori strains vary cell shape and flagellum number to maintain robust motility in viscous environments, Mol. Microbiol. 99 (2016) 88.
[108] T.N. Mayadas, D.D. Wagner, Vicinal cysteines in the prosequence play a role in von
Willebrand factor multimer assembly, Proc. Natl. Acad. Sci. U. S. A. 89 (8) (1992)
3531–3535.
[109] T.J. McMaster, M. Berry, A.P. Corfield, M.J. Miles, Atomic force microscopy of the
submolecular architecture of hydrated ocular mucins, Biophys. J. 77 (1) (1999)
533–541.
[110] M. Menárguez, L.M. Pastor, E. Odeblad, Morphological characterization of different
human cervical mucus types using light and scanning electron microscopy, Hum.
Reprod. 18 (9) (2003) 1782–1789, https://doi.org/10.1093/humrep/deg382.
[111] J.F. Ménétret, R.S. Hegde, S.U. Heinrich, P. Chandramouli, S.J. Ludtke, T.A. Rapoport,
C.W. Akey, Architecture of the ribosome-channel complex derived from native
membranes, J. Mol. Biol. 348 (2) (2005) 445–457.
[112] R.R. Mercer, M.L. Russell, J.D. Crapo, Mucous lining layers in human and rat airways,
Am. Rev. Respir. Dis. 145 (1992) 355.
[113] P.B. Molinoff, A.J. Atkinson, Peptic Ulcer Disease: Mechanisms & Management,
HealthPress, Rutherford, N.J, 1990.
[114] Moran, Rowley, website: http://visualhistology.com/store/default/classic-histology-power-point-images/powerpoint2-epithelial-tissue-individual-cd.html 2009
http://www.visualhistology.com/products/atlas/VHA_Chpt1_Cells.html Link for
goblet cell TEM http://www.visualhistology.com/products/atlas/VisualHistology_
Atlas_2-0-23_1.jpg.
[115] M.R. Neutra, J.F. Forstner, Gastrointestinal mucus: synthesis, secretion, and function, in: L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Raven Press,
New York 1987, p. 975.
[116] T.M. Newman, A. Robichaud, D.F. Rogers, Microanatomy of secretory granule release from guinea pig tracheal goblet cells, Am. J. Respir. Cell Mol. Biol. 15 (4)
(1996) 529–539.
[117] T. Nguyen, W.C. Chin, P. Verdugo, Role of Ca2+/K+ ion exchange in intracellular
storage and release of Ca2+, Nature 395 (6705) (1998) 908–912.
[118] A.V. Nikonov, G. Kreibich, Organization of translocon complexes in ER membranes,
Biochem. Soc. Trans. 31 (6) (2003) 1253–1256 (Review).
[119] S. Nunn, R.S. Gilmore, J.A. Dodge, K.E. Carr, Exudate variation in the rabbit gastrointestinal tract: a scanning electron microscope study, J. Anat. 170 (1990) 87.
[120] K.J. Palmer, P. Watson, D.J. Stephens, The role of microtubules in transport between
the endoplasmic reticulum and Golgi apparatus in mammalian cells, Biochem. Soc.
Symp. 72 (2005) 1–13.
[121] T. Pelaseyed, J.H. Bergström, J.K. Gustafsson, A. Ermund, G.M. Birchenough, A.
Schütte, S. van der Post, F. Svensson, A.M. Rodríguez-Piñeiro, E.E. Nyström, C.
Wising, M.E. Johansson, G.C. Hansson, The mucus and mucins of the goblet cells
and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system, Immunol. Rev. 260 (1) (2014) 8–20.
[122] J. Perez-Vilar, R.L. Hill, Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the
CGLCG motif in the D1- and D3-domains, J. Biol. Chem. 273 (51) (1998)
34527–34534 (PubMed PMID: 9852122).
[123] J. Perez-Vilar, R.L. Hill, The structure and assembly of secreted mucins, J. Biol. Chem.
274 (45) (1999) 31751–31754.
[124] J. Perez-Vilar, A.E. Eckhardt, A. DeLuca, R.L. Hill, Porcine submaxillary mucin forms
disulfide-linked multimers through its amino-terminal D-domains, J. Biol. Chem.
273 (23) (1998) 14442–14449 (PubMed PMID: 9603957).
[125] J. Perez-Vilar, S.H. Randell, R.C. Boucher, C-mannosylation of MUC5AC and MUC5B
Cys subdomains, Glycobiology 14 (4) (2004) 325–337.
[126] J. Perez-Vilar, J.C. Olsen, M. Chua, R.C. Boucher, pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells, J. Biol. Chem. 280 (17)
(2005) 16868–16881.
[127] A. Popov, E. Enlow, J. Bourassa, H. Chen, Mucus-penetrating nanoparticles made
with “mucoadhesive” poly(vinyl alcohol), Nanomed.: Nanotechnol., Biol. Med. 12
(7) (2016) 1863–1871, https://doi.org/10.1016/j.nano.2016.04.006.
[128] J.C. Probst, E.M. Gertzen, W. Hoffmann, An integumentary mucin (FIM-B.1) from
Xenopus laevis homologous with von Willebrand factor, Biochemistry 29 (26)
(1990) 6240–6244.
[129] E.A. Rakha, R.W.G. Boyce, D. Abd El-Rehim, T. Kurien, A.R. Green, E.C. Paish, J.F.
Robertson, I.O. Ellis, Expression of mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC
and MUC6) and their prognostic significance in human breast cancer, Mod. Pathol.
18 (2005) 1295–1304, https://doi.org/10.1038/modpathol.3800445 (published
online 24 June 2005).
[130] K.A. Ramsey, Z.L. Rushton, C. Ehre, Mucin agarose gel electrophoresis: western
blotting for high-molecular-weight glycoproteins, J. Vis. Exp. 14 (112)
(2016)https://doi.org/10.3791/54153.
[131] C. Robbe, C. Capon, C. Flahaut, J.C. Michalski, Microscale analysis of mucin-type Oglycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and
mass spectrometry approach, Electrophoresis 24 (4) (2003) 611–621.
[132] C. Robbe, J.C. Michalski, C. Capon, Structural determination of O-glycans by tandem
mass spectrometry, Methods Mol. Biol. 347 (2006) 109–123.
[133] D.F. Rogers, Physiology of airway mucus secretion and pathophysiology of hypersecretion, Respir. Care 52 (9) (2007) 1134–1146 (discussion 1146-1139).
[134] M.C. Rose, J.A. Voynow, Respiratory tract mucin genes and mucin glycoproteins in
health and disease, Physiol. Rev. 86 (1) (2006) 245–278.
[135] Y. Rossez, E. Maes, D.T. Lefebvre, P. Gosset, C. Ecobichon, J. Chevalier, M. Curt, I.G.
Boneca, J.C. Michalski, C. Robbe-Masselot, Almost all human gastric mucin O-glycans harbor blood group A, B or H antigens and are potential binding sites for
Helicobacter pylori, Glycobiology 22 (9) (2012) 1193–1206, https://doi.org/10.
1093/glycob/cws072 (Epub 2012 Apr 21).
[136] J. Roth, C. Zuber, B. Guhl, J.-y. Fan, M. Ziak, The importance of trimming reactions on
asparagine-linked oligosaccharides for protein quality control, Histochem. Cell Biol.
117 (2) (2002) 159–169.
[137] P. Roussel, P. Delmotte, The diversity of epithelial secreted mucins, Curr. Org.
Chem. 8 (5) (2004) 413–437.
[138] L. Royle, A. Roos, D.J. Harvey, M.R. Wormald, D. van Gijlswijk-Janssen, el-R.M.
Redwan, I.A. Wilson, M.R. Daha, R.A. Dwek, P.M. Rudd, Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems, J. Biol.
Chem. 278 (22) (2003) 20140–20153 (Epub 2003 Mar 10).
[139] B.E. Sands, D.K. Podolsky, The trefoil peptide family, Annu. Rev. Physiol. 58 (1996)
253–273.
[140] C.S. Schneider, Q. Xu, N.J. Boylan, J. Chisholm, B.C. Tang, B.S. Schuster, A. Henning,
L.M. Ensign, E. Lee, P. Adstamongkonkul, B.W. Simons, S.-Y.S. Wang, X. Gong, T.
Yu, M.P. Boyle, Jung Soo Suk, J. Hanes, Nanoparticles that do not adhere to mucus
provide uniform and long-lasting drug delivery to airways following inhalation,
Sci. Adv. (05 Apr 2017), E1601556. https://doi.org/10.1126/sciadv.1601556.
[141] J.C. Seagrave, H. Albrecht, Y.S. Park, B. Rubin, G. Solomon, K.C. Kim, Effect of
guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells, Exp. Lung Res. 37 (2011) 606–614, https://
doi.org/10.3109/01902148.2011.623116.
[142] M.H. Sedaghat, M.M. Shahmardan, M. Norouzi, M. Nazari, P.G. Jayathilake, On the
effect of mucus rheology on the muco-ciliary transport, Math. Biosci. 272 (2016)
44–53.
[143] J.L. Shaw, C.R. Smith, E.P. Diamindis, Proteomic analysis of human cervico-vaginal
fluid, J. Proteome Res. 6 (7) (2007) 2859–2865.
[144] J.K. Sheehan, K. Oates, I. Carlstedt, Electron microscopy of cervical, gastric and
bronchial mucus glycoproteins, Biochem. J. 239 (1) (1986) 147–153.
[145] R. Shogren, T.A. Gerken, N. Jentoft, Role of glycosylation on the conformation and
chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin, Biochemistry 28 (13) (1989) 5525–5536.
[146] A. Silberberg, On mucociliary transport, Biorheology 27 (3–4) (1990) 295–307.
[147] M.P. Simula, R. Cannizzaro, M.D. Marin, A. Pavan, G. Toffoli, V. Canzonieri, V. De Re,
Two-dimensional gel proteome reference map of human small intestine, Proteome
Sci. 7 (10) (2009)https://doi.org/10.1186/1477-5956-7-10.
[148] J.D. Smart, The basics and underlying mechanisms of mucoadhesion, Adv. Drug
Deliv. Rev. 57 (2005) 1556–1568, https://doi.org/10.1016/j.addr.2005.07.001.
[149] D. Snary, A. Allen, R.H. Pain, The structure of pig gastric mucus. Conformational
transitions induced by salt, Eur. J. Biochem. 24 (1) (1971) 183–189.
[150] P. Stanley, N. Taniguchi, M. Aebi, N-Glycans, in: A. Varki, R.D. Cummings, J.D. Esko,
P. Stanley, G.W. Hart, M. Aebi, A.G. Darvill, T. Kinoshita, N.H. Packer, J.H. Prestegard,
R.L. Schnaar, P.H. Seeberger (Eds.), Essentials of Glycobiology [Internet], 3rd
editionCold Spring Harbor Laboratory Press, Cold Spring Harbor (NY), 2017
(2015-2017. Chapter 9).
[151] S. Subramanian, N.W. Ross, S.L. MacKinnon, Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine
glutinosa L.), Fish Shellfish Immunol. 25 (2008) 625–632, https://doi.org/10.
1016/j.fsi.2008.08.012.
[152] P.Y. Tam, P. Verdugo, Control of mucus hydration as a Donnan equilibrium process,
Nature 292 (5821) (1981) 340–342.
[153] T. Tanaka, Collapse of gels and the critical endpoint, Phys. Rev. Lett. 40 (12) (1978)
820, https://doi.org/10.1103/PhysRevLett.40.820 (https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.40.820).
[154] L.J. Tang, F. De Seta, F. Odreman, P. Venge, C. Piva, S. Guaschino, R.C. Garcia, Proteomic analysis of human cervical-vaginal fluids, J. Proteome Res. 6 (7) (2007)
2874–2883 (Epub 2007 Jun 1).
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023
R. Bansil, B.S. Turner / Advanced Drug Delivery Reviews xxx (2017) xxx–xxx
[155] C. Taylor, A. Allen, P.W. Dettmar, J.P. Pearson, Two rheologically different gastric
mucus secretions with different putative functions, Biochim. Biophys. Acta 1674
(2) (2004) 131–138.
[156] K.G. Ten Hagen, T.A. Fritz, L.A. Tabak, All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, Glycobiology 13 (1) (2003) 1R–16R.
[157] L. Thim, F. Madsen, S.S. Poulsen, Effect of trefoil factors on the viscoelastic properties of mucus gels, Eur. J. Clin. Investig. 32 (7) (2002) 519–527.
[158] D.J. Thornton, K. Rousseau, M.A. McGuckin, Structure and function of the polymeric
mucins in airways mucus, Annu. Rev. Physiol. 70 (2008) 459–486.
[159] E. Tian, K.G. Ten Hagen, Recent insights into the biological roles of mucin-type Oglycosylation, Glycoconj. J. 26 (3) (2009) 325–334, https://doi.org/10.1007/
s10719-008-9162-4 (Epub 2008 Aug 10. Review).
[160] D.T. Tran, K.G. Ten Hagen, Mucin-type O-glycosylation during development, J. Biol.
Chem. 288 (10) (2013) 6921–6929.
[161] I. Urban, L.B. Santurio, A. Chidichimo, H. Yu, X. Chen, J. Mucci, F. Agüero, C.A.
Buscaglia, Molecular diversity of the Trypanosoma cruzi TcSMUG family of mucin
genes and proteins, Biochem. J. 438 (2) (2011) 303–313, https://doi.org/10.
1042/BJ20110683.
[162] E.S. Vasquez, J. Bowser, C. Swiderski, K.B. Walters, S. Kundu, Rheological characterization of mammalian lung mucus, RSC Adv. 4 (2014) 34780–34783, https://doi.
org/10.1039/C4RA05055J.
[163] A. Vassilakos, M. Michalak, M.A. Lehrman, D.B. Williams, Oligosaccharide binding
characteristics of the molecular chaperones calnexin and calreticulin, Biochemistry
37 (10) (1998) 3480–3490.
[164] P. Verdugo, Goblet cells secretion and mucogenesis, Annu. Rev. Physiol. 52 (1990)
157–176.
[165] P. Verdugo, Mucin exocytosis, Am. Rev. Respir. Dis. 144 (3 Pt 2) (1991) S33–37.
[166] P. Verdugo, Supramolecular dynamics of mucus, Cold Spring Harb. Perspect. Med. 2
(11) (2012) a009597, https://doi.org/10.1101/cshperspect.a009597.
[167] P. Verdugo, M. Aitken, L. Langley, M.J. Villalon, Molecular mechanism of product
storage and release in mucin secretion. II. The role of extracellular Ca++,
Biorheology 24 (6) (1987) 625–633.
[168] P. Verdugo, I. Deyrup-Olsen, M. Villalon, D. Johnson, Molecular mechanism of
mucin secretion: I. The role of intragranular charge shielding, J. Dent. Res. 66 (2)
(1987) 506–508.
[169] L.E. Vinall, A.S. Hill, P. Pigny, W.S. Pratt, N. Toribara, J.R. Gum, Y.S. Kim, N. Porchet, J.P. Aubert, D.M. Swallow, Variable number tandem repeat polymorphism of the
mucin genes located in the complex on 11p15.5, Hum. Genet. 102 (3) (1998)
357–366.
[170] H.H. Wandall, F. Irazoqui, M.A. Tarp, E.P. Bennett, U. Mandel, H. Takeuchi, K. Kato, T.
Irimura, G. Suryanarayanan, M.A. Hollingsworth, H. Clausen, The lectin domains of
polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for
GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation, Glycobiology 17 (4) (2007) 374–387, https://doi.org/
10.1093/glycob/cwl082.
13
[171] S. Wei, X. Zhu, M. Liu, L. Li, J. Zhong, W. Sun, Z. Zhang, Y. Huang, Overcoming the
diffusion barrier of mucus and absorption barrier of epithelium by self-assembled
nanoparticles for oral delivery of insulin, ACS Nano 9 (3) (2015) 2345–2356,
https://doi.org/10.1021/acsnano.5b00028ISSN 0025-5564 (http://dx.doi.org/
10.1016/j.mbs.2015.11.010).
[172] J.G. Widdicombe, Role of lipids in airway function, Eur. J. Respir. Dis. Suppl. 153
(1987) 197–204.
[173] O.W. Williams, A. Sharafkhaneh, V. Kim, B.F. Dickey, C.M. Evans, Airway mucus:
from production to secretion, Am. J. Respir. Cell Mol. Biol. 34 (5) (2006) 527–536.
[174] N.A. Wright, Interaction of trefoil family factors with mucins: clues to their mechanism of action? Gut 48 (3) (2001) 293–294.
[175] M.M. Wu, M. Grabe, S. Adams, R.Y. Tsien, H.-P.H. Moore, T.E. Machen, Mechanisms
of pH regulation in the regulated secretory pathway, J. Biol. Chem. 276 (35) (2001)
33027–33035.
[176] G.E. Yakubov, A. Papagiannopoulos, E. Rat, R.L. Easton, T.A. Waigh, Molecular structure and rheological properties of short-side-chain heavily glycosylated porcine
stomach mucin, Biomacromolecules 8 (11) (2007) 3467–3477.
[177] K. Yamashita, S. Hara-Kuge, T. Ohkura, Intracellular lectins associated with Nlinked glycoprotein traffic, Biochim. Biophys. Acta 1473 (1) (1999) 147–160
(Review).
[178] A. Yan, W.J. Lennarz, Unraveling the mechanism of protein N-glycosylation, J. Biol.
Chem. 280 (5) (2005) 3121–3124 (Epub 2004 Dec 7. Review. PubMed PMID:
15590627).
[179] H.M. Yildiz, C.A. McKelvey, P.J. Marsac, R.L. Carrier, Size selectivity of intestinal
mucus to diffusing particulates is dependent on surface chemistry and exposure
to lipids, J. Drug Target. 23 (7–8) (2015) 768–774, https://doi.org/10.3109/
1061186X.2015.1086359 (PubMed PMID: 26453172; PubMed Central PMCID:
PMC4874559).
[180] T. Yu, G.P. Andrews, D.S. Jones, Mucoadhesion and characterization of
mucoadhesive properties, in: J. das Neves, B. Sarmento (Eds.), Mucosal Delivery
of Biopharmaceuticals, © Springer Science + Business Media, New York,
2014https://doi.org/10.1007/978-1-4614-9524-6_2.
[181] Y.F. Zhou, T.A. Springer, Highly reinforced structure of a C-terminal dimerization
domain in von Willebrand factor, Blood 123 (12) (2014) 1785–1793, https://doi.
org/10.1182/blood-2013-11-523639 (Epub 2014 Jan 6. PubMed PMID:
24394662; PubMedCentral PMCID: PMC3962159).
[182] I. Ziment, Historic overview of mucoactive drugs, in: P.C. Braga, L. Alegra (Eds.),
Drugs in Bronchial Mucology, Raven Press, New York, NY 1989, pp. 1–33.
[183] E. Zmuda-Trzebiatowska, PPS'97 Project, Glycosylation, http://www.cryst.bbk.ac.
uk/pps97/assignments/projects/emilia/N-glycosylation.HTM 1997 N-Linked Oligosaccharides (picture) http://www.cryst.bbk.ac.uk/pps97/assignments/projects/
emilia/typ.GIF.
Please cite this article as: R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv. Drug Deliv. Rev. (2017),
https://doi.org/10.1016/j.addr.2017.09.023