o-galnac glycans - essentials of glycobiology 2nd...

Chapter9–O-GalNAcGlycans-EssentialsofGlycobiology2ndeditionIntestinalmucosalbarrierfunctioninhealthanddisease.Turner,JRNatureRev.Immunology9:799(2009)FromMucinstoMucusThorntonDJandSheehanJK,ProcAmThoracSoc.,1:54(2004)Mucinstructure,aggregation,physiologicalfunctionsandbiomedicalapplicationsBansilR.andTurnerBSCurrOpininColloid&InterfaceSci,11:164(2006)MucinsinthemucosalbarriertoinfectionLindenetal,NatureImmunology1:183(2008)Thecancerglycocalyxmechanicallyprimesintegrin-mediatedgrowthandsurvival.Paszeketal,Nature511:319(2014)

Chapter9–O-GalNAcGlycans

EssentialsofGlycobiology2ndedition

4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf

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NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring

Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

Chapter 9O-GalNAc Glycans

Inka Brockhausen, Harry Schachter, and Pamela Stanley.

Correction

O-glycosylation is a common covalent modification of serine and threonine residues of mammalian

glycoproteins. This chapter describes the structures, biosynthesis, and functions of glycoproteins that are

often termed mucins. In mucins, O-glycans are covalently α-linked via an N-acetylgalactosamine

(GalNAc) moiety to the -OH of serine or threonine by an O-glycosidic bond, and the structures are named

mucin O-glycans or O-GalNAc glycans. The focus of this chapter is on mucins and mucin-like

glycoproteins that are heavily O-glycosylated, although glycoproteins that carry only one or a few O-

GalNAc glycans are also briefly discussed. There are also several types of nonmucin O-glycans, including

α-linked O-fucose, β-linked O-xylose, α-linked O-mannose, β-linked O-GlcNAc (N-acetylglucosamine),

α- or β-linked O-galactose, and α- or β-linked O-glucose glycans (discussed in Chapters 12 and 16–18).

In this chapter, however, the term O-glycan refers to mucin O-glycans, unless otherwise specified. Mucin

glycoproteins are ubiquitous in mucous secretions on cell surfaces and in body fluids.

DISCOVERY AND BACKGROUND

In 1865, E. Eichwald, a Russian physician working in Germany, provided the first chemical evidence that

mucins are proteins conjugated to carbohydrate. He also showed that mucins are widely distributed in the

animal body. In 1877, F. Hoppe-Seyler discovered that mucins produced by the epithelial cells of mucous

membranes and salivary glands contain an acidic carbohydrate, identified many years later to be sialic

acid. Mucins are present at many epithelial surfaces of the body, including the gastrointestinal,

genitourinary, and respiratory tracts, where they shield the epithelial surfaces against physical and

chemical damage and protect against infection by pathogens. Mucin O-glycans begin with an α-linked N-

acetylgalactosamine residue linked to serine or threonine. The N-acetylgalactosamine may be extended

with sugars including galactose, N-acetylglucosamine, fucose, or sialic acid, but not mannose, glucose, or

xylose residues. There are four common O-GalNAc glycan core structures, designated cores 1 through 4

and an additional four designated cores 5 though 8 (Table 9.1). Mucin O-glycans can be branched, and

many sugars or groups of sugars on mucin O-glycans are antigenic (Table 9.1). Important modifications

of mucin O-glycans include O-acetylation of sialic acid and O-sulfation of galactose and N-

acetylglucosamine. Thus, mucin O-glycans are often very heterogeneous, with hundreds of different

chains being present in some mucins. Numerous chemical, enzymatic, and spectroscopic methods are

applied to analyze the sugar composition and the linkages among sugars of mucin O-glycans.

MUCIN GLYCOPROTEINS

Mucins are heavily O-glycosylated glycoproteins found in mucous secretions and as transmembrane

glycoproteins of the cell surface with the glycan exposed to the external environment. The mucins in

mucous secretions can be large and polymeric (gel-forming mucins) or smaller and monomeric (soluble



mucins). Many epithelial cells produce mucin, but gel-forming mucins are produced primarily in the

goblet or mucous cells of the tracheobronchial, gastrointestinal, and genitourinary tracts. In goblet cells,

mucins are stored intracellularly in mucin granules from which they can be quickly secreted upon

external stimuli.

The hallmark of mucins is the presence of repeated peptide stretches called “variable number of tandem

repeat” (VNTR) regions that are rich in serine or threonine O-glycan acceptor sites and have an

abundance of clustered mucin O-glycans that may comprise 80% of the molecule by weight. The tandem

repeats are usually rich in proline residues that appear to facilitate O-GalNAc glycosylation. Mucins may

have hundreds of O-GalNAc glycans attached to serine or threonine residues in the VNTR regions. The

clustering of O-GalNAc glycans causes mucin glycoproteins to adopt an extended “bottle brush”

conformation (Figure 9.1).

About 20 different mucin genes have been cloned, and they are expressed in a tissue-specific fashion. For

example, different mucin genes are expressed in different regions of the gastrointestinal tract, suggesting

that they serve specific functions. The first mucin gene to be cloned encoded a transmembrane mucin

termed MUC1. Mucins that span the plasma membrane are known to be involved in signal transduction,

to mediate cell–cell adhesion, or to have an antiadhesive function. Cell-surface mucins contain an

extracellular domain with a central VNTR region that carries O-GalNAc glycan chains, a single

transmembrane domain, and a small cytoplasmic tail at the carboxyl terminus.

The mucin genes, their transcripts, the resulting mucin proteins, and the attached O-GalNAc glycans all

exhibit extreme variability. Different mucins vary in the number and composition of the peptide repeats in

their VNTR regions. Within the same mucin, the repeats usually vary in their amino acid sequences. It

has not yet been possible to determine exactly which of the many different O-GalNAc glycan structures

isolated from a purified mucin preparation are attached to specific serine or threonine residues in the

tandem repeats. However, in some less densely glycosylated glycoproteins, specific O-GalNAc

glycosylation sites have been identified. The structures of O-glycans at these defined sites are usually

heterogeneous and any mucin includes a range of glycoforms. One of the exceptions is the Antarctic fish

antifreeze glycoprotein, which carries only Galβ1-3GalNAc- at threonine residues of alanine-alanine-

threonine repeat sequences.

Some mucins contain a hydrophobic transmembrane domain that serves to insert the molecule into the

cell membrane. Secreted mucins have cysteine-rich regions and cystine knots that are responsible for their

polymerization and the formation of extremely large molecules of several million daltons. Mucins may

also have protein domains similar to motifs present in other glycoproteins. For example, D domains found

in mucins are also found in von Willebrand factor, a glycoprotein important in blood clotting. The D

domain may regulate mucin polymerization (Figure 9.1).

Mucin-like glycoproteins such as GlyCAM-1, CD34, and PSGL-1 are less densely O-glycosylated

membrane-associated glycoproteins that mediate cell–cell adhesion. Most glycoproteins with O-GalNAc

glycans carry several O-glycans, although some, such as interleukin-2 and erythropoietin, carry a single

O-GalNAc glycan. The expression of mucin genes is regulated by a large number of cytokines and

growth factors, differentiation factors, and bacterial products.

The purification of gel-forming mucins is achieved by density-gradient centrifugation. Purified mucins

have distinct viscoelastic properties that contribute to the high viscosity of mucous secretions. They are

hydrophilic and contain charges that attract water and salts. Bacteria, viruses, and other microbes are

trapped by mucins and sometimes adhere to specific O-GalNAc glycans that serve as receptors (see

Tables 34.1 and 34.2). The ciliary action of the mucous membrane epithelium aids in the removal of

microbes and small particles.



Mucins hydrate and protect the underlying epithelial cells, but they have also been shown to have roles in

fertilization, blastocyst implantation, and the immune response. Cell-surface MUC1 and mucin-like

glycoproteins have roles in cell adhesion (see Chapter 31). A number of diseases are associated with

abnormal mucin gene expression and abnormal mucin carbohydrate structures and properties. These

include cancer, inflammatory bowel disease, lung disease, and cystic fibrosis. The expression of

underglycosylated MUC1 is often increased in individuals with cancer (see Chapters 43 and 44).

O-GalNAc GLYCAN STRUCTURES

The simplest mucin O-glycan is a single N-acetylgalactosamine residue linked to serine or threonine.

Named the Tn antigen, this glycan is often antigenic. The most common O-GalNAc glycan is Galβ1-

3GalNAc-, and it is found in many glycoproteins and mucins (Table 9.1, Figure 9.2). It is termed a core 1

O-GalNAc glycan because it forms the core of many longer, more complex structures. It is antigenic and

is also named the T antigen. Both Tn and T antigens may be modified by sialic acid to form sialylated-Tn

or -T antigens, respectively.

Another common core structure contains a branching N-acetylglucosamine attached to core 1 and is

termed core 2 (Table 9.1, Figure 9.2). Core 2 O-GalNAc glycans are found in both glycoproteins and

mucins from a variety of cells and tissues. Linear core 3 and branched core 4 O-GalNAc glycans (Table

9.1, Figure 9.2) have been found only in secreted mucins of certain mucin-secreting tissues, such as

bronchi, colon, and salivary glands. Core structures 5–8 have an extremely restricted occurrence. Mucins

with core 5 have been reported in human meconium and intestinal adenocarcinoma tissue, whereas core 6

structures are found in human intestinal mucin and ovarian cyst mucin. Core 8 has been reported in

human respiratory mucin. Bovine submaxillary mucin was shown to contain O-GalNAc glycans with

relatively short chains, including those containing a core 7 structure (Table 9.1).

All of the core structures can be sialylated. However, only cores 1–4 and core 6 have been shown to occur

as extended, complex O-glycans that carry antigens such as the ABO and Lewis blood group

determinants, the linear i antigen (Galβ1-4GlcNAcβ1-3Gal-), and the GlcNAcβ1-6 branched I antigens

(Table 9.1). Both type 1 (based on the Galβ1-3GlcNAc sequence) and type 2 (based on the Galβ1-

4GlcNAc sequence) extensions can be repeated several times in poly-N-acetyllactosamine units, which

provide a scaffold for the attachment of additional sugars or functional groups. The terminal structures of

O-GalNAc glycans may contain fucose, galactose, N-acetylglucosamine, and sialic acid in α-linkages, N-acetylgalac-tosamine in both α- and β-linkages, and sulfate. Many of these terminal sugar structures are

antigenic or represent recognition sites for lectins. In particular, the sialylated and sulfated Lewis antigens

are ligands for selectins (see Chapter 31). Poly-N-acetyllactosamine units and terminal structures may

also be found on N-glycans and glycolipids (see Chapter 13).

ISOLATION, PURIFICATION, AND ANALYSIS OF MUCIN O-

GLYCANS

All O-GalNAc glycans may be released from the peptide by an alkali treatment called β-elimination. The

N-acetylgalactosamine residue attached to serine or threonine is reduced to N-acetylgalactosaminitol by

borohydride as it is released. Conversion to N-acetylgalactosaminitol prevents the rapid alkali-catalyzed

degradation of the released O-GalNAc glycan by a “peeling” reaction. Glycoproteins that contain both N-

and O-glycans will retain their N-glycans and specifically lose O-glycans when subjected to β-

elimination. However, harsh alkali treatment will also disrupt peptide bonds and degrade the mucin.

Unsubstituted N-acetylgalactosamine residues may also be released from serine or threonine by a specific

N-acetylgalactosaminidase, and unsubstituted Galβ1-3GalNAc (core 1) can be released with an enzyme



termed O-glycanase. For core 1 O-GalNAc glycans substituted with sialic acids, the sialic acid must first

be removed by treatment with sialidase or mild acid before O-glycanase treatment will be successful. No

enzymes are known to release larger O-glycans from the peptide, in contrast to the availability of several

enzymes that release N-glycans from asparagine (see Chapter 8).

Released O-GalNAc glycans may be separated into different species by chromatographic methods such as

gel filtration, anion-exchange chromatography, and high-pressure liquid chromatography. Chemical

derivatization of purified reduced O-glycans helps in the analysis of sugar composition and the

determination of linkages between sugars by gas chromatography and mass spectrometry (see Chapter

47). With improvements in the sensitivity of mass spectrometry, it is now possible to analyze small

amounts and identify structures on the basis of their mass in complex mixtures. Structural analysis by

nuclear magnetic resonance (NMR) spectroscopy is more precise, but requires more material and highly

purified compounds. The NMR spectra of hundreds of O-glycans have been published and are used as

references to identify known and new O-glycan structures. Other tools to analyze the structures of O-

GalNAc glycans include specific exoglycosidases that remove terminal sugars, antibodies to N-acetylgalactosamine (anti-Tn) and core 1 (anti-T), and lectins such as peanut agglutinin (which binds to

core 1) and Helix pomatia agglutinin (which binds to terminal N-acetylgalactosamine) (see Chapter 45).

O-GalNAc glycans in a specific cell line or tissue may be predicted based on the presence of active

glycosyltransferases required for their synthesis.

BIOSYNTHESIS OF O-GalNAc GLYCANS

Polypeptide-N-Acetylgalactosaminyltransferases

The first step of mucin O-glycosylation is the transfer of N-acetylgalactosamine from UDP-GalNAc to

serine or threonine residues, which is catalyzed by a polypeptide-N-acetyl-galactosaminyltransferase

(ppGalNAcT). There are at least 21 polypeptide-N-acetylgalactosaminetransferases (ppGalNAcT-1 to

-21) that differ in their amino acid sequences and are encoded by different genes. Homologs of

ppGalNAcT genes are expressed in all eukaryotic organisms and a high degree of sequence identity exists

between mouse and human ppGalNAcTs.

Immunohistochemistry studies have demonstrated that some ppGalNAcTs localize to the cis-Golgi in

submaxillary glands (see Chapter 3). However, more recent studies suggest that in human cervical cancer

cells, several enzymes of this family are located throughout the Golgi. The subcellular localization of

ppGalNAcTs and other glycosyltransferases involved in O-glycosylation has a critical role in determining

the range of O-glycans synthesized by a cell. Thus, a ppGalNAcT localized to a late compartment of the

Golgi will act just before the secretion of a glycoprotein and is likely to produce a number of

“incomplete” shorter O-GalNAc glycans, a factor that will contribute to the glycan heterogeneity of a

mucin.

ppGalNAcT expression levels vary considerably between cell types and mammalian tissues. All

ppGalNAcTs bind UDP-GalNAc (the donor of N-acetylgalactosamine), but they often differ in the protein

substrates to which they transfer N-acetylgalactosamine. Such differences allow ppGalNAcTs to be

distinguished. Many ppGalNAcTs do not efficiently transfer N-acetylgalactosamine to serine, but only to

threonine in peptide acceptors used in in vitro assays. Nevertheless, the VNTR regions of mucins appear

to be fully O-glycosylated. Many ppGalNAcTs appear to have a hierarchical relationship with one

another, such that one enzyme cannot attach an N-acetylgalactosamine until an adjacent serine or

threonine is glycosylated by a different ppGalNAcT. Thus, coexpression in the same cell of ppGalNAcTs

with complementary, partly overlapping acceptor substrate specificities probably ensures efficient O-

GalNAc glycosylation.



Although defined amino acid sequons that accept N-acetylgalactosamine have not been identified, certain

amino acids are preferred in the substrate. Proline residues near the site of N-acetylgalactosamine addition

are usually favorable to mucin O-glycosylation, whereas charged amino acids may interfere with

ppGalNAcT activity. It is possible that the role of proline is to expose serine or threonine residues in a β-

turn conformation, leading to more efficient O-glycosylation. Databases are available that estimate the

likelihood of O-glycosylation at specific sites based on known sequences around O-glycosylation sites of

several hundreds of glycoproteins (e.g., OGLYCBASE and NetOGlyc).

The domain structure of ppGalNAcTs is that of a type II membrane protein, typical of all Golgi

glycosyltransferases (see Chapter 3). In addition, a distinct lectin-like domain has been discovered at the

carboxyl terminus of several ppGalNAcTs. The lectin domain is used for binding to N-acetylgalactosamine residues that have already been added to the glycoprotein. In vitro studies using O-

glycosylated peptides as acceptor substrates have shown that further addition of N-acetylgalactosamine is

strongly influenced by the presence of existing N-acetylgalactosamine and larger O-glycans in the

acceptor substrate, suggesting a molecular mechanism for the hierarchical relationships of ppGalNAcTs.

Purification of ppGalNAcTs has been achieved using mucin peptide and nucleotide sugar derivatives as

affinity reagents. The recent elucidation of the crystal structure of ppGalNAcT-1 showed that the catalytic

site of the enzyme is spatially separated from the lectin-binding site, and that there is a nucleotide sugar-

and Mn++

-binding groove containing an aspartate-X-histidine motif. The unique structure of ppGalNAcT-

1 suggests that this transferase can accommodate a number of different glycosylated acceptor substrates.

Synthesis of O-GalNAc Glycan Core Structures

The transfer of the first sugar from UDP-GalNAc directly to serine or threonine in a protein initiates the

biosynthesis of all O-GalNAc glycans. Subsequently, with the addition of the next sugar, different mucin

O-glycan core structures are synthesized (Figure 9.2). In contrast to the initial reactions of N-

glycosylation and O-mannosylation, no lipid-linked intermediates are involved in O-GalNAc glycan

biosynthesis, and no glycosidases appear to be involved in the processing of O-GalNAc glycans within

the Golgi.

The O-glycans of mucins produced in a specific tissue predict the enzyme activities that are present in

that tissue. The glycosyltransferases that are involved solely in the assembly of mucin O-GalNAc glycans

are listed in Table 9.2. The many other enzymes that may be involved also contribute to the synthesis of

N-glycans and glycolipids. The first sugar (N-acetylgalactosamine) added to the protein creates the Tn

antigen (GalNAc-Ser/Thr), which is uncommon in normal mucins, but is often found in mucins derived

from tumors. This suggests that the extension of O-GalNAc glycans beyond the first sugar is blocked in

some cancer cells. Another common cancer-associated structure found in mucins is the sialyl-Tn antigen,

which contains a sialic acid residue linked to C-6 of N-acetylgalactosamine (Table 9.1). O-Acetylation of

the sialic acid residue masks the recognition of sialyl-Tn by anti-sialyl-Tn antibodies. No other sugars are

known to be added to the sialyl-Tn antigen.

There are eight O-GalNAc glycan core structures (Table 9.1), most of which may be further substituted

by other sugars. N-Acetylgalactosamine is converted to core 1 (Galβ1-3GalNAc-) by a core 1 β1-3

galactosyltransferase termed T synthase or C1GalT-1 (Figure 9.3). This activity is present in most cell

types. However, to be exported from the endoplasmic reticulum in vertebrates, T synthase requires a

specific molecular chaperone called Cosmc. Cosmc is an ER protein that appears to bind specifically to T

synthase and ensures its full activity in the Golgi. Lack of core 1 synthesis can be due to either defective

T synthase or the absence of functional Cosmc chaperone. The result is high expression of Tn and sialyl-

Tn antigens. Immunoglobulin A nephropathy in humans is associated with low expression of Cosmc, and

a mutation in the Cosmc gene gives rise to a condition called the Tn syndrome.



In many serum glycoproteins and mucins, the T antigen is substituted by sialic acid at C-3 of galactose

and at C-6 of N-acetylgalactosamine (Figure 9.3). These substitutions add a negative charge to the O-

GalNAc glycan. They also prevent other modifications of core 1. The cell surfaces of many leukemia and

tumor cells contain large numbers of sialylated core 1 O-GalNAc glycans. On rare occasions, core 1

remains unsubstituted, leaving the T antigen exposed, for example, in cancer and inflammatory bowel

disease. In these cases, it is likely that there is an abnormality in either the sialylation of core 1 or further

extension and branching of core 1.

Core 2 mucin O-glycans are branched core 1 structures that are produced in many tissues, including the

intestinal mucosa. The synthesis of core 2 O-GalNAc glycans is regulated during activation of

lymphocytes, cytokine stimulation, and embryonic development. Leukemia and cancer cells and other

diseased tissues have abnormal amounts of core 2 O-GalNAc glycans. The synthesis of core 2 O-GalNAc

glycans has been correlated with tumor progression. Because of their branched nature, core 2 O-glycans

can block the exposure of mucin peptide epitopes. The enzyme responsible for core 2 synthesis is core 2

β1-6 N-acetylglucosaminyltransferase or C2GnT (Figure 9.3). At least three genes encode this subfamily

(C2GnT-1 to -3) of a larger family of β1-6 N-acetylglucosaminyltransferases that catalyze the synthesis of

GlcNAcβ1-6-linked branches. There are two major types of core 2 β1-6 N-

acetylglucosaminyltransferases. The L type (leukocyte type, C2GnT-1 and -3) synthesizes only the core 2

structure, whereas the M type (mucin type, C2GnT-2) is also involved in the synthesis of core 4 and other

GlcNAcβ1-6-linked branches. The L enzyme is active in many tissues and cell types, but the M enzyme

is found only in mucin-secreting cell types. The expression and activity of both the L and M enzymes are

altered in certain tumors.

The synthesis of core 3 O-GalNAc glycans appears to be restricted mostly to mucous epithelia from the

gastrointestinal and respiratory tracts and the salivary glands. The enzyme responsible is core 3 β1-3 N-

acetylglucosaminetransferase (C3GnT; Figure 9.4). Although in vitro assays of this enzyme suggest that it

is relatively inefficient, the enzyme must act efficiently in vivo in colonic goblet or mucous cells because

secreted colonic mucins mainly carry core 3 and few core 1, core 2, or core 4 O-GalNAc glycans. The

activity of C3GnT is especially low in colonic tumors and virtually absent from tumor cells in culture.

The synthesis of core 4 by the M-type β1-6 N-acetylglucosaminetransferase (C2GnT-2; see above)

requires the prior synthesis of a core 3 O-GalNAc glycan (Figure 9.4). The glycosyltransferase

synthesizing core 5 must exist in colonic tissues and in colonic adenocarcinoma because these tissues

produce mucin with core 5. The enzymes synthesizing cores 5 to 8 remain to be characterized.

Synthesis of Complex O-GalNAc Glycans

The elongation of the galactose residue of core 1 and core 2 O-GalNAc glycans is catalyzed by a β1-3 N-

acetylglucosaminyltransferase (Table 9.2) that is specific for O-GalNAc glycans. These O-glycans can

also be extended by N-acetylglucosaminyltransferases and galactosyltransferases to form repeated

GlcNAcβ1-3Galβ1-4 (poly-N-acetyllactosamine) sequences that represent the little i antigen. Less

common elongation reactions are the formation of GalNAcβ1-4GlcNAc- (LacdiNAc) and Galβ1-

3GlcNAc- sequences. Linear poly-N-acetyllactosamine units can be branched by members of the β1-6 N-

acetylglucosaminyltransferase family, resulting in the large I antigen. Some of these branching reactions

are common to other O- and N-glycans and glycolipids (see Chapter 13).

The ABO and other glycan-based blood groups as well as sialic acids, fucose, and sulfate are common

terminal structures in mucins as in other glycoconjugates. The families of glycosyltransferases that

catalyze the addition of these terminal structures are described in more detail in Chapters 13 and 14. In

contrast to N-glycans, O-GalNAc glycans do not have NeuAcα2-6Gal linkages, although the NeuAcα2-

6GalNAc moiety is common, for example, in the sialyl-Tn antigen and in elongated core 1 and 3

structures. Thus, in most mammalian mucin-producing cells, α2-6 sialyltransferases act on N-



acetylgalactosamine, and α2-3 sialyltransferases act on galactose. Some of the sialyltransferases and

sulfotransferases prefer O-GalNAc glycans as their substrate (Table 9.2), but many of these enzymes have

an overlapping specificity and also act on N-glycan structures as acceptor substrates.

Control of O-GalNAc Glycan Synthesis

The acceptor specificities of glycosyltransferases and sulfotransferases are the main factors determining

the structures of O-GalNAc glycans found in mucins, and these specificities restrict the high number of

theoretically possible O-glycans to “only” a few hundred. The specificities also direct the pathways that

are feasible. For example, the sialylated core 1 structure NeuAcα2-6(Galβ1-3)GalNAc- can be

synthesized only by adding a sialic acid residue to core 1, but not by adding a galactose residue to the

sialyl-Tn antigen, because the sialic acid prevents the action of core 1 β1-3 galactosyltransferase. The

disialylated core 1 structure NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAc- is synthesized by the addition of

the α2-3-linked sialic acid first to core 1, followed by the addition of the α2-6-linked sialic acid to N-

acetylgalactosamine. These enzymes can therefore recognize at least two sugar residues and are often (but

not always) blocked by the presence of sialic acid. Expression of sialyltransferases is consequently often

associated with shorter O-GalNAc glycans. When a core 1 O-GalNAc glycan is sialylated to form

NeuAcα2-3Galβ1-3GalNAc-, core 2 cannot be synthesized because the L-type core 2 β1-6 N-

acetylglucosaminyltransferase requires an unsubstituted core 1 structure as the acceptor substrate.

Detailed specificity studies suggest that the L-type core 2 β1-6 N-acetylglucosaminyltransferase

recognizes unsubstituted C-4 and C-6 hydroxyl groups of both the galactose and N-acetylgalactosamine

residues of a core 1 O-GalNAc glycan.

Another important control factor is that the relative activities of the glycosyltransferases determine the

relative amounts of O-GalNAc glycans in mucins. Because of partially overlapping localization in the cis-and medial-Golgi compartments of breast cancer cells, core 2 β1-6 N-acetylglucosaminyltransferase

(C2GnT-1) competes with α2-3 sialyltransferase for the common core 1 substrate (Figure 9.3). Therefore,

the relative activities of these two enzymes determine the nature of the O-GalNAc glycans synthesized

and the antigenic properties of the cell surface. Depending on which activity predominates, the cell-

surface mucin MUC1 may be highly sialylated and its O-glycans relatively small or its O-glycans may be

large and more complex due to the presence of extended, branched core 2 O-GalNAc glycans. In MUC1

from breast cancer cells, the presence of these sialylated small O-glycans leads to the exposure of

underlying mucin peptide epitopes.

In vitro studies have shown that the activities of transferases are controlled by many factors such as metal

ions and membrane components. Only two activities involved in O-glycan biosynthesis have been found

to be regulated by the presence of specific binding proteins. The first is β1-4 galactosyltransferase 1

(β4GalT-1), which can bind to α-lactalbumin in mammary gland to change its preferred acceptor

substrate from N-acetylglucosamine to glucose, thereby forming lactose. The second enzyme is core 1 β1-

3 galactosyltransferase (C1GalT-1), which requires the coexpression of the molecular chaperone Cosmc.

The first step of O-GalNAc glycosylation is clearly regulated by the amino acid sequence of the acceptor

substrate. Although individual ppGalNAcTs may prefer certain sites in the acceptor substrate, a

combination of enzymes expressed in the same cell compartment ensures efficient mucin O-

glycosylation. Several enzymes of the O-glycosylation pathway recognize their substrates in the context

of the underlying peptide. The sequence of the peptide moiety of the acceptor substrate may direct the

synthesis of cores 1, 2, and 3 as well as the extension of N-acetylglucosamine-terminating core structures

by galactose (Figures 9.3 and 9.4).

It appears that glycosyltransferases are arranged in a diffuse “assembly line” within the Golgi

compartments. This intracellular localization of enzymes allows them to act on mucin substrates in a



specific sequence. Thus, early-acting enzymes synthesize the substrates for the next reactions. Because

several enzymes can often use the same substrate, and there is extensive overlap of both localization and

specificities, large numbers of different O-glycan structures are synthesized, leading to the heterogeneity

seen in mucins. The concentrations of nucleotide sugar and sulfate-donor substrates within the Golgi and

the rate of substrate transport throughout the Golgi are additional important control factors (see Chapter

4). The sensitive and complex balance of these various factors appears to be altered upon cell

differentiation, cytokine stimulation, and in disease states.

FUNCTIONS OF O-GalNAc GLYCANS

O-GalNAc glycosylation is probably an essential process because all mammalian cell types studied to

date express ppGalNAcTs. However, when the ppGalNAcT-1 gene was deleted in mice the animals

appeared to be unaffected, possibly due to the fact that another ppGalNAcT replaces the function of

ppGalNAcT-1. In the secreted mucins of the respiratory, gastrointestinal, and genitourinary tracts, as well

as those of the eyes, the O-GalNAc glycans of mucous glycoproteins are essential for their ability to

hydrate and protect the underlying epithelium. Mucins also trap bacteria via specific receptor sites within

the O-glycans of the mucin. Some sugar residues or their modifications can mask underlying antigens or

receptors. For example, O-acetyl groups on the sialic acid residue of the sialyl-Tn antigen prevent

recognition by anti-sialyl-Tn antibodies. Gut bacteria enzymatically remove this blocking group. Bacteria

can cleave sulfate with sulfatases or terminal sugars with glycosidases. Because the O-glycans are

hydrophilic and usually negatively charged, they promote binding of water and salts and are major

contributors to the viscosity and adhesiveness of mucus, which forms a physical barrier between lumen

and epithelium. The removal of microbes and particles trapped in mucus is an important physiological

process. However, in diseases such as cystic fibrosis, the abnormally high viscosity of the mucus leads to

obstruction and life-threatening tissue malfunction.

O-GalNAc glycans, especially in the highly glycosylated mucins, have a significant effect on the

conformation of the attached protein. Depending on the size and bulkiness of O-glycans, underlying

peptide epitopes can be variably recognized by antibodies. O-glycosylation of mucins provides almost

complete protection from protease degradation, and it is possible that the sparse O-glycosylation of some

secreted glycoproteins, such as the single O-GalNAc glycan on interleukin-2, has a similar protective

role.

Cell lines defective in specific O-glycosylation pathways can be used as models to study the role of O-

glycosylation (see Chapter 46). For example, due to a defect in UDP-Glc-4-epimerase that reversibly

converts UDP-GlcNAc to UDP-GalNAc, the ldlD cell line does not produce GalNAc-Ser/Thr linkages

unless N-acetylgalactosamine is added to the cell medium. The addition of galactose as well as N-acetylgalactosamine results in complete O-GalNAc glycans. This model has been used to demonstrate

that O-glycans of cell-surface receptors may regulate receptor stability and expression levels.

O-GalNAc glycans change during lymphocyte activation and are abnormal in leukemic cells where an

increase in core 2 and a decrease in core 1 O-glycans are often seen. The ligands for selectin-mediated

interactions between endothelial cells and leukocytes are commonly based upon sialyl Lewisx epitopes

attached to core 2 O-GalNAc glycans. This type of selectin–glycan interaction is important for the

attachment of leukocytes to the capillary endothelium during homing of lymphocytes or the extravasation

of leukocytes during the inflammatory response (see Chapter 31). Removal of core 2 O-GalNAc glycans

by eliminating the C2GnT-1 gene in mice results in a severe deficiency in the immune system,

particularly in the selectin-binding capability of leukocytes (see Chapter 50).

Cancer cells often express sialyl Lewisx epitopes and may thus use the selectin-binding properties of their



cell surface as a mechanism to invade tissues. The critical role of O-glycans in this process has been

studied in cell lines treated with the O-glycan extension inhibitor benzyl-α-GalNAc (see Chapter 50).

This compound acts as a competitive substrate for the synthesis of core 1, core 2, core 3, and core 4 O-

glycans in cells and thus causes a reduction in the synthesis of complex O-GalNAc glycans. As a

consequence, mucins carry a higher number of unmodified N-acetylgalactosamine residues and shorter O-

GalNAc glycans. Inhibitor-treated cancer cells lose the ability to bind to E-selectin and endothelial cells

in vitro.

Reproductive tissues produce mucins and O-glycosylated glycoproteins that may have important roles in

fertilization. Specific terminal O-glycan structures have been shown to form the ligands for sperm-egg

interactions in several species (see Chapter 25).

The changes of O-glycans commonly observed in diseases can be due to the actions of cytokines or

growth factors that affect cell growth, differentiation, and cell death and alter the expression of

glycosyltransferase genes. Although these glycosylation changes may be considered a “side effect” of a

pathological condition, they can also significantly contribute to the ultimate pathology and the course of

disease. In cancer, the biosynthesis of O-GalNAc glycans is often abnormal due to either decreased or

increased expression and activities of specific glycosyltransferases. An altered cell-surface glycocalyx

may then affect the biology and survival of the cancer cell.

FURTHER READING

1. Tabak LA. In defense of the oral cavity: Structure, biosynthesis, and function of salivary mucins.

Annu Rev Physiol. 1995;57:547–564. [PubMed: 7778877]

2. van Klinken BJW, Dekker J, Büller HA, Einerhand AWC. Mucin gene structure and expression:

Protection vs. adhesion. Am J Physiol. 1995;269:G613–627. [PubMed: 7491952]

3. Hansen JE, Lund O, Nielsen JO, Brunak S. O-GLYCBASE: A revised database of O-glycosylated

proteins. Nucleic Acids Res. 1996;24:248–252. [PMC free article: PMC145605] [PubMed:

8594592]

4. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochim. Biophys. Acta.

1999;1473:67–95. [PubMed: 10580130]

5. Fukuda M. Roles of mucin-type O-glycans in cell adhesion. Biochim. Biophys. Acta.

2002;1573:394–405. [PubMed: 12417424]

6. Brockhausen I. Sulphotransferases acting on mucin-type oligosaccharides. Biochem Soc Trans.

2003;31:318–325. [PubMed: 12653628]

7. Lowe JB, Marth J. A genetic approach to mammalian glycan function. Annu Rev Biochem.

2003;72:643–691. [PubMed: 12676797]

8. ten Hagen KG, Fritz TA, Tabak LA. All in the family: The UDP-GalNAc:polypeptide N-

acetylgalactosaminyltransferases. Glycobiology. 2003;13:1R–16R. [PubMed: 12634319]

9. Hollingsworth MA, Swanson BJ. Mucins in cancer: Protection and control of the cell surface. Nat.

Rev. Cancer. 2004;4:45–60. [PubMed: 14681689]

10. Robbe C, Capon C, Coddeville B, Michalski JC. Structural diversity and specific distribution of O-

glycans in normal human mucins along the intestinal tract. Biochem J. 2004;384:307–316. [PMC

free article: PMC1134114] [PubMed: 15361072]

11. Wandall HH, Irazoqui F, Tarp MA, Bennett EP, Mandel U, Takeuchi H, Kato K, Irimura T,

Suryanarayanan G, Hollingsworth MA, Clausen H. 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.

2007;17:374–387. [PubMed: 17215257]

12. Hattrup CL, Gendler SJ. Structure and function of the cell surface (tethered) mucins. Annu Rev



Physiol. 2008;70:431–457. [PubMed: 17850209]

Figures

FIGURE 9.1. A simplified model of a large secreted mucin.

FIGURE 9.1

A simplified model of a large secreted mucin. The VNTR (variable number of tandem repeat) region rich

in serine, threonine, and proline is highly O-glycosylated and the peptide assumes an extended “bottle

brush” conformation. Hundreds of O-GalNAc glycans with many different structures may be attached to

serine or threonine residues in the VNTR domains. The cysteine-rich regions at the ends of the molecules

are involved in disulfide bond formation to form large polymers of several million daltons. D domains

have similarity to von Willebrand factor and are also involved in polymerization.

FIGURE 9.2. Complex O-GalNAc glycans with different core structures.

FIGURE 9.2

Complex O-GalNAc glycans with different core structures. Representative examples of complex O-

GalNAc glycans with extended core 1, 2, or 4 structures from human respiratory mucins and an O-

GalNAc glycan with an extended core 3 structure from human colonic mucins. All four core structures (in

boxes) can be extended, branched, and terminated by fucose in various linkages, sialic acid in α2-3

linkage, or blood group antigenic determinants (Table 9.1). Core structures 1 and 3 may also carry sialic

acid α2-6-linked to the core N-acetylgalactosamine.

Symbol Key: FIGURE 9.2. Complex O-GalNAc glycans with different core structures.

FIGURE 9.3. Biosynthesis of core 1 and 2 O-GalNAc glycans.

FIGURE 9.3

Correction

Biosynthesis of core 1 and 2 O-GalNAc glycans. Shown is the biosynthesis of some extended core 1 and

core 2 O-GalNAc glycans. The linkage of N-acetylgalactosamine to serine or threonine to form the Tn

antigen, catalyzed by polypeptide-N-acetylgalactosaminyltransferases (ppGalNAcTs), is the basis for all

core structures. Core 1 β1-3 galactosyltransferase (C1GalT-1) synthesizes core 1 (T antigen) and requires

a specific molecular chaperone, Cosmc. Core 1 may be substituted with sialic acid or N-

acetylglucosamine (as shown) or by fucose or sulfate (not shown). Substitution of core 1 by a β1-3-linked

N-acetylglucosamine prevents the synthesis of core 2 by core 2 β1-6 N-acetylglucosaminyltransferase

(C2GnT), an enzyme that is highly specific for unmodified core 1 as a substrate. Red cross marks blocked

pathway. In breast cancer cells, C2GnT and α2-3 sialyltransferase (ST3Gal I) compete for the common

core 1 substrate. If sialyltransferase activity is high, chains will be mainly short with sialylated core 1

structures; if C2GnT activity is high, chains will be complex and large. The Galβ1-3 residue of the core 1

O-GalNAc glycan and both N-acetylglucosamine and galactose branches of the core 2 O-GalNAc glycan

may be extended and carry terminal blood group and Lewis antigens (see Table 9.1).



Symbol Key: FIGURE 9.3. Biosynthesis of core 1 and 2 O-GalNAc glycans.

FIGURE 9.4. Biosynthesis of core 3 and core 4 O-GalNAc glycans.

FIGURE 9.4

Biosynthesis of core 3 and core 4 O-GalNAc glycans. N-Acetylgalactosamine is substituted by a β1-3 N-

acetylglucosamine residue to form core 3. This reaction is catalyzed by core 3 β1-3 N-acetyl-

glucosaminyltransferase (C3GnT), which is active in colonic mucosa. Core 3 may be substituted with

sialic acid in α2-6 linkage to N-acetylgalactosamine or with galactose in β1-4 linkage to N-

acetylglucosamine. These substitutions prevent the synthesis of core 4 by core 2/4 β1-6 N-

acetylglucosaminyltransferase (C2GnT-2). Red crosses mark blocked pathways. Unmodified core 3 can

be branched by C2GnT-2 to form core 4. Both core 3 and core 4 O-GalNAc glycans can be extended to

complex chains carrying determinants similar to those of core 1 and core 2 O-GalNAc glycans.

Symbol Key: FIGURE 9.4. Biosynthesis of core 3 and core 4 O-GalNAc glycans.

Tables

TABLE 9.1

Structures of O-glycan cores and antigenic epitopes found in mucins

O-Glycan Structure

Core

Tn antigen GalNAcαSer/Thr

Sialyl-Tn antigen Siaα2-6GalNAcαSer/Thr

Core 1 or T antigen Galβ1-3GalNAcαSer/Thr

Core 2 GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr

Core 3 GlcNAcβ1-3GalNAcαSer/Thr

Core 4 GlcNAcβ1-6(GlcNAcβ1-3)GalNAcαSer/Thr

Core 5 GalNAcα1-3GalNAcαSer/Thr

Core 6 GlcNAcβ1-6GalNAcαSer/Thr

Core 7 GalNAcα1-6GalNAcαSer/Thr

Core 8 Galα1-3GalNAcαSer/Thr

Epitope

Blood groups O, H Fucα1-2Gal-

Blood group A GalNAcα1-3(Fucα1-2)Gal-

Blood group B Galα1-3(Fucα1-2)Gal-

Linear B Galα1-3Gal-

Blood group i Galβ1-4GlcNAcβ1-3Gal-

Blood group I Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Gal-

Blood group Sd(a), Cad GalNAcβ1-4(Siaα2-3)Gal-



Blood group Lewisa Galβ1-3(Fucα1-4)GlcNAc-

Blood group Lewisx

Galβ1-4(Fucα1-3)GlcNAc-

Blood group sialyl-Lewisx

Siaα2-3Galβ1-4(Fucα1-3)GlcNAc-

Blood group Lewisy

Fucα1-2Galβ1-4(Fucα1-3)GlcNAc-

TABLE 9.2

Glycosyltransferases specific for mucin O-GalNAc glycans

Enzyme Short form

Polypeptide N-acetylgalactosaminyltransferase ppGalNAcT-1 to -24

Core 1 β1-3 galactosyltransferase C1GalT-1 or T synthase

Core 2 β1-6 N-acetylglucosaminyltransferase C2GnT-1, C2GnT-3

Core 3 β1-3 N-acetylglucosaminyltransferase C3GnT-1

Core 2/4 β1-6 N-acetylglucosaminyltransferase C2GnT-2

Elongation β1-3 N-acetylglucosaminyltransferase elongation β3GnT-1 to -8

Core 1 α2-3 sialyltransferase ST3Gal I, ST3Gal IV

α2-6 sialyltransferase ST6GalNAc I, II, III or IV

Core 1 3-O-sulfotransferase Gal3ST4

Secretor gene α1-2 fucosyltransferase FucT-I, FucT-II

Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.

Bookshelf ID: NBK1896PMID: 20301232

Contents

< PrevNext >

Intestinalmucosalbarrierfunctioninhealthanddisease.Turner,JRNatureRev.Immunology9:799(2009)

Complex multicellular organisms interface with their external environments at multiple sites, including mucosae of the airways, oral cavity, digestive tract and genitourinary tract, and the skin. Although the skin is the most visible site of interface, the combined area of the mucosal surfaces is much greater than that of the skin. Mucosae are also the primary sites at which the mucosa-associated lymphoid tissue (MALT) is exposed to and interacts with the external environment. The magni-tude of these interactions is greatest in the gastrointesti-nal tract, which is the largest mucosal surface and is also in continuous contact with dietary antigens and diverse microorganisms. Thus, mucosal surfaces, particularly in the intestines, are crucial sites of innate and adaptive immune regulation.

A central mediator of interactions between MALT and the external environment, including the intesti-nal lumen, is the epithelium that covers the mucosa. Epithelial cells establish and maintain the barrier, which in the skin is a tight, although not impermeant, seal. By contrast, most mucosal epithelial cells form leaky barriers. This is necessary to support universal functions that include fluid exchange, which occurs at nearly all mucosal surfaces, as well as essential tissue-specific functions. Thus, the precise permea-bility characteristics of each barrier type differ based on the functions supported. For example, the ion-selective properties of the barrier vary considerably along the length of a nephron to promote or restrict transport of specific ions in each segment1. Similarly, the ability of tight junctions to discriminate between and restrict passage of solutes based on size (a characteristic known

as size selectivity) varies with location in the intestine, as permeability to larger solutes decreases from the crypt to the villus2. In addition to these fixed differences, mucosal permeability of many tissues is adaptable and may be regulated in response to extracellular stimuli, such as nutrients, cytokines and bacteria.

Recent advances have uncovered some of the mech-anisms by which physiological and immunological stimuli affect cellular and extracellular components of the intestinal barrier. In this article I review our current understanding of the mechanisms that regulate intesti-nal barrier integrity, and discuss the hypothesis that the mucosal barrier can shape pro-inflammatory and immuno-regulatory responses in the context of homeostasis and disease.

Although they may affect barrier function, den-dritic cells (DCs), which extend dendritic processes across the tight junctions in the distal small intestine3,4, and intraepithelial lymphocytes are not discussed here. In addition, the functions of M cells, which are spe-cialized epithelial cells that deliver antigens directly to intra epithelial lymphocytes and to subepithelial lymphoid tissues by transepithelial vesicular transport from the gut lumen, are reviewed elsewhere5. Finally, detailed analyses of the interactions between epithelial cells and luminal microorganisms, as well as the intri-cacies of mucosal immune regulation, have also been recently reviewed elsewhere6,7. This Review therefore focuses on the roles of the epithelial cell barrier in health and disease, with particular emphasis on the junctional complexes between intestinal epithelial cells, which have a crucial role in barrier regulation.

Department of Pathology, The University of Chicago, 5841 South Maryland, MC 1089, Chicago, Illinois 60637, USA.e-mail: [email protected]:10.1038/nri2653

Mucosa-associated lymphoid tissue(MALT). The collections of B cells, T cells, plasma cells, macrophages and other antigen-presenting cells found in the mucosal linings of organs including the gastrointestinal tract, lungs, salivary glands and conjuctiva.

Tight junctionAlso known as the zonula occludens, this is a site of close apposition of adjacent epithelial cell membranes — kiss points — that create a barrier against the free diffusion of water and solutes.

Intestinal mucosal barrier function in health and diseaseJerrold R. Turner

Abstract | Mucosal surfaces are lined by epithelial cells. These cells establish a barrier between sometimes hostile external environments and the internal milieu. However, mucosae are also responsible for nutrient absorption and waste secretion, which require a selectively permeable barrier. These functions place the mucosal epithelium at the centre of interactions between the mucosal immune system and luminal contents, including dietary antigens and microbial products. Recent advances have uncovered mechanisms by which the intestinal mucosal barrier is regulated in response to physiological and immunological stimuli. Here I discuss these discoveries along with evidence that this regulation shapes mucosal immune responses in the gut and, when dysfunctional, may contribute to disease.

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nATuRE REvIEwS | Immunology voLuME 9 | novEMBER 2009 | 799

© 2009 Macmillan Publishers Limited. All rights reserved

mailto:[email protected]

E-cadherin

F-actinMyosinOccludin

Desmoglein

Keratin

Desmoplakin

Desmocollin

β-catenin

α-catenin 1

a b

ClaudinZO1

MLCK

Tight junction

Desmosome

Adherensjunction

Nature Reviews | Immunology

Microvilli

Mucus

Goblet cell

Unstirred layer

Epithelial cells

Intraepithelial lymphocyte

Basement membrane

Plasma cell

Apicaljunctional complex

Lamina proprialymphocyte

MucinsA family of heavily glycosylated proteins that are secreted as large aggregates by mucous epithelial cells.

Unstirred layerA thin layer of fluid at epithelial cell surfaces that is separated from the mixing forces created by luminal flow and, in the intestine, peristalsis.

Coeliac diseaseA chronic inflammatory condition of the upper small intestine in humans that is caused by immunological hypersensitivity to the α-gliadin component of wheat gluten. It can cause severe villous atrophy, which can lead to malabsorption and malnutrition if gluten-containing foods are not removed from the diet.

Anatomy of mucosal barriersExtracellular components of the barrier. Most mucosal surfaces are covered by a hydrated gel formed by mucins (FIG. 1a). Mucins are secreted by specialized epithelial cells, such as gastric foveolar mucous cells and intestinal goblet cells, and create a barrier that prevents large par-ticles, including most bacteria, from directly contacting the epithelial cell layer8. The importance of mucus gel hydration is shown by cystic fibrosis, in which the pro-duction of hyperviscous mucus contributes to pulmo-nary, pancreatic and intestinal disease9. Defective mucus production has also been reported in various immune-mediated diseases, and spontaneous colitis develops in mice that lack specific mucin genes10.

Although small molecules pass through the heavily glycosylated mucus layer with relative ease, bulk fluid flow is limited and thereby contributes to the develop-ment of an unstirred layer of fluid at the epithelial cell sur-face. As the unstirred layer is protected from convective mixing forces, the diffusion of ions and small solutes is slowed. In the stomach, this property of the unstirred layer works with epithelial cell bicarbonate secretion to main-tain a zone of relative alkalinity at the mucosal surface11. The unstirred layer of the small intestine slows nutrient

absorption by reducing the rate at which nutrients reach the transporting protein-rich microvillus brush border, but may also contribute to absorption by limiting the extent to which small nutrients released by the activities of brush border digestive enzymes are lost by diffusion into the lumen. unstirred layer defects have not been linked to specific diseases. However, it is interesting to note that increased unstirred layer thickness has been reported in coeliac disease12, in which it may contribute to nutrient malabsorption.

Cellular components of the mucosal barrier. The pri-mary responsibility for mucosal barrier function resides with the epithelial cell plasma membrane, which is impermeable to most hydrophilic solutes in the absence of specific transporters. Accordingly, direct epithelial cell damage, such as that induced by mucosal irritants or cytotoxic agents, including some drugs used for can-cer chemotherapy, results in a marked loss of barrier function. However, in the presence of an intact epithe-lial cell layer, the paracellular pathway between cells must be sealed. This function is mediated by the apical junc-tional complex, which is composed of the tight junction and subjacent adherens junction (FIG. 1b). Both tight and

Figure 1 | Anatomy of the mucosal barrier. a | The human intestinal mucosa is composed of a simple layer of columnar epithelial cells, as well as the underlying lamina propria and muscular mucosa. Goblet cells, which synthesize and release mucin, as well as other differentiated epithelial cell types, are present. The unstirred layer, which cannot be seen histologically, is located immediately above the epithelial cells. The tight junction, a component of the apical junctional complex, seals the paracellular space between epithelial cells. Intraepithelial lymphocytes are located above the basement membrane, but are subjacent to the tight junction. The lamina propria is located beneath the basement membrane and contains immune cells, including macrophages, dendritic cells, plasma cells, lamina propria lymphocytes and, in some cases, neutrophils. b | An electron micrograph and corresponding line drawing of the junctional complex of an intestinal epithelial cell. Just below the base of the microvilli, the plasma membranes of adjacent cells seem to fuse at the tight junction, where claudins, zonula occludens 1 (ZO1), occludin and F‑actin interact. E‑cadherin, α‑catenin 1, β‑catenin, catenin δ1 (also known as p120 catenin; not shown) and F‑actin interact to form the adherens junction. Myosin light chain kinase (MLCK) is associated with the perijunctional actomyosin ring. Desmosomes, which are located beneath the apical junctional complex, are formed by interactions between desmoglein, desmocollin, desmoplakin and keratin filaments.

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Paracellular pathwayThe route of transepithelial transport that involves passive movement through the space between adjacent cells.

Adherens junctionAlso known as the zonula adherens, this junction is immediately subjacent to the tight junction and requires the activity of lineage-specific Ca2+-dependent adhesion proteins, termed cadherins.

DesmosomeAn adhesive junction that connects adjacent epithelial cells. These junctions are composed of multiple protein subunits and are the points where keratin filaments attach to the plasma membrane.

ClaudinFrom the Latin claudere, meaning ‘to close’, members of this family of transmembrane proteins are variably expressed by specific epithelial cell types and thereby contribute to the unique barrier properties of different tissues.

OccludinThe first transmembrane tight junction protein identified. The function of occludin remains controversial but it is likely to have roles in barrier regulation and tumour suppression. It also serves as a cofactor in hepatitis C virus entry.

Zonula occludens 1A peripheral membrane, or plaque, protein containing multiple protein interaction domains that, along with the related protein zonula occludens 2, is required for tight junction assembly.

Transcellular pathwayThe route of transepithelial transport that involves active or passive movement across cell membranes, usually as a result of the action of specific transport channels.

Transepithelial transportThe sum of transport through the transcellular and paracellular pathways.

adherens junctions are supported by a dense perijunc-tional ring of actin and myosin that, as discussed below, can regulate barrier function.

As implied by the name, the adherens junctions, along with desmosomes, provide the strong adhesive bonds that maintain cellular proximity and are also a site of intercellular communication. Loss of adherens junctions results in disruption of cell–cell and cell–matrix contacts, ineffective epithelial cell polariza-tion and differentiation, and premature apoptosis13. Adherens junctions are composed of cadherins, a family of transmembrane proteins that form strong, homotypic interactions with molecules on adjacent cells. The cyto-plasmic tail of the epithelial cadherin, E-cadherin (also known as cadherin-1), interacts directly with catenin δ1 (also known as p120 catenin) and β-catenin. In turn, β-catenin binds to α-catenin 1, which regulates local actin assembly and contributes to development of the perijunctional actomyosin ring.

Adherens junctions are required for assembly of the tight junction, which seals the paracellular space. Tight junctions are multi-protein complexes composed of transmembrane proteins, peripheral membrane (scaf-folding) proteins and regulatory molecules that include kinases. The most important of the transmembrane proteins are members of the claudin family, which define several aspects of tight junction permeability, as dis-cussed below. Claudins are expressed in a tissue-specific manner, and mutation or deletion of individual family members can have profound effects on organ function. The role of occludin, a transmembrane tight junction protein that interacts directly with claudins and actin, is less well understood. Peripheral membrane proteins, such as zonula occludens 1 (Zo1) and Zo2, are crucial to tight junction assembly and maintenance, partly owing to the fact that these proteins include multiple domains for interaction with other proteins, including claudins, occludin and actin.

The tight junction limits solute flux along the para-cellular pathway, which is typically more permeable than the transcellular pathway. The tight junction is, there-fore, the rate-limiting step in transepithelial transport and the principal determinant of mucosal permeability. Thus, it is important to understand the specific barrier properties of the tight junction, which can be defined in terms of size selectivity and charge selectivity.

At least two routes allow transport across the tight junction, and emerging data suggest that the relative contributions of these types of paracellular transport may be regulated independently2,14,15. one route, the leak pathway, allows paracellular transport of large solutes, including limited flux of proteins and bacterial lipopolysaccharides14,15. Although the size at which par-ticles are excluded from the leak pathway has not been precisely defined, it is clear that materials as large as whole bacteria cannot pass. As might be expected for a route that allows large solutes to cross, the leak path-way does not show charge selectivity. Flux across the leak pathway may be increased by cytokines, including interferon-γ (IFnγ) in vitro and tumour necrosis factor (TnF) in vitro and in vivo15–17.

A second pathway is characterized by small pores that are thought to be defined by tight junction-associated claudin proteins, which are also primary determinants of charge selectivity18–20. These pores have a radius that excludes molecules larger than 4 Å14,15. Expression of spe-cific claudins varies between organs and even within dif-ferent regions of a single organ and, as detailed below, can be modified by external stimuli, such as cytokines. Thus, tight junctions show both size selectivity and charge selec-tivity, and these properties may be regulated individually or jointly by physiological or pathophysiological stimuli.

Interdependence of transport routesTransepithelial transport requires a selectively permeable barrier. The vectorial nature of transcellular transport, which is required for effective absorption and secre-tion, generates a transepithelial concentration gradient. Transepithelial transport would, therefore, be ineffective without a tight junction barrier, as diffusion would allow equalization of concentrations on both sides of the epith-elium. Thus, active transcellular transport depends on the presence of an intact tight junction barrier. The selective permeability of the tight junction barrier also allows trans-epithelial gradients to drive passive paracellular transport of ions and water17,20. For example, claudin-16, which is necessary for tight junction cation selectivity, is mutated in the human disease familial hypomagnesaemia with hyper-calciuria and nephro calcinosis20. Paracellular absorption of Mg2+ and Ca2+ in the thick ascending limb of Henle requires a cation-selective tight junction barrier, the absence of which results in urinary loss of Mg2+ and Ca2+ (ReF. 20). Similarly, apical na+–H+-exchange protein 3 (nHE3) is required for transcellular na+ absorption in the renal tubule and intestine, and also contributes to the trans-epithelial na+ gradient that drives paracellular water absorption (BOX 1). The tight junction barrier to para-cellular na+ efflux prevents dissipation of the gradient established by nHE3-mediated transcellular transport.

Tight junction barrier regulation modifies absorption. In addition to providing the driving force for paracel-lular transport, transcellular transport can activate intra-cellular signalling events that regulate the tight junction barrier. The best studied physiological example of this is the increased intestinal paracellular permeability that is induced by apical na+–nutrient co-transport21. This allows passive paracellular absorption of nutrients and water to amplify transcellular nutrient absorption, partic-ularly when high luminal nutrient concentrations exceed the capacity of apical na+–nutrient co-transporters22. ultrastructural analyses of intestinal epithelial cells during na+–nutrient co-transport revealed condensation of the perijunctional actomyosin ring, suggesting that cytoskel-etal contraction could be involved in this physiological process21. Subsequent studies confirmed this hypothesis and showed that the Ca2+–calmodulin-dependent serine–threonine protein kinase myosin light chain kinase (MLCK) is essential for na+–nutrient co-transport-induced tight junction regulation23,24. Moreover, MLCK activation alone is sufficient to increase tight junction perme ability, both in vitro and in vivo25,26. The mechanisms that mediate this

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http://www.uniprot.org/uniprot/P12830

http://www.uniprot.org/uniprot/O60716



http://www.uniprot.org/uniprot/Q07157

http://www.uniprot.org/uniprot/Q9UDY2



http://www.uniprot.org/uniprot/Q9Y5I7



Thick ascending limb of HenleThe portion of the nephron just proximal to the distal tubule. This is a site of active Na+, K+ and Cl– reabsorption, which generates an electrochemical gradient that drives paracellular reabsorption of Mg2+ and Ca2+.

Myosin light chain kinaseThe Ca2+–calmodulin-dependent kinase that phosphorylates myosin II regulatory light chain at serine 19 and threonine 18 to activate myosin ATPase.

MLCK-dependent tight junction regulation have been studied in detail owing to their contributions to nutri-ent and water absorption under normal physiological conditions and to the diarrhoea associated with acute barrier loss (BOX 1).

Barrier regulation by immune stimuli The ability of cytokines, such as TnF and IFnγ, to regulate the function of the tight junction barrier was first described 20 years ago27. Since then, increased tight junction protein transcription, vesicular removal of pro-teins from the tight junction, tight junction protein deg-radation, kinase activation and cytoskeletal modulation have all been proposed to mediate cytokine-induced loss of tight junction barrier function. Although extensive apoptosis of epithelial cells may also cause barrier loss, the relevance of single-cell apoptosis to barrier dysfunc-tion remains controversial owing to differing results in diverse experimental systems.

Tight junction regulation through the cytoskeleton. TnF and IFnγ modify tight junction barrier function in intestinal27,28, renal29, pulmonary30 and salivary gland31 epithelia as well as between endothelial cells32. The effects of TnF on barrier integrity have been best studied in the gut, where this cytokine has a central role in many diseases associated with intestinal epith elial barrier dysfunction, including inflammatory bowel disease33, intestinal ischaemia28,34 and graft-versus- host disease35. For example, although the effect of therapy

with TnF-specific antibodies may be largely due to the overall reduction in inflammation, it is notable that this treatment corrects barrier dysfunction in patients with Crohn’s disease36. Increased mucosal TnF production may also contribute to increased intestinal permeabil-ity and susceptibility to colitis in mice with defective mucin biosynthesis10,37.

MLCK has been shown to have a central role in TnF-induced epithelial and endothelial barrier dysregulation, both in vitro and in vivo16,38–41. Similar to na+–nutrient co-transport, TnF-induced MLCK activation seems to increase paracellular flux through the leak pathway16–17,23. This MLCK activation occurs as a result of increased enzymatic activity and increased MLCK transcription and translation, both in vitro and in vivo16,40,42. Similarly, MLCK expression and activity are increased in intesti-nal epithelial cells of patients with inflammatory bowel disease43. The degree to which MLCK expression and activity are increased correlates with local disease activ-ity, suggesting that these processes may be regulated by local cytokine signalling in these patients43. MLCK is also a fundamental intermediate in barrier dysfunction induced by the TnF family member LIGHT (also known as TnFSF14)17,44, interleukin-1β (IL-1β)45, enteropatho-genic Escherichia coli infection39, Helicobacter pylori infection46, giardiasis47, lipopolysaccharide48,49 and the ethanol metabolite acetaldehyde50. Thus, MLCK activa-tion can be viewed as a common final pathway of acute tight junction regulation in response to a broad range of immune and infectious stimuli.


Nodiarrhoea

H2O

Na+

Na+

a Normal homeostasisH2O

Na+

b Barrier disruption only

Milddiarrhoea

H2O

Na+

Na+

c Na+ malabsorption only

H2O

H2O

Na+

Na+

d Na+ malabsorption and barrier disruption

Nodiarrhoea

Copiousdiarrhoea

Na+

Box 1 | Coordination of transcellular and paracellular transport

The signal transduction pathway that enhances paracellular permeability following initiation of Na+–glucose co-transport has been characterized and includes activation of mitogen-activated protein kinase cascades, trafficking of Na+–H+-exchange protein 3 (NHE3) to the apical membrane and myosin light chain kinase (MLCK) activation23–24,107. Increased NHE3 activity at the apical membrane enhances Na+ absorption, which (along with the Na+ absorbed as a result of Na+–glucose co-transport) increases the transcellular Na+ gradient and promotes paracellular water absorption (see the figure, part a). The MLCK-dependent increase in tight junction permeability enhances paracellular absorption of water and small solutes, such as glucose, that are concentrated in the unstirred layer. This might contribute to the observation that rehydration after infectious diarrhoea, or even exercise, is more effective when oral rehydration solutions contain Na+ and carbohydrates108 (see the figure, part b). By contrast, tumour necrosis factor activates both protein kinase Cα (PKCα) and MLCK to cause diarrhoea. NHE3 inhibition, mediated by PKCα, reduces the transcellular Na+ gradient that normally drives water absorption (see the figure, part c). This synergizes with MLCK- dependent increases in tight junction permeability to allow water to flow into the lumen, thereby causing large-volume diarrhoea17 (see the figure, part d).

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a b

c d

CaveolaeSpecialized flask-shaped invaginations of the plasma membrane that contain the protein caveolin-1 and cholesterol. These proteins mediate uptake of some extracellular materials and are involved in cell signalling.

Although it is clear that MLCK phosphorylates myosin II regulatory light chain (MLC) within the peri-junctional actomyosin ring to activate myosin ATPase activity, the subsequent molecular events that cause increased permeability are poorly defined. However, recent work suggests that Zo1, which interacts directly with actin, occludin, claudins and other proteins, may be an essential effector of perijunctional actomyosin ring-mediated tight junction regulation25,51,52. Endocytic removal of the transmembrane protein occludin from the tight junction is also common in actomyosin-dependent, cytokine-mediated tight junction regulation38,53 (FIG. 2). In vitro studies of tight junction regulation induced by LIGHT suggest that occludin endocytosis occurs via caveolae and that inhibition of this process can prevent loss of barrier integrity despite MLCK activa-tion44. Caveolar endocytosis of occludin has also been associated with loss of epithelial and endothelial tight junction barrier function in response to actin disrup-tion54 and chemokine signalling55, respectively, in vitro. However, other mechanisms of occludin removal may also be involved as in vitro studies have shown that IFnγ-induced occludin internalization is mediated by myosin ATPase-dependent macropinocytosis53,56, and occludin cleavage may even modify the barrier to enhance transepithelial migration of inflamma-tory cells in the lung57. nevertheless, further work is necessary to define the contributions of occludin and endocytosis to in vivo tight junction regulation. This is particularly important as, despite numerous in vitro studies demonstrating a role for occludin in tight junction function, intestinal barrier function is intact in occludin-deficient mice58. Future analyses of the response of occludin-deficient mice to stress, with particular reference to intestinal tight junction

function, as well as studies of potential compensatory changes in other, perhaps not yet discovered, proteins that allow these mice to avoid intestinal disease will be of great interest.

Although MLCK activation is clearly important, it is not the only means of cytoskeletal tight junction regulation. other mediators include myosin ATPase51, the activity of which is regulated by MLC phosphory-lation59–60; members of the Rho kinase family41,51,53,61, which can both phosphorylate MLC directly 59 and inhibit MLC phosphatase62; and AMP-activated protein kinase52, which is activated during stress and can also directly phosphorylate MLC63. Moreover, Rho kinases and AMP-activated protein kinase each have diverse effects that are separate from myosin function, and it is likely that at least some of these contribute to tight junction regulation.

Tight junction permeability and claudin expression. As discussed above, regulation of perijunctional acto-myosin provides a means of rapidly and reversibly regulating the paracellular leak pathway. By contrast, synthesis and trafficking of claudin proteins provides a means of regulating tight junction pores over longer periods. The perijunctional actomyosin ring does not seem to be directly involved in this mechanism of bar-rier regulation, which is consistent with the fact that claudins do not interact with actin directly. Expression of specific claudin proteins changes during develop-ment, differentiation and disease and in response to stressors — including cytokines — in intestinal64, renal65 and alveolar66 epithelial cells. In addition to affecting barrier function, altered patterns of claudin expression may have other consequences. For example, claudin proteins have been associated with control of cell and organ growth. It may be, therefore, that some changes in claudin protein expression enhance cell proliferation and regeneration, as might be necessary to compensate for cell loss in colitis. This may explain the increased claudin-1 expression by intestinal epithelial cells of patients with inflammatory bowel disease67. However, claudin-1 has also been shown to enhance neoplastic transformation, tumour growth and metastasis in exper-imental models68. Thus, changes in claudin expression may have undesired consequences.

one common change in claudin expression that directly affects barrier function is the increased claudin-2 expression by intestinal epithelial cells in animal models of colitis (FIG. 2) and patients with inflammatory bowel disease64. Consistent with this, IL-13 and IL-17, which are increased in the mucosa of patients with colitis64,69, reduce barrier function and increase claudin-2 expres-sion in cultured intestinal epithelium monolayers64,70. In vitro studies have shown that claudin-2 expression increases the number of pores that allow paracellular flux of cations, predominantly na+, and small mole-cules with radii less than 4 Å14,71, and recent analyses have provided new insight into the structure of these pores72. However, it is not clear whether increased claudin-2 expression contributes to disease progres-sion or, as proposed above for claudin-1, is an adaptive

Figure 2 | Differential effects of cytokines on tight junction structure and function. a | Occludin is normally concentrated at the tight junction (arrow) in jejunal villus epithelium. The perijunctional actomyosin ring (red) and nuclei (blue) are shown for reference. b | Exogenous tumour necrosis factor increases myosin light chain kinase activity, which causes perijunctional myosin II regulatory light chain phosphorylation and triggers occludin (green) endocytosis (arrow). This increases flux across the tight junction leak pathway and enhances paracellular permeability to large solutes. c | Claudin‑2 expression (green) is limited to crypt epithelial cells; it is not expressed by epithelial cells at the mucosal surface in the normal colon. F‑actin (red) and nuclei (blue) are shown for reference. d | Interleukin‑13 can stimulate claudin‑2 expression (green) in surface epithelial cells (arrows). This increases flux across small tight junction pores, thereby enhancing paracellular cation permeability.

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SAMP1/yit miceAn outbred mouse strain that spontaneously develops a chronic intestinal inflammation similar to human Crohn’s disease.

response that promotes homeostasis. nevertheless, the consistency of intestinal epithelial claudin-2 upregula-tion in disease suggests that this should be a subject of further study.

Barrier function and immunityBarrier loss is associated with disease risk. A large body of circumstantial evidence suggests that intestinal bar-rier dysfunction is associated with the pathogenesis of Crohn’s disease. This includes the observation that a sub-set of first-degree relatives of patients with Crohn’s dis-ease has increased intestinal permeability despite being completely healthy73. Interestingly, genetic analyses have linked increased intestinal permeability in these healthy relatives to the Crohn’s disease-associated frameshift

insertion at nucleotide 3020 of the gene encoding the cytoplasmic sensor nucleotide-binding oligomerization domain-containing 2 (NOD2)74 (TABLe 1), and the same NOD2 mutation has been associated with decreased IL-10 production by peripheral blood mononuclear cells75. Thus, one explanation for the increased intes-tinal permeability observed in some of these healthy relatives is that they have subclinical mucosal immune activation, perhaps with increased TnF production, that leads to barrier dysfunction without overt disease. This hypothesis would also explain why IL-10-deficient and SAMP1/yit mice, which develop spontaneous colitis and enteritis, respectively, show increased intestinal per-meability before disease onset76,77. The suggestion that permeability is merely a sensitive indicator of mucosal

Table 1 | Barrier defects associated with intestinal disease

Disease or model Cause of barrier defect

Timing of barrier defect

Effects on tight junction proteins and cytoskeleton

Role of commensal microorganisms

Refs

Crohn’s disease Unknown, associated with a frameshift insertion at nucleotide 3020 of NOD2

Before clinical onset and before clinical relapse

Claudin‑2 upregulation, MLCK activation and occludin downregulation

Antibiotics can be helpful in maintaining remission

43,64,109

Ulcerative colitis Unknown Not well studied Claudin‑2 upregulation, MLCK activation and occludin downregulation

Not defined 43,64,109

IL‑10‑deficient mice Immune signalling (IL‑10 deficiency)

Before clinical onset Not defined Eradication of microorganisms prevents disease

76,97

Coeliac disease Not well studied Not well studied Not defined No demonstrated role 110,111

Systemic T cell activation

MLCK activation Associated with acute diarrhoea

MLCK activation and occludin endocytosis

Not defined 38

CD4+CD45RBhi T cell adoptive transfer model of colitis

Cytokine release and epithelial cell damage

With clinical onset MLCK activation, claudin‑2 upregulation and occludin endocytosis

Eradication of microorganisms reduces severity

112–114

SAMP1/yit mice Unknown Before clinical onset Claudin‑2 upregulation and occludin downregulation

Not defined 77

DSS‑induced colitis Epithelial cell damage After DSS treatment but before clinical onset

Not defined Eradication of microorganisms exacerbates disease

80

TNBS‑induced colitis Immune signalling Not well studied Claudin‑18 upregulation Probiotics can reduce disease severity

92

Mucin‑2‑deficient mice

Not well studied Not well studied Not defined Not defined 10,37

MDR1A‑deficent mice

Unknown, follows increased CCL2 production

After immune cell activation

Reduced occludin phosphorylation

Increased epithelial cell response to LPS precedes disease

115,116

JAM‑A‑deficient mice

JAM‑A deficiency Not well studied JAM‑A deficiency Not defined 89,90

Clostridium difficile‑induced colitis

Actomyosin disruption and glucosylation of Rho proteins

With release of toxin and disease onset

Loss of ZO1 and ZO2 Antibiotics predispose to disease

117

EPEC infection Type III secretion (of bacterial proteins)

After infection MLCK activation and occludin endocytosis

Not defined 118

Graft‑versus‑host disease

Associated with elevated TNF production

After clinical onset Not defined Eradication of microorganisms limits disease

35

CCL2, CC‑chemokine ligand 2; DSS, dextran‑sulphate sodium; EPEC, enteropathogenic Escherichia coli; IL‑10, interleukin‑10; JAM‑A, junctional adhesion molecule‑A; LPS, lipopolysaccharide; MDR1A, multidrug resistance protein 1a; MLCK, myosin light chain kinase; NOD, nucleotide‑binding oligomerization domain; TNF, tumour necrosis factor; TNBS, trinitrobenzene sulphonic acid; ZO, zonula occludens.

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CD4+CD45RBhi T cell adoptive transfer colitis modelA well-characterized model of chronic colitis induced by transfer of CD4+CD45RBhi (naive) T cells from healthy wild-type mice into immunodeficient syngeneic recipients.

immune activation would also explain the observa-tion that, during clinical remission, increased intestinal permeability is a predictor of relapse in patients with Crohn’s disease78. However, barrier dysfunction must also be regarded as a potential contributor to disease progression.

The classic experiment showing a role for epithelial cell function in maintaining mucosal immune homeo-stasis used chimeric mice in which dominant-negative cadherin was expressed in some intestinal epithelial stem cells, resulting in disruption of the adherens junctions13. This led to profound epithelial cell defects that included incomplete polarization, brush border and actin cytoskel-etal disruption, accelerated crypt–villus migration and premature apoptosis13. Moreover, the mucus layer over-lying epithelial cells expressing dominant-negative cad-herin was disrupted and numerous adherent bacteria were present at these sites13. Patchy, transmural enteritis developed in these mice and was limited to regions where epithelial cells expressed dominant-negative cadherin79. In addition, epithelial cell dysplasia was occasionally present in these areas79. This study has often been cited as evidence that tight junction barrier loss is sufficient to cause inflammatory bowel disease and, although tight junctions were not specifically examined, they were likely to be defective. However, although this important study shows that apical junctional complex disruption causes enteritis, the broad range of epithelial cell defects induced makes it impossible to draw conclusions regarding the specific contributions of the tight junction to homeostasis or disease. Similarly, although dextran-sulphate sodium (DSS)-induced colitis is associated with increased intes-tinal permeability, this is the result of widespread epithe-lial cell damage80 rather than targeted dysfunction of the tight junction barrier. Altered sensitivity of genetically modified mice to DSS must therefore be viewed in the context of epithelial cell injury and repair81–83 and cannot be interpreted as a function of disrupted tight junction permeability alone.

Isolated barrier loss is insufficient to cause disease. one study has reported an in vivo model of barrier dys-function induced by direct activation of endogenous tight junction regulatory mechanisms26. Although no overt developmental defects or disease developed in mice in which intestinal epithelial cells transgenically expressed constitutively active MLCK, increased intes-tinal tight junction permeability was observed26. This increase in paracellular permeability was quantitatively similar to that induced by activation of na+–glucose co-transport and was not an indiscriminate loss of barrier function26, as occurs with exposure to DSS or dominant-negative cadherin expression. In addition, MLCK inhibition normalized MLC phosphorylation in epithelial cells and paracellular permeability26, sug-gesting that compensatory changes in pathways that regulate MLC phosphorylation or barrier function were not induced. Moreover, growth of these mice was normal, and intestinal morphology, enterocyte struc-ture, tight junction and adherens junction organiza-tion, actomyosin and brush border architecture, and

epithelial proliferation, migration and apoptosis were all similar to that observed in wild-type mice26. This suggests that the effects of constitutively active MLCK expression in these mice were specific to its effect on the tight junction. Thus, consistent with the presence of barrier dysfunction in healthy relatives of patients with Crohn’s disease, these mice provide evidence to suggest that increased tight junction permeability in the absence of more extensive epithelial cell dysfunc-tion is insufficient to cause intestinal disease. Further analysis of mice expressing constitutively active MLCK did, however, provide evidence of mucosal immune cell activation, including increased numbers of lamina pro-pria T cells, enhanced mucosal IFnγ, TnF and IL-10 transcription and repositioning of CD11c+ DCs to the superficial lamina propria26. Thus, despite being insuf-ficient to cause disease, chronic increases in intestinal permeability as a result of continuous activation of a physiological pathway of tight junction regulation does lead to mucosal immune cell activation.

Because a subset of healthy first-degree relatives of patients with Crohn’s disease will ultimately develop the disease, and because a similar fraction of these healthy relatives have increased intestinal permeability, it has been suggested that tight junction barrier dysfunction is one factor that contributes to the development of inflammatory bowel disease84. This hypothesis is consist-ent with a case report documenting increased intestinal permeability 8 years prior to disease onset in a healthy relative of a patient with Crohn’s disease85. However, given that the subject of the case report had two first-degree relatives with Crohn’s disease and was, therefore, at increased risk of developing the disease regardless of intestinal permeability measures, this report does not provide evidence that barrier dysfunction itself is related to disease onset.

The relationship between barrier dysfunction and disease has been assessed using mice with a constitutively active MLCK transgene, which have increases in permea-bility that are quantitatively and qualitatively similar to those in healthy relatives of patients with Crohn’s disease. Recombination-activating gene 1-knockout mice (which lack mature B and T cells) expressing constitutively active MLCK, as well as littermates that did not express constitutively active MLCK, received CD4+CD45RBhi naive T cells from wild-type mice (the CD4+CD45RBhi T cell adoptive transfer colitis model). Both groups of mice developed colitis. However, the disease developed more rapidly and was more severe, in clinical, biochemical and histological terms, in the mice that expressed constitu-tively active MLCK26. Although one could argue that this accelerated disease progression was due to effects of constitutively active MLCK expression beyond tight junction regulation, other epithelial cell defects were not apparent in these mice (see above), perhaps because con-stitutively active MLCK targets an endogenous signalling pathway. Thus, targeted increases in intestinal epithelial tight junction permeability through constitutive activa-tion of the pathophysiologically relevant MLCK pathway are sufficient to accelerate the onset and enhance the severity of immune-mediated colitis.

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Latency-associated peptideA small peptide derived from the N-terminal region of the TGFβ precursor protein; it can modulate TGFβ signalling.

Conversely, the data above also suggest that inhibi-tion of MLCK, which is sufficient to reverse acute TnF-induced barrier loss38, may have therapeutic benefit in immune-mediated colitis. This notion is supported by a preliminary in vivo report using mice with a knock-out of non-muscle MLCK86. However, as non-muscle MLCK has many functions, including an essential role in neutrophil transendothelial migration87, the results of this report may reflect changes beyond tight junc-tion barrier preservation. Another recent study showed that a peptide capable of enhancing small-intestinal barrier function reduced the extent of mucosal inflam-mation in IL-10-deficient mice88. However, this result must be interpreted with caution, as the peptide used (which is thought to antagonize the putative extra-cellular pathway signalling molecule zonulin) activates an undefined regulatory pathway and the overall physi-ology of peptide-treated mice has not been examined in detail. Thus, although the finding is intriguing, further investigation is necessary to determine whether barrier preservation, by MLCK inhibition or other means, can prevent or reverse intestinal disease.

Two studies have recently reported that junctional adhesion molecule-A (JAM-A)-deficient mice have reduced intestinal barrier function and increased rates of intestinal epithelial cell proliferation and apopto-sis89,90. These mice also have intestinal mucosal immune cell activation as defined by increased numbers of neutrophils in the intestinal mucosa89,90. In addition, one study reported an increase in the number of dis-tal-colonic lymphoid aggregates in JAM-A-deficient mice90, although neither lymphoid aggregates nor enhanced mucosal cytokine expression was detected in a subsequent analysis89. However, as JAM-A is widely expressed by epithelial cells, endothelial cells, platelets, antigen-presenting cells, circulating neutrophils, mono-cytes, lymphocytes and platelets, and as the mice studied were not epithelial cell-specific knockouts, it is not clear whether the observed effects were due to altered tight junction integrity, changes in epithelial cell shape91, an increased rate of epithelial cell apoptosis90 or trapping of neutrophils within mucosal vessels owing to the loss of endothelial cell-expressed JAM-A. nevertheless, it is intriguing that JAM-A-deficient mice showed increased sensitivity to DSS-induced colitis89,90. By contrast, the sensitivity of mice with an endothelium-specific JAM-A deficiency to DSS was similar to that of wild-type control mice, indicating that the response of mice with universal JAM-A deficiency was not solely due to loss of endothelial JAM-A89. unfortunately, the use of mice with a universal JAM-A deficiency and the presence of other epithelial cell abnormalities, particularly increased epi-thelial cell turnover, in the absence of exogenous stimuli limits interpretation of these studies. Even so, data from JAM-A-deficient mice do support the hypothesis that mucosal barrier loss can enhance the severity of colitis.

overall, data from both human subjects and mouse experimental models show that defects in tight junc-tion barrier function are insufficient to cause disease. However, several lines of evidence suggest that increased paracellular permeability can increase mucosal immune

activity, enhance disease progression and severity and, possibly, be a risk factor for development of disease. Finally, although much more investigation is needed, early reports indicate that restoration of tight junction barrier function may be effective, either alone or in combination with other agents, in preventing disease in at-risk individuals or maintaining remission in patients with inflammatory bowel disease.

Barrier loss activates immunoregulatory processes. why is tight junction barrier dysfunction alone insufficient to cause disease? Endoscopic mucosal resection, which removes the epithelium and mucosa completely and therefore causes barrier loss far greater than that caused by targeted tight junction dysfunction, is insufficient to cause chronic disease. Therefore, immuno regulatory mechanisms must be induced by the host following barrier loss to prevent inappropriate inflammatory responses, as mucosal damage is a daily occurrence in the gastrointestinal tract. Moreover, these immuno-regulatory mechanisms must be robust because, even in patients with inflammatory bowel disease, endoscopic mucosal resection is insufficient to initiate a relapse to active disease.

The increased transcription of IL-10 in the intestinal mucosa of mice with a constitutively active MLCK trans-gene may provide a clue to which immuno regulatory processes are triggered by barrier dysfunction. In addi-tion, responses to transient barrier loss have been used to explore mucosal immunoregulation92. In this model, intrarectal administration of ethanol caused transient epithelial cell damage, mucosal erosion and barrier loss. The induction of barrier loss was followed by an increase in the numbers of IFnγ- and IL-10-producing lamina propria mononuclear cells and lamina propria CD4+CD25+ T cells that express latency-associated peptide (LAP) on their surface92. Remarkably, such ethanol administration conferred protection from subsequent trinitrobenzene sulphonic acid (TnBS)-induced coli-tis, and this protection required the presence of LAP+ T cells92. The induction of these LAP+ T cells was shown to depend on CD11c+ DCs, Toll-like receptor 2 signalling and a normal luminal microorganism population92.

Although further study is needed to understand the mechanisms of LAP+ T cell induction by transient mucosal damage, it is interesting that in different experi-mental systems increased intestinal epithelial cell tight junction permeability increased the number of CD11c+ DCs in the superficial lamina propria86, and that inter-actions with intestinal epithelial cells enhance the abil-ity of bone marrow-derived CD11c+ DCs to induce the differentiation of regulatory T cells93. Together, these observations support the hypothesis that the interactions between CD11c+ DCs and luminal materials are regu-lated by tight junction permeability and are also central to mucosal immune homeostasis.

other data also support important roles for luminal material, particularly microorganisms and their prod-ucts, in mucosal immune regulation94. For example, antibiotics can be helpful in the management of Crohn’s disease95. Although the mechanisms by which antibiotics

R E V I E W S



http://www.uniprot.org/uniprot/Q9Y624



Epithelial cell

Laminapropria

Bacterial products and dietary antigens

MLCK

Endosome

Occludin

MHC

TCR

TReg cell

T cell TH1 cell

Antigen

IFNγTNF

TGFβRetinoic acid

IL-10 and TGFβ

IL-13

TH2 cell

Na+

Small solutes

Na+

Small solutes

Claudin Claudin-2

LAP

Actomyosinring

TLR

APC

promote maintenance of remission in patients with Crohn’s disease are not defined, they may be related to the observation that luminal microorganisms are required in experimental models of inflammatory bowel disease that include IL-10-deficient mice96,97 and adop-tive transfer colitis. Microorganisms are also necessary for the development of increased intestinal permea-bility before the onset of overt disease in IL-10-deficient mice76. Although this observation is not entirely under-stood, one interpretation is that the limited flux of microbial products that normally occurs across intact intestinal epithelial tight junctions is sufficient to trig-ger mucosal immune activation in IL-10-deficient mice. This mucosal immune activation could, in turn, result in the release of cytokines that cause increased intestinal permeability and accelerate disease progression (FIG. 3). However, in addition to immune cells, epithelial cells can respond to foreign materials. Lipopolysaccharide, for

example, enhances paracellular permeability in pulmo-nary and intestinal epithelium48,49. Conversely, epithelial Toll-like receptor 2 activation may restore barrier func-tion98. It will therefore be of interest to define the micro-bial products, dietary components and other factors that influence mucosal immune status following alterations in intestinal epithelial tight junction permeability.

Tight junctions integrate mucosal homeostasisone interpretation of the available data is that the tight junction barrier integrates the relationship between lumi-nal material and mucosal immune function (FIG. 3). In most individuals, this is a healthy relationship in which regulated increases in tight junction permeability or transient epithelial cell damage trigger the release of pro-inflammatory cytokines, such as TnF and IFnγ, as well as immunoregulatory responses. Immunoregulatory responses may include DC conditioning by epithelial cell-derived transforming growth factor-β (TGFβ) and retinoic acid, both of which can enhance regulatory T cell differentiation99,100. This precarious balance between pro-inflammatory and immunoregulatory responses can fail if there are exaggerated responses to pro-inflammatory cytokines, as may be associated with mutations in the endoplasmic reticulum stress response transcription factor X-box-binding protein 1 (XBP1) (ReF. 101), insuf-ficient IL-10 production (as in IL-10-deficient mice and, possibly, in patients with IL10 promoter polymor-phisms102) or inadequate immune tolerance to luminal antigens and microbial products94 (potentially because of NOD2 mutations in patients with inflammatory bowel disease103–106). As a result, mucosal immune cell activation may proceed unchecked and the release of cytokines, including TnF and IL-13, may enhance bar-rier loss that, in turn, allows further leakage of luminal material and perpetuates the pro-inflammatory cycle. This model highlights the roles of a susceptible host and defective epithelial cell barrier function as key compo-nents of the pathogenesis of intestinal inflammatory disease and explains the crucial role of the epithelial barrier in moulding mucosal immune responses.

ConclusionsSignificant progress has been made in understanding the processes by which physiological and pathophysio-logical stimuli, including cytokines, regulate the tight junction. Early reports suggest that restoration of tight junction barrier function may have benefit38,86. However, the mechanisms of tight junction regulation will have to be defined in greater detail if they are to be viable pharmacological targets. Conversely, recent data have emphasized the presence of immune mechanisms that maintain mucosal homeostasis despite barrier dysfunction, and some data suggest that the epithelium orchestrates these immunoregulatory events through direct interactions with innate immune cells. Future elucidation of the processes that integrate mucosal bar-rier function, or dysfunction, and immune regulation to prevent or perpetuate disease may lead to novel thera-peutic approaches for diseases associated with increased mucosal permeability.

Figure 3 | The epithelium and tight junction as integrators of mucosal homeostasis. Minor barrier defects allow bacterial products and dietary antigens to cross the epithelium and enter the lamina propria. This can lead to disease or homeostasis. If the foreign materials are taken up by antigen‑presenting cells (APCs), such as dendritic cells, that direct the differentiation of T helper 1 (T

H1) or T

H2 cells, disease can develop. In this

process, APCs and TH1 cells can release tumour necrosis factor (TNF) and interferon‑γ

(IFNγ), which signal to epithelial cells to increase flux across the tight junction leak pathway, thereby allowing further leakage of bacterial products and dietary antigens from the lumen into the lamina propria and amplifying the cycle of inflammation. This may, ultimately, culminate in established disease. Alternatively, interleukin‑13 (IL‑13) released by T

H2 cells increases flux across small cation‑selective pores, potentially contributing to

ongoing disease. Conversely, homeostasis may dominate if APCs promote regulatory T (T

Reg) cell differentiation, which can be enhanced by epithelial cell‑derived transforming

growth factor‑β (TGFβ) and retinoic acid. The TReg

cells display latency‑associated peptide (LAP) on their surfaces and may secrete IL‑10 and TGFβ to prevent disease. MLCK, myosin light chain kinase; TLR, Toll‑like receptor; TCR, T cell receptor.

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AcknowledgementsI am indebted to current and past members of my laboratory for discussions and investigations that contributed to this article. Thanks also to A. M. Marchiando and C. R. Weber for the images used in figures 1 and 2. Work in my laboratory is currently supported by the US National Institutes of Health (DK061931, DK068271, DK67887 and HL091889), the University of Chicago Cancer Center (CA14599), the University of Chicago Digestive Disease Research Core Center (DK042086 ) , t he US Depar tmen t o f De f ense (W81XWH-09-1-0341), the Broad Medical Research Program (IBD-0272) and the Chicago Biomedical Consortium. I apologize to colleagues whose work and publications could not be referenced owing to space constraints.

DATABASESUniProtKB: http://www.uniprot.orgα‑catenin 1 | β‑catenin | catenin δ1 | claudin‑1 | claudin‑2 | claudin‑16 | E‑cadherin | IFNγ | IL‑1β | IL‑10 | JAM‑A | LAP | LIGHT | MLCK | NHE3 | TNF | XBP1 | ZO1 | ZO2

FURTHER INFORMATIONJerrold R. Turner’s homepage: http://pathologyweb.bsd.uchicago.edu/faculty/JTurner.html

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FromMucinstoMucus.ThorntonDJandSheehanJK,ProcAmThoracSoc.,1:54(2004)

From Mucins to MucusToward a More Coherent Understanding of This Essential Barrier

David J. Thornton and John K. Sheehan

Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, United Kingdom; andDepartment of Biophysics and Biochemistry, Cystic Fibrosis Research Center, University of North Carolina, Chapel Hill, North Carolina

Mucus is essential for protection of the airways; however, in chronicairway disease mucus hypersecretion is an important factor in mor-bidity and mortality. The properties of the mucus gel are dictatedin large part by the oligomeric mucins and, over the past decade,we have gained a better understanding of the molecular nature ofthese complex O-linked glycoproteins. We know now that MUC5ACmucins, as well as different glycoforms of the MUC5B mucin, arethe predominant gel-forming glycoproteins in airways mucus. Fur-thermore, the amount, molecular size, and morphology of theseglycoproteins can be altered in disease. From more recent data, ithas become clear that oligomeric mucins alone do not constitutemucus, and other mucin and nonmucin components must be impor-tant contributors to mucus organization and hence airways defense.Therefore, the challenge over the coming decade will be to investi-gate how the oligomeric mucins are organized to yield “functional”mucus. Such studies will provide a clearer perception of airwaysmucosal protection and may highlight specific components as po-tential targets for therapeutic strategies for the treatment of hyper-secretory disease.

Keywords: mucus; sputum; MUC5AC; MUC2; MUC5B

The respiratory mucus gel performs a number of essential func-tions that collectively lead to the protection of the airways. Thishighly hydrated gel, in conjunction with ciliated epithelial cells,forms the mucociliary “escalator,” which, along with cough, isessential for the maintenance of sterile and unobstructed air-ways. By contrast, overproduction of mucus with altered rheo-logic properties is an important factor in the morbidity and mor-tality of chronic airways disease (e.g., asthma, cystic fibrosis [CF],and chronic obstructive pulmonary disease [COPD]) (1–3). As aresult of the change in physical properties of the gel, mucociliaryclearance is impaired or abolished with the attendant problemsof poor gas exchange and/or inflammation and/or bacterial colo-nization. Thus, production of mucus with the “correct” proper-ties for airways protection is paramount for health.

A variety of factors have been found to trigger mucus hyper-secretion, including proinflammatory cytokines (e.g., interleu-kin-4, interleukin-13, epidermal growth factor, and tumor necro-sis factor-�), ATP, bacterial exoproducts, and host proteases(4–9), but the mechanisms underlying mucus biogenesis andsecretion, and its molecular composition and supramolecular or-ganization, are still obscure. The major gel-forming componentsof the airways mucus gel are the oligomeric mucins, and thehypothesis that drives our research is that these glycoproteins

(Received in original form June 19, 2003; accepted in final form September 30, 2003)

Supported by the Wellcome Trust and the National Asthma Campaign.

Correspondence and requests for reprints should be addressed to David J. Thorn-ton, B.Sc., Ph.D., Wellcome Trust Centre for Cell-Matrix Research, School ofBiological Sciences, University of Manchester, 2.205 Stopford Building, Manches-ter, M13 9PT, UK. E-Mail: [email protected]

Proc Am Thorac Soc Vol 1. pp 54–61, 2004DOI: 10.1513/pats.2306016Internet address: www.atsjournals.org

play the vital role in determining the physical properties of thisessential barrier in health and disease. However, it should alsobe pointed out, especially in pathologic conditions, that othermolecules such as proteoglycans and DNA may contribute tothe physical properties of airways mucus.

Over the last decade, great strides have been made in identi-fying and characterizing the major oligomeric mucins present inairways sputa. However, there have been no studies to date thatrelate the ensemble of their physical properties (i.e., length,charge density, macromolecular architecture) to a specific set ofrheologic parameters for a mucus gel, never mind its optimiza-tion to a specific function, such as ciliary transport. In this article,we have been given the task of reviewing our own contributionsto this area. We have also suggested further avenues of investiga-tion that will be necessary to gain a better understanding of thebiology and pathobiology of this essential barrier.

MUCINS—WHAT ARE THEY?

Mucins are currently defined as high-Mr glycoproteins that con-tain at least one and sometimes multiple protein domains thatare sites of extensive O-glycan attachment (mucin-like domains).Thus, the composition of these glycoproteins is dominated bycarbohydrate, which can total in some cases as much as 80% ofthe weight of the molecule. There appear to be two major typesof mucin, one thought to be monomeric and, primarily, but notexclusively, located at the cell surface, and the other oligomeric.This latter type is secreted and thought to be responsible forthe rheologic properties of mucus. The mucin-like domains areenriched in the amino acids serine and threonine, which are thesites for the attachment to the linkage sugar N-acetylgalactos-amine. In addition to N-acetylgalactosamine, mucin oligosaccha-rides may also contain N-acetylglucosamine, galactose, fucose,sialic acids, and sulfate. The oligosaccharides have remarkablestructural diversity and all of their specific roles remain to befully understood. However, a consequence of their attachmentto the polypeptide is to cause it to stiffen, resulting in a largeexpansion of the volume domain of the molecule, which givesmucins their characteristic space-filling, and thus gel-making,properties (10). Furthermore, it has long been known that certainbacteria bind specific oligosaccharide ligands (for a review see[11]). It is likely that the primary function of the oligosaccharidediversity is to enhance the possibility that bacteria bind to mucus,thus facilitating their removal by mucociliary transport. More-over, by providing competing receptors for cell-surface glycocon-jugates, mucins may trap bacteria and make them less successfulin their attempts to colonize the epithelium. Thus, the array ofoligosaccharides expressed on the mucins of an individual mayplay a key role in governing the susceptibility to infection.

The studies we shall summarize have been concerned solelywith the gel-forming mucins. From our studies in collaborationwith Ingemar Carlstedt in the early 1990s, it was clear that the gel-forming mucins in airways mucus consisted of a heterogeneousmixture of glycoproteins that was physically similar and extremelylarge (12–16). The mucus gel could be almost completely solubi-lized by noncovalent bond–breaking agents (e.g., 6M guanidi-

Thornton and Sheehan: From Mucins to Mucus 55

Figure 1. Electron micrographs of (A ) an intact oligomeric mu-cin, along with a generic model for oligomeric mucin organiza-tion and (B ) reduced mucin subunits. (A ) Transmission electronmicrograph of a respiratory mucin preparation showing a singleoligomeric mucin chain. Mucins were isolated from sputum bysolubilization with 6 M guanidinium chloride (GuCl) followedby purification by two stages of cesium chloride (CsCl) isopynicdensity gradient centrifugation, first in 4 M GuCl/CsCl andthen in 0.2 M GuCl/CsCl (12). Also shown is a cartoon of anexpanded view of a section of the mucin chain highlightingthe intermolecular, end-to-end, disulfide (S-S)-mediated as-sembly of the mucin subunits (monomers). The polypeptideconsists of alternating stretches of dense O-linked glycosylation(mucin-like domains) and folded, intramolecular disulfide bondstabilized regions (depicted as knots). A single monomer spansthe two S-S linkages. (B ) Transmission electron micrograph ofreduced mucin subunits generated after treatment of oligo-meric mucins (prepared as in A ) with a reducing agent (e.g.,10 mM dithiothreitol). The reduced subunits are approximately600 nm in length. Also shown is a cartoon of the reducedmucin subunit highlighting the unfolding of the intramoleculardisulfide stabilized domains situated at the ends and inter-spersed between the mucin-like domains. SH represents re-duced cysteine residues.

nium chloride), a process usually achieved overnight but in somecases taking days. Thus, entanglement of the long mucin chainswas considered to be the primary mechanism for gel formationand, as a consequence, the size, concentration, and chemical natureof the mucins are important factors for determining the proper-ties of the gel. Physical characterization of the mucins isolatedfrom sputum using light scattering and electron microscopyshowed them to be polydisperse in both mass (2–40 mD) and size(0.5–10 �m in length). Furthermore, these studies demonstratedthat the component mucin monomers or subunits (2–3 mD) areassembled linearly and held together by disulfide bonds (17).Figure 1 shows electron micrographs of oligomeric respiratorymucins before and after treatment with a reducing agent, alongwith the model proposed for their structure. It is important tonote that for these studies the mucins were isolated and purifiedin highly chaotropic solvents (e.g., 4–6 M guanidinium chloride)to preserve their primary structure. Thus, the observed architec-ture may not reflect their “native” conformation in mucus. Amore realistic view of their native conformation will require char-acterization of the mucins after isolation using nonchaotropicagents.

WHICH MUCINS ARE IMPORTANT IN THE AIRWAYSAND WHAT ARE THEY LIKE?

It is now apparent that these oligomeric glycoproteins are mem-bers of a larger family of mucins. Of the 13 mucin genes (MUC)currently identified, only 4 (MUC2, MUC5AC, MUC5B, andMUC6) contain the cysteine-rich motifs in their C- and N-termi-nal domains necessary for oligomerization (18–21). Three ofthese genes, MUC2, MUC5AC, and MUC5B, are expressed inthe airways. A number of important studies on airways mucinshave concentrated on messenger RNA expression, in particularfocusing on MUC2 and MUC5AC, and highlight specific disease-related factors that upregulate these two mucins (4–9, 22–24).However, the fact that these macromolecules are synthesizedand then stored awaiting the appropriate stimulus for secretionimplies that messenger RNA studies may be misleading for theamount of mucin in mucus. This is borne out by biochemical data,which have demonstrated that the products of the MUC5AC and

MUC5B genes are predominant in sputum (25–29) and that theMUC2 mucin is barely detectable (see subsequent text). Thus,there seems to be a discrepancy between MUC2 messenger RNAlevels and the occurrence of the mature MUC2 mucin in sputum.This may be due to the apparent “insolubility” of this glycopro-tein, as has been reported for MUC2 isolated from small-intesti-nal mucus (30), causing it to be retained in the airways. Whileour data do not rule this out, in the single study we have per-formed, where the mucus was physically removed from an asth-matic lung postmortem, there was still little evidence for thismucin (3, 31).

Matching the polypeptides of the different mucins present inmucus to their respective genes has been a long process, whichwas eventually achieved by fractionation of the mucins (afterreduction of their disulfide bonds) by anion exchange chroma-tography and agarose gel electrophoresis, and then by a combina-tion of biochemical analyses (i.e., amino acid composition, pep-tide sequencing, and peptide fingerprinting) as well as reactivitywith mucin-specific antisera (for more details see Figure 2).

The analysis of multiple sputum samples revealed that thetwo major mucin gene products, MUC5AC and MUC5B havedifferent chemical properties. The MUC5AC mucin has a morehomogeneous charge distribution than MUC5B, which occursin differently charged forms (26). In most samples analyzed, twodifferent MUC5B forms (glycoforms) are found, and while theircharge density is not identical between individuals it is apparentthat a “high” and “low” charge form occurs in most cases (Fig-ure 3). These glycoforms also exhibit different buoyant densitiesand, as a result, can be partially separated by isopycnic densitycentrifugation where there is a direct correlation between theircharge and buoyant densities (17). As far as we can tell, each gly-coform is found in separate oligomeric mucins and is not partof a heterooligomer. This separation, coupled with the distinctivepattern of glycosylation, at least at the level of charge density,associated with each mucin species, may reflect their differentcellular origins (see Where Are Mucins Synthesized?).

Studies performed on the intact MUC5AC and MUC5B mu-cins suggest that these mucins have different macromolecularcharacteristics. Sedimentation studies have indicated that bothare polydisperse in size, and this may arise from the oligomeriza-

56 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 1 2004

Figure 2. Identifying the major oligomeric mucins in sputum. Sputum is a mixture of oligomeric mucins (MUC2, MUC5AC, and MUC5B), whichare polydisperse in size and share similar biochemical and biophysical properties. Therefore, purification of the individual mucin species is notpossible with intact mucins but can be achieved by working with preparations of reduced mucins. (A ) A typical anion exchange chromatogramof a reduced and alkylated respiratory mucin preparation. In this case, the separation matrix was a Mono Q HR 5/5 column, which was elutedwith a linear salt gradient of 0–0.25 lithium perchlorate containing 6 M urea (dashed line). The mucin distribution (solid line) was monitored withthe periodic acid Schiff’s (PAS) reagent that detects the glycan component of the molecule. In this example, the mucin distribution was pooledinto three populations with increasing charge density (I, II, and III), which were further purified by rerunning on the Mono Q column. Fractionsfrom the rerun were pooled on the basis of their electrophoretic mobility on a 1% agarose gel and their reactivity with a number of lectins (datanot shown) to yield 3 distinct populations. (B ) The 3 resultant pools were subjected to 1% agarose gel electrophoresis and a Western blot of theagarose gel was probed with an antiserum that recognizes oligomeric mucins (64). Other Western blots (not shown) were probed with antiseraspecific for MUC2, MUC5AC, and MUC5B mucins (25, 26, 44) and the bands that were reactive with these probes are labeled. Two of the bandswere reactive with the MUC5B antiserum, and these correspond to populations I and III, which had very different charge densities as assessed byanion exchange chromatography (see Fig. 2A). Therefore, these bands are labeled as high- and low-charge variants of MUC5B. The third bandcorresponding to the intermediate charge species (population II) on the Mono Q column was reactive with the MUC5AC antiserum. None of thebands were stained with the MUC2 antiserum. The identification of mucins was not based solely on antisera reactivity but also relied on chemicalanalyses. For example, (C ) shows the MALDI-TOF MS peptide fingerprint after trypsin treatment of a reduced and alkylated MUC5B mucinpreparation. The four major peptides highlighted (peptides 1–4) were purified by reverse-phase chromatography on a C18 column and theirsequence determined by N-terminal sequencing (26). Their sequences and locations within the MUC5B polypeptide are shown in the boxedschematic diagram. This represents the central portion of the MUC5B apoprotein, showing the cysteine-rich domains (cys1–7, black boxes) andthe R and R-end domains (hatched boxes) corresponding to the glycosylated mucin-like domains (adapted from [20]). The four major peptidesare located in some of the seven cys-domains, which share a similar, repetitive sequence. The 62 amino acid sequence shown is found in threeof these domains (cys 4, 5, and 6) and the 2 peptides with masses 1,132.5 D (peptide 2) and 1,687.7 D (peptide 3) are also found in cys 7. Themultiple occurrences of these four peptides explain their dominance of the MUC5B tryptic fingerprint. Amino acid composition can also be usedto identify mucins. Shown in (D ) are the predicted contents (calculated from published deduced amino acid sequences) of serine, threonine, andproline in the mucin-like domains of the MUC2, MUC5AC, and MUC5B mucins. This analysis highlights the uniqueness of the MUC2 mucin inthis regard. Also shown are the experimentally derived values for these three amino acids generated from amino acid analysis of the highlyglycosylated fragments of the mucin (corresponding to the mucin-like domains) obtained after trypsin digestion of population I (MUC5B) andpopulation II (MUC5AC) from the Mono Q column. These data confirm the absence of significant amounts of MUC2 in a typical mucin preparation.For greater experimental detail concerning the separation and characterization of the different mucin populations see Reference (65).

tion of a variable number of their constituent monomers and/oras a consequence of specific proteolytic processing and/or degra-dation. Moreover, these studies showed that the MUC5AC mucinsexhibited a lower average sedimentation rate than the MUC5B

glycoprotein, which was taken to indicate that MUC5AC wassmaller than MUC5B (27, 32). However, this is probably not thecase, as we have shown that MUC5AC is in fact highly oligomer-ized and the apparent difference in size may be explained by a


Figure 3. Agarose gel electrophoresis of reduced sputum samples.Sputa, NaCl-induced from healthy airways (lanes 1, 6 and 7 ) and sponta-neous secretions from diseased airways, were solubilized in 6 M GuCland then dialyzed into 6 M urea. The samples were reduced with 10 mMdithiothreitol, treated with 25 mM iodoacetamide to alkylate-free thiolgroups and then subjected to electrophoresis on a 0.7% agarose gel(for more details see Reference [65]). After electrophoresis, gels weretransferred to nitrocellulose and probed with antisera specific for (A )MUC5AC and (B ) MUC5B mucins (section from the data presented inReference [39]). These blots were also stained with an antiserum directedagainst MUC2, but no bands were visualized (data not shown). Theresults show the greater variation in electrophoretic migration of thereduced MUC5B mucins compared with MUC5AC; furthermore, withthe exception of one sample (lane 6), all the others exhibit two differentMUC5B charge forms. These data also demonstrate that there is a largervariation in electrophoretic mobility between low-charge populationsthan there is between highly charged forms.

difference in physical characteristics of these two mucins (33).The MUC5AC polymer has a low mass per unit length, suggestingsmall oligosaccharides, and adopts a very stiff extended confor-mation in solution, whereas the MUC5B mucin appears to adopta more compact structure. Further evidence that these two mu-cins have different macromolecular features is indicated by theirbehavior on agarose gel electrophoresis. Unreduced MUC5ACpreparations exhibit a ladder-like banding pattern and oligo-meric species containing 16 monomers can be discerned (33).By contrast, unreduced MUC5B mucins barely enter the gel(Thornton and coworkers, unpublished observation).

It is clear that MUC5AC and MUC5B are the predominantgel-forming mucins in the airways and that they have distinctchemical and physical features. What effect these differencesbetween the mucins have on the gel properties has yet to be de-termined. Moreover, at an even more fundamental level, we donot know whether MUC5AC and MUC5B mucins are blendedtogether to form the mucus gel or if they form the basis ofseparate gels. The latter has been suggested in gastric mucus,where laminated layers containing different mucins have beenreported (34).

WHERE ARE MUCINS SYNTHESIZED?

Immunolocalization studies on normal airways, using mucin-specific antisera, have shown that MUC5AC mucins are pro-duced predominantly by goblet cells in the surface epithelium.MUC5B mucins on the other hand, originate mainly, but not

exclusively, from the mucous cells of the submucosal glands (17,27, 32), and this distribution is consistent with messenger RNAexpression as shown by in situ hybridization (35). While antiserato MUC5AC and MUC5B have been used to localize theirrespective cellular sources, we do not have, and may never have,probes to the polypeptide that are specific for each of the glyco-forms of the MUC5B mucin. Thus, we do not know if they havedifferent sites of synthesis. We have previously shown in salivarymucin preparations that the highly charged variant of MUC5Bwas reactive with a monoclonal antibody F2 that is specific for thecarbohydrate epitope, the sulfo-Lewisa antigen (SO3-3Gal�1–3GlcNAc) (36). This antibody has been shown to stain a subset ofglandular mucous cells in normal human trachea (27). Whereasthis suggests different cellular locations for the MUC5B variants,immunolocalization data from carbohydrate-directed probes arenot conclusive owing to potential cross-reactivity with other gly-coproteins. Thus, polypeptide probes would be desirable andmight allow us to conclusively pinpoint their cellular sources;however, unless we can find differences in their polypeptidemoieties, this will not be possible.

It is interesting to speculate that the spatial separation ofmucin production in the normal airways (i.e., MUC5AC on thesurface and MUC5B in the glands), coupled to the fact that thetwo sites are controlled by different secretory mechanisms, mayallow for fine-tuning of the mucus composition to modulate mu-cus properties depending on the challenge in the airways.

On the basis of the immunolocalization and expression data,it seemed logical at that time to believe that the mucin composi-tion of mucus would inform us on the contribution of the variouscellular sources to the secretion and, therefore, might throw uppossible cellular targets for therapies aimed at modulating mucin,and hence mucus production in hypersecretory diseases. How-ever, in hypersecretory disease, this marked spatial separationof synthesis does not seem to hold. Such diseases are associatedwith significant hyperplasia and metaplasia of mucin secretingcells, and it has been shown that MUC5AC production can alsooccur in glandular mucous cells, while some MUC5B and MUC2synthesis can be detected in surface goblet cells (37, 38). Whetherthese mucins can be coexpressed in the same cell or arise fromdifferent cells has not yet been determined. Because we do notknow the relative change in expression of the mucins at thedifferent locations, we cannot be certain that their occurrenceis not a valid indicator of the major source of their production.Thus, there is an obvious need for further, more in-depth studieson mucin expression in disease.

ARE AIRWAYS MUCINS ALTERED IN DISEASE?

In a series of studies, we have shown that the gel-forming mucinscan be changed in amount, type, and size in airways disease(3, 12, 13, 31). For example, in the viscid mucus obstructing theairways of an individual who died in status asthmaticus, therewere changes in all three parameters. There was an approxi-mately seven-fold increase in mucin concentration comparedwith healthy secretions. Furthermore, we demonstrated that theonly mucin type responsible for the aberrant physical propertiesof this gel was a low-charge glycoform of the MUC5B mucinwith unusual structural features and extreme size (3, 31). Theclinical outcome in this case demonstrated the dramatic conse-quences of alterations in the mucin component of mucus andhighlighted the need to determine the molecular profile of mu-cins in sputum.

Therefore, in order to gain a more detailed picture of mucintype (gene product and glycoform) and quantity in normal andpathologic respiratory mucus, we developed a quantitative West-ern blotting assay to measure the levels of MUC2, MUC5AC,


and the different glycoforms of the MUC5B mucins directly fromsputum (39). Data from 44 samples (15 NaCl-induced normal, 10asthma, 10 CF, and 9 COPD) showed, as our previous nonquanti-tative studies had suggested, that MUC5AC and MUC5B arethe major gel-forming mucins in sputum. By contrast, the MUC2mucin was present in only trace amounts, confirming our earlierobservations that MUC2 is only a minor component in spu-tum. Moreover, this study showed that airways mucus is not asingle substance and is comprised of variable amounts of MUC5ACand MUC5B glycoproteins. Somewhat surprisingly, the variationin content was particularly marked in the “normal” samples. A keyobservation to come from this work was that, compared withsecretions from normal subjects and individuals with asthma,there was more MUC5B in CF and COPD sputa and, mostnotably, there was a significant increase in the amount of thelow charge form of the MUC5B mucin in the diseased, possiblyinfected sputa. This might suggest a link between infection/in-flammation and MUC5B production. The functional conse-quences of mucus with different mucin compositions, and inparticular of the different charge forms of the MUC5B mucin,are at present completely obscure. We would suggest the generalhypothesis that MUC5AC, being primarily the product of gobletcells, may have the major mechanical function of facilitatingciliary clearance of mucus, whereas MUC5B emanating fromthe glands may form the basis of a gel, the primary role ofwhich is to help with the clearance of specific pathogens or otherirritants. The abnormal mucin composition of the gel in CF andCOPD airways may provide an insight into the altered physicalnature of these secretions and ultimately suggest potential cellu-lar targets for therapy. These observations again highlight theneed for more studies on mucin expression in disease, and inparticular on MUC5B expression, which has so far been largelyignored. Furthermore, these findings reemphasize the need todevelop reliable probes for the MUC5B mucin glycoforms toestablish their cellular origin.

GEL-FORMING MUCINS PRODUCED BY AIRWAYSCELLS IN AIR–LIQUID INTERFACE CULTURE

Studies of human airways secretions have been, and will continueto be, essential to highlight changes associated with disease.However, these investigations are fraught with difficulty relatedto mucus collection, as well as possible changes in the mucinsbeing disguised by nonspecific postsecretion processing. Further-more, the effects of extraneous environmental influences aredifficult to control and, for ethical reasons, it is impossible toperform in vivo studies of mucin/mucus biogenesis and secretion.Thus, to progress our understanding of both normal and diseasedairways, there is an urgent need for in vitro systems that mimicin vivo mucus production. For this purpose a number of labora-tories have developed airways epithelial cell cultures (40–43).The normal human tracheobronchial air–liquid interface cul-tures instigated by Nettesheim and colleagues are of particularinterest (40). When cultured in the presence of retinoic acid,normal human tracheobronchial cells grow and differentiate intoa mucociliary epithelium. They secrete functional mucus ontotheir apical surface (40) that is clearly capable of ciliary transport.This culture system appears to mimic in vivo airways mucin pro-duction in that all three oligomeric airways mucins, MUC5AC,MUC5B, and MUC2 are expressed. Furthermore, their basal lev-els of expression can be modulated by physiologically relevantregulators (e.g., thyroid hormone and epidermal growth factor)(44–46). We have investigated the apical secretions from suchcultures and have shown that MUC5AC and MUC5B are thepredominant mucins stored and secreted by normal human tra-cheobronchial cells (passage 2). Moreover, these mucins have

similar macromolecular properties to their counterparts in hu-man airways (44). However, while the MUC5AC mucin exhibitsa similar charge profile to MUC5AC in vivo, by contrast, theMUC5B mucin produced is more homogeneous, with evidenceof only a single (perhaps high-charge) glycoform. Thus, the mu-cin phenotype and hence the resultant properties of the secre-tions may not be a truly accurate reflection of in vivo mucus.Nonetheless, such culture systems provide a powerful and manip-ulable experimental system and seem set to form the basis of everwidening studies that should revolutionize our understanding ofthe biology and pathobiology of mucus over the next few years.For example, work from Boucher and collaborators using cul-tures derived from cells with the CF defect has shown that thedynamics of the attached mucus layer are altered and, in thesecompared with normal cultures, mucus transport is reduced oreven abolished (41).

AIRWAYS MUCUS GEL FORMATION—OLIGOMERICMUCINS ARE NOT THE WHOLE STORY!

While size and concentration of the mucins are key factors con-trolling gel formation, concentrated solutions of mucins alonedo not reproduce all the physical properties of the gel (47). Thisis emphasized in our recent findings where we have shown, usingconfocal fluorescence recovery after photobleaching, that thenetwork properties of mucus cannot be recapitulated by concen-trated mucin solutions even at higher than in vivo mucin concen-tration (48). Unlike some other methods for looking at mucusproperties, confocal fluorescence recovery after photobleachingis nondestructive and provides a powerful approach to investigat-ing the properties of macromolecules in concentrated solution,including complex mixtures, such as in mucus, under equilibriumconditions, and in the absence of concentration gradients and flowand shear forces (48, 49). We have used it to determine lateraltracer diffusion of fluorescent molecular probes (i.e., bovine serumalbumin and mucins) and of rigid polystyrene microspheres ofdifferent size in mucus at physiological concentrations. It is im-portant to note that the tracer diffusion measurements aloneare not a measure of gel formation, which involves polymer cross-links and entanglement and occurs in concentrated solutionsof all flexible polymers. However, they provide a quantitativecomparison of the “network” caused by gel-formation, or entan-glement, that impedes the free diffusion of other macromoleculesin a concentration-dependent way. Our initial confocal fluores-cence recovery after photobleaching analysis of MUC5B (ex-tracted and purified in the presence of 4–6 M guanidinium chlo-ride) showed that in concentrated solutions its properties resultedfrom polymer entanglement with no evidence of protein–proteinself-association or glycan-mediated interactions (48). A direct com-parison of concentrated guanidinium chloride-purified MUC5Bmucin solutions with the native saliva from which it was preparedrevealed that the MUC5B mucin did not replicate the propertiesof the parent mucus. At similar MUC5B concentrations to thosefound in saliva, the guanidinium chloride–purified mucin wasapproximately 20 times more permeable, suggesting that therewas a higher order structure in the native mucus, which mayinvolve other components in the secretion (48). Further analyseshave implicated calcium as a key regulator of MUC5B mucinsupramolecular organization in salivary mucus (50). This studydemonstrated a reversible, calcium-dependent interaction be-tween MUC5B mucins that was sensitive to denaturation bychaotropic solvents (e.g., 6 M guanidinium chloride), treatmentwith reducing agents and the proteinase trypsin (50). This studyalso highlighted the need for alternative, nondenaturing solventsto disassemble mucus gels for investigation of their functions.

The fact that gel-forming mucins alone do not constitute mu-


cus may not be totally surprising because it is a complex mixtureof ions, mucins, glycoproteins, proteins, and lipids and, like themucins, the total amount of these other components is increasedin hypersecretory disease (51–54). Some of these componentscontribute to airways protection by targeting pathogens (e.g.,secretory IgA, proline-rich proteins, defensins, lysozyme, andtransferrin), and others may act to modulate the organizationand hence the properties of the gel. For example, in the gastro-intestinal tract the mitogenic/reparative trefoil peptides are inti-mately associated with mucus and have been suggested to bindto oligomeric mucins and increase mucus viscosity (55). Workfrom our laboratory has demonstrated that a large glycoprotein,gp-340, is found in complexes with MUC5B in airways mucus(56). It is interesting to speculate on the consequences of aninteraction of gp-340 with the mucin network. Gp-340 clearlyhas an important antibacterial role in mucus, either via its directbinding to bacteria (57) or by its association with the bacterial-binding collectin surfactant protein-D (58). Thus, binding ofgp-340, and likely other protective factors, to the gel networkincreases the functionality of the mucus by facilitating clearanceof sequestered bacteria from the respiratory tract via the muco-ciliary escalator or cough.

It is clear from these two examples that nonmucin glycopro-teins and proteins play key roles in mucosal protection andmucus organization; but while there have been many studies onthe molecular components of mucus, we do not yet know theirfull complement or which of these are associated with the mucinsand what function(s) they perform. These other components maybe over- or under-expressed in disease conditions, which wouldhave an effect on the hosts ability to prevent colonization orthe rheologic properties of mucus may be compromised withattendant problems. Thus proteomics has a key role to play inidentifying the different molecular species impacting on mucusstructure and function.

A final but no less important factor that will have a majorinfluence on the properties of airways mucus is the availabilityof water on the epithelial surface. Currently, the relationshipbetween mucin hydration and mucus properties is not well un-derstood. We know that oligomeric mucins are preassembledinto large oligomers within the cell, where they are stored withingranules in a largely dehydrated form. However, after secretionthe requirements of a specific mucin to hydrate are unknownbut are likely affected by a variety of factors, including theirsize, charge density, and organization. Furthermore, their hydra-tion will be intimately coupled with the ionic composition andwater availability of the environment into which they are se-creted. This will in turn be affected by the other biomolecules al-ready present in that environment. Aberrant mucin hydrationmay be of particular importance in the airways of patients withCF. In CF, the competition for water on the airways epitheliumappears to be more severe, and the water imbalance in theepithelial fluid (41, 59, 60) may well have a strong negativeimpact on mucin unpackaging and hydration. This may explainthe abnormal properties of the mucus, which are a feature ofthis disease.

OTHER AIRWAYS MUCINS

While this article has focused on the oligomeric mucins, theseare not the only mucins expressed in the airways. Other membersof the mucin family (MUC1, MUC4, MUC7, MUC11, andMUC13) are expressed and these mucins, with the exception ofMUC7, are thought primarily, though not exclusively, to occur intransmembrane forms, which can arise in mucus due to sheddingfrom the cell surface (61–63). However, in the case of MUC1 andMUC4, there are alternatively spliced forms that are secreted

(61–63). There is little biochemical characterization of thesemucins, and their amounts in mucus are unknown. Furthermore,their role in mucus has yet to be defined and there is clearly anurgent need to understand their function in mucus.

Conclusions

As far as the oligomeric mucins are concerned, an extensivearray of mucin-specific probes are available, the methodologiesnecessary for their separation and characterization have beendeveloped, and the air–liquid cultures offer a more physiologi-cally relevant in vitro system for their study. Thus, we are wellplaced to perform new studies of the cell and structural biologyof the mucins and mucus, which is essential for a more coherentpicture of how this essential barrier functions to protect theairways. Finally, there are currently few effective therapies toalleviate mucus hypersecretion, and these studies may highlightspecific components as potential targets for future therapeuticstrategies for the treatment of hypersecretory conditions.

Acknowledgment : The authors acknowledge the substantial contributions to theresearch reviewed here by Ingemar Carlstedt (University of Lund, Sweden), PaulRichardson (St. Georges Hospital, London, UK), Paul Nettesheim, Tom Gray, andPeter Koo (NIEHS, U.S.A.), and Sara Kirkham, Marj Howard, Nagma Khan, DavidKnight, Bertrand Raynal, and Tim Hardingham (University of Manchester, UK).

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Mucinstructure,aggregation,physiologicalfunctionsandbiomedicalapplications.BansilR.andTurnerBSCurrOpininColloid&InterfaceSci,11:164(2006)

www.elsevier.com/locate/cocis

Current Opinion in Colloid & Interface S

Mucin structure, aggregation, physiological functions and

biomedical applications

Rama Bansil a,*, Bradley S. Turner b,c,1

a Department of Physics and Center for Polymer Studies, Boston University, Boston, MA 02215, United Statesb Molecular Biology, Cell Biology and Biochemistry, Boston University, Boston, MA 02215, United States

c Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, United States

Available online 18 January 2006

Abstract

An understanding of the basic structure, viscoelastic properties and interactions of mucin glycoproteins is of considerable interest to food

science because of the important protective role that these macromolecules play in gastric physiology. The polymeric/colloidal behavior of mucins

is complicated due to their large size (2–50 MDa) and complex structure with domains involving hydrophilic/hydrophobic, hydrogen bonds and

electrostatic interactions, and their propensity to aggregate and form complexes with other polymers. These properties are also of direct relevance

to the numerous diseases involving mucins, and to the problems of uptake of nutrients and delivering drugs through the mucus barrier. In this

review we describe the current state of understanding of the relevant properties, with an emphasis on recent research. The findings described in

this review are of direct relevance to the uptake of nutrients in digestion.

D 2005 Published by Elsevier Ltd.

Keywords: Mucin; Aggregation; Gelation; Gastric mucus; Mucoadhesion; Glycoprotein; Biorheology

1. Introduction

Mucus is a complex viscous adherent secretion synthesized

by specialized goblet cells in the columnar epithelium that lines

all of the organs that are exposed to the external environment.

This includes the respiratory tract, the gastrointestinal tract, the

reproductive tract, and the oculo-rhino-otolaryngeal tracts. It

serves many functions in those locations, among which are

lubrication for the passage of objects, maintenance of a hydrated

layer over the epithelium, a barrier to pathogens and noxious

substances and as a permeable gel layer for the exchange of

gases and nutrients with the underlying epithelium [1,2].

In addition to its protective functions, mucus is also

involved in many disease processes. Mucus also is the first

barrier with which nutrients and enteric drugs must interact and

diffuse through, in order to be absorbed and gain access to the

circulatory system and their target end organs.

1359-0294/$ - see front matter D 2005 Published by Elsevier Ltd.

doi:10.1016/j.cocis.2005.11.001

* Corresponding author. Tel.: +1 617 353 2969.

E-mail addresses: [email protected] (R. Bansil),

[email protected] (B.S. Turner).1 Tel.: +1 617 667 1215.

2. Molecular biology and biochemical structure of mucins

2.1. Composition of mucus

Mucus is composed primarily of water (¨95%), but also

contains salts, lipids such as fatty acids, phospholipids and

cholesterol [1], proteins which serve a defensive purpose such

as lysozyme, immunoglobulins, defensins, growth factors and

trefoil factors. However, the main component that is respon-

sible for its viscous and elastic gel-like properties is the

glycoprotein mucin.

2.2. Mucins

Mucins are large, extracellular glycoproteins with molecular

weights ranging from 0.5 to 20 MDa. Both membrane bound

mucins, and secreted mucins [3&] share many common features.

They are both highly glycosylated consisting of ¨80%

carbohydrates primarily N-acetylgalactosamine, N-acetylglu-

cosamine, fucose, galactose, and sialic acid (N-acetylneu-

raminic acid) and traces of mannose and sulfate. The

oligosaccharide chains consisting of 5–15 monomers, exhibit

moderate branching and are attached to the protein core by

cience 11 (2006) 164 – 170

http://dx.doi.org/10.1016/j.cocis.2005.11.001



R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170 165

O-glycosidic bonds to the hydroxyl side chains of serine and

threonines and arranged in a ‘‘bottle brush’’ configuration about

the protein core.

The protein core, making up the remaining 20% of the

molecular mass (¨200–500 kDa), is arranged into distinct

regions. First, a central glycosylated region, which is com-

prised of a large number of tandem repeats that are rich in

serine, threonine and proline (STP repeats), which can make up

greater than 60% of the amino acids. Second, located at the

amino and carboxy terminals, and sometimes interspersed

between the STP-repeats, are regions with an amino acid

composition more representative of globular proteins, relatively

little O-glycosylation and a few N-glycosylation sites [4] and a

high proportion of cysteine (>10%). These cysteine rich

regions contain domains that possess sequence similarity to

von Willebrand factor (vWF) C and D domains, and C-terminal

cystine knot domains [5&,6,7], and have been shown to be

involved in dimerization via disulfide bond formation, and

subsequent polymerization of the dimers to form multimers [8]

(Fig. 1).

2.3. Mucin genes

Currently, approximately 19 mucin (designated MUC)

genes have been identified cloned and partially sequenced in

the human, and homologs to many of them have been

identified in the mouse and rat [5&]. Only three MUC

(MUC1, MUC2 and MUC5B) genes have been totally

sequenced due to the large size of the central tandem repeats,

which are difficult to accurately assemble. Many of the smaller

membrane bound mucins are not considered ‘‘true’’ mucins

since they only share the STP repeats and glycosylation with

other mucins [3&]. Of the remaining ‘‘true’’ mucins (MUC1–7),

the secreted mucins (MUC 2, 5AC, 5B and 6) are located in a

Fig. 1. (a) A schematic drawing of the pig gastric mucin (PGM) monomer consisting

The symbols indicate the different domains in the sketch in (a). (This representation i

we show (c) a dimer formed by two monomeric subunits linked via disulfide bonds in

form higher multimers. This gives rise to the high molecular weight and polydisper

from human airway secretions by Sheehan et al. [8]. (The bottom part of the figure

cluster within ¨500 kb on the short arm of chromosome 11

(11p15) [9]. It is thought that they represent homologs that

diverged from a common ancestor gene on chromosome 11

[10]. The STP-repeat sequences for each MUC gene are unique

to each species, whereas the cys-rich regions share a large

degree of similarity [5&]. Different collections of mucin genes

are expressed in different tissues. Such repeats and super

repeats are relatively uncommon in normal proteins.

3. Physical properties of mucin in dilute solution

Mucin has been difficult to characterize, owing to its large

molecular weight, polydispersity and high degree of glycosyl-

ation. Earlier biophysical studies, reviewed by Bansil et al. [11&]and Harding [12&] primarily using light scattering methods

showed that mucin was a somewhat stiffened random coil with a

radius of gyration around 100 nm. NMR studies of MUC1

[13,14&] revealed very little alpha helix, a small amount of beta

and mostly random coil. The large size of mucin has, on the

other hand, turned out to be of great advantage in imaging the

molecule directly. Earlier transmission electron micrographic

studies by Fiebrig et al. [15] revealed long fibers approximately

400 nm long in pig gastric mucin (PGM). More recent Atomic

Force Microscopy (AFM) studies of ocular mucin [16&] showindividual fibers with a broad distribution of contour lengths.

While most of the fibers are between 200–600 nm long, the tail

of the distribution extends to 1500 nm. They also estimated a

persistence length of about 36 nm from these images, which

confirms the extended nature of the polymer. In another AFM

study of ocular mucin, Brayshaw et al. [17] demonstrated the

multimeric nature of mucin, by observing in situ depolymer-

ization on treatment with DTT (dithiothreitol). Longer fibers, up

to 2 Am in length, were observed in PGM by Deacon et al. [18].

McMaster et al. [19] examined ocular mucin using tapping

of glycosylated regions flanked by regions with relatively little glycosylation. (b)

s based in part on Figs. 1 and 2 of Dekker et al. [3&]). At the bottom of the figure

the non-glycosylated regions and in (d) dimers that are further disulfide linked to

sity of secretory mucins. Polymers>16-mers have been described in MUC5AC

is adapted from Fig. 8 of Ref. [8].) Reprinted with permission from [21].

R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170166

mode AFM under a buffer and observed regular variations in

height along the length of the fiber which they interpret as

glycosylated regions of the mucin molecules. Round et al. [20]

correlated the conformations with differing amounts of glyco-

sylation by imaging different fractions obtained on a CsCl

gradient. However the sugar chains are too mobile in their

solvated state to be seen fully extended in an AFM image,

which requires a high degree of immobilization on the substrate,

the AFM tip might have penetrated the brush without sensing it.

AFM images of PGM which was about 50% deglycosylated

showed that the deglycosylated portions re-folded forming

compact globular structures [21]. Thus the sugars are important

for maintaining the extended conformation of mucin.

The conformation of mucin also depends on factors such as

pH and ionic strength. For example, dynamic light scattering

(DLS) studies [22] have shown that as the pH is lowered in

dilute solutions (<5 mg/ml) PGM undergoes a conformational

change from an isotropic random coil (with end to end length

of 390 nm and persistence length of 8–10 nm) at pH 7 to an

anisotropic extended random coil (with end-to-end length of

490 nm, persistence length 43 nm) at pH 2. This is paralleled

by an increase in the sedimentation coefficient from 11S at pH

7 to greater than 31S at pH 3 [23]. In a recent paper the

conformation of commercially available PGM from Sigma in

dilute solution examined by viscosity and circular dichroism

measurements reveals similar changes as function of pH which

are attributed to the unfolding of hydrophobic domains at low

pH [24]. These authors also measured the zeta (1) potential ofmucin and found that its isoelectric point lies between 2 and 3,

suggesting that the charge on the molecule varies as pH is

lowered.

4. Colloidal properties of mucin

4.1. Aggregation and gelation

Mucins exhibit a tendency to aggregate and form gels.

Taylor et al. [23] used rheological techniques to investigate the

structure and formation of the pig gastric mucus gel and

showed that both transient and non-transient interactions are

responsible in maintaining the gel matrix. Purified mucin also

has been shown to form gels. Human tracheobronchial mucin

forms a gel below 30 -C at concentrations above 14 mg/mL

[25]. Gelation was also observed in canine submaxillary mucin

in the chaotropic (denaturing) solvent guanidine HCl [26].

These authors suggest that since non-covalent interactions are

destabilized by guanidine HCl, gelation in these high molecular

weight fractions involves the interpenetration of the carbohy-

drate side chains [26].

Our laboratory has focused on gelation of gastric mucin at

low pH, which has implications for the physiologically relevant

question of how the stomach is prevented from being digested

by the acidic gastric juice it secretes. Bhaskar et al. [27] showed

a reversible increase in viscosity of aqueous solutions of pig

gastric mucin (PGM) at pH 2. DLS studies [22] show that at

concentrations above 10 mg/ml gelation occurs below pH 4. At

low pHs we also observed an increase in the hydrophobicity of

the protein core of PGM as indicated by its increased binding of

the fluorescent hydrophobic dye 1-anilinonaphthyl-8-sulfonic

acid (ANS). Atomic force microscopy images confirm that

while PGM exists as single molecules at pH 6 (about 400 nm in

length), it forms aggregates below pH 4 [21,28]. The viscosity

increase and gelation are not observed if PGM is broken into its

subunits (by treating with enzymes such as pronase or disulfide

reduction with DTT) or salt concentration is increased.

Commercial preparations of PGM available from Sigma

chemicals (which include protease treatment during purifica-

tion) also do not gel [28].

4.2. Model for pH induced gelation of PGM

This accumulated data showing a sharp increase in the

observed changes at pH 4 and the lack of gelation in PGM

which is broken into the subunits has led us to suggest that

PGM gelation involves interactions of side chains of amino

acids with pKs around 4, such as Asp or Glu from the non-

glycosylated regions of the molecule. At neutral pH of 6–7

these non-glycosylated Cys-rich regions of PGM are in

conformations with hydrophobic domains hidden in folds of

stabilized by salt bridges between negatively charged carbox-

ylates and positively charged amino groups. At acidic pH 2 the

carboxylates of the salt bridges are protonated, breaking the salt

bridges and allowing the unfolding and exposure of hydro-

phobic regions of protein (Fig. 2). Circular dichroism studies

reported by Lee et al. [24] on pig gastric mucin from Sigma

also confirm the conformational transition at pH 4 and below.

The hydrophobic domains on adjacent molecules are then able

to associate acting as the crosslinks of a gel. Hydrophobic

interactions among protein domains were also shown to be

involved in aggregation of human tracheobronchial mucin [25].

The gelation of mucin is a complex problem, involving the

interplay of electrostatic and hydrophobic interactions, some-

what reminiscent of the association of multiblock copolymers

in solvents that have differential solubility to the component

blocks. The entanglement of the sugar side chains further

contributes to the high viscosity of mucin solutions (Fig. 2).

4.3. Liquid crystalline properties of mucin

Mucin glycoprotein has also been shown to exhibit liquid

crystalline order. Viney, et al. [30] had shown that slug mucin

forms nematic liquid crystals. A detailed study of the

temperature and concentration dependence of the liquid

crystalline behavior of commercially available mucin from

Sigma was reported by Davis et al. [31] who suggest that the

interactions of interdigitated sugar side chains are responsible

for the nematic behavior. Waigh et al. [32] confirmed these

observations by neutron scattering, comparing Sigma mucin at

high concentrations in the presence and absence of an external

magnetic field to produce orientation. Purified PGM, on the

other hand, did not show this liquid crystalline orientation,

suggesting that the Sigma mucin, which consists of a single

mucin monomer, is more rigid and easier to orient than the

multimeric form of native PGM in which the non-glycosylated

Fig. 2. A model showing how the breaking of electrostatic interactions around pH 4 and below can produce a conformational change in PGM from a random coil to a

rod (or stiff worm-like chain) and lead to gelation at high concentrations at low pH by exposing hydrophobic regions which were folded and thus sequestered in the

interior at neutral pH. [Reproduced from Ref. [29], PhD dissertation of Xingxiang Cao, Boston University, 1997, with permission of the author].


portions serve as flexible links between the rigid, glycosylated,

monomeric domains.

4.4. Adhesive properties of mucin

The well know tendency of other substances to adhere to

mucin, known as mucoadhesivity, is not surprising given that

this glycoprotein exhibits electrostatic, hydrophobic, and H-

bonding interactions [33]. By the same token the molecule can

be anti-adhesive, for example to negatively charged species.

Thus mucin coated AFM tips adhered to mica in presence of

divalent cations [34] but did not adhere to mica coated with

mucin, presumably due to the polyelectrolytic charge repul-

sion. Bovine submaxillary mucin from Sigma could be

adsorbed to hydrophobic polystyrene surface rendering it

hydrophilic [35]. Mucin lipid interactions are well known,

reflecting the presence of hydrophobic domains [36]. In this

context, it is worth noting that since mucin is negatively

charged it binds to positive ions. For example, Chromium III

binding has been shown to cause significant conformational

changes and it can lead to aggregation of mucin [37].

4.5. Diffusion of macromolecules and particles and fluid flow

through mucin

In view of the protective function of mucus studying the

diffusion of other molecules through it is of considerable

physiological interest. It is also an important factor in the

design of drugs which have to diffuse through the mucus layer,

or kept from entering it. While many small molecules diffuse

readily through mucus, the diffusion of larger particles depends

both on size and muco-adhesive interactions. The diffusion of

latex particles and lipid vesicles in purified gall bladder mucin

has been examined using DLS [38] and fluorescence recovery

after photobleaching [39] methods. Olmsted et al. [40] showed

that while many small viruses (20–200 nm) could diffuse

through cervical mucus, others such as the Herpes Simplex

virus got stuck to the mucus. Celli et al. [41] have exploited the

movement of latex particles through mucin solutions to

measure its rheological properties as a function of pH using

microscope based DLS.

Finally, it is worth noting that the transport of water and

other fluids through mucus and purified mucin solutions is a

very interesting problem in fluid dynamics. Due to its high

viscosity, water and other low viscosity fluids injected under a

pressure gradient across an unstirred mucin solution do not

simply diffuse, but are transported by a viscous fingering

mechanism [42,43]. This transport of HCl through ‘‘channels’’

produced by the viscous fingering mechanism has also been

demonstrated in vivo in rats [44]. The mucus in the

immediate vicinity of the surface of the acid channel will

gel, thereby confining the acid in a ‘‘tube’’. Similarly the

mucus layer on the luminal surface of the stomach will gel as

the acid is emptied into the lumen. The negatively charged

mucin gel prevents back diffusion of H+ via the mechanism

of Donnan equilibrium.

& Of special interest.&& Of outstanding interest.


5. Biomedical function and applications

5.1. Mucus and disease

The physical state of the mucus, change in the concentra-

tion of secreted mucin, and the strong dependence of its

physicochemical properties on environmental factors such as

ionic strength and pH play an important role in many diseases.

For example, besides serving as a barrier to bacteria, many

bacteria reside within the mucus and possess specific adhesins

that specifically bind to it [45]. This includes pathogenic

strains of Pseudomonas, Streptococcus and Pneumococcus.

Helicobacter pylori particularly resides in the mucus layer of

the stomach, and is a common cause of ulcers [46]. Some

parasitic organisms also produce their own layers of mucus to

evade the immune system [47]. Second, overproduction of

mucus is involved in cystic fibrosis [48&], bronchitis, asthma

[49] and in middle ear infections, and mucus gels serve as the

matrix in which gallstones are nucleated and grow [50].

Thirdly, mucus underproduction is present in dry eye

syndromes [51&] and in some forms of ulcer disease. Finally,

mucus expression and composition is altered in cancers of

epithelial origin [52&&].

5.2. Mucus and pharmacology

Mucus is the first barrier with which nutrients and enteric

drugs must interact and diffuse through, in order to be absorbed

and gain access to the circulatory system and their target end

organs. There is great interest in methods to optimize these so-

called muco-adhesive interactions for improved drug delivery.

Various molecular interactions have been exploited to enhance

mucoadhesion, including, polyelectrolytic interactions (chito-

sans, poly-acrylic acid, etc.) hydrogen bonds (hydrogels), [33]

and disulfide binding (thiomers) [53]. Efforts are underway to

design pH sensitive drug carriers such as gels which will not

release the drug in the acidic environment of the stomach but

will do so in the basic environment of the intestine and colon.

In this context, we emphasize recent studies [54] which show

that the resting stomach has a pH close to 4 which drops to 2

during active acid secretion. Mucin can be used as a high

molecular weight additive to improve the adherence of artificial

tear drops in treating dry eye syndrome [55]. Efforts to develop

nanoparticles for mucosal DNA vaccines and gene therapy are

also being considered [56]. The abnormality of the sugars in

cell membrane bound mucins from cancerous cells has also

been targeted as a potential for cancer vaccine development.

Excellent reviews of oral mucosal drug delivery are collected in

Ref. [57&].

6. Conclusions

We have reviewed recent, as well as some pertinent older

literature describing the biophysical properties of mucin in

solution and its properties of interest in colloid science. The

picture that emerges is that of a macromolecule with a complex

organization into domains with different structures. From the

polymeric viewpoint mucin can be considered as a multiblock

copolymer with alternating polyelectrolytic domains having a

grafted sugar brush, connected by flexible regions with less

glycosylation and the tendency to form multimers. The

molecule has both hydrophobic and hydrophilic regions with

the ability to form H-bonds, and electrostatic interactions. This

wide range of interactions causes it to aggregate gel and form

mucoadhesive interactions with other substances. These

physical properties are of direct relevance to the physiological

functions of mucus in normal and diseased states. An

understanding these properties is of considerable current

interest because of the many biomedical applications.

[Note: Topics that are not covered in this review such as

mucin glycosylation [58], signal transduction [59], and

secretion [60], can be found in the indicated references.]

Acknowledgements

The authors would like to thank Dr. Nezam H. Afdhal, Dr.

K. Ramakrishnan Bhaskar, Dr. XingXiang Cao, Prof. Bernard

Chasan, and Dr. Zhenning Hong for our long-standing

collaboration on studies of mucin. Our research on mucin has

been supported by grants from NIH (P.I. Dr. N.H. Afdhal) and

NSF (P.I. R.B.).

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Mucinsinthemucosalbarriertoinfection.Lindenetal,NatureImmunology1:183(2008)

MucosalImmunology | VOLUME 1 NUMBER 3 | MAY 2008 183

nature publishing group REVIEW

MUCINS — AN INTEGRAL PART OF THE MUCOSAL BARRIER Mucosal epithelial tissues have evolved multiple mechanisms

of defense in response to their vulnerability to microbial attack

due to their exposure to the external environment. The mucosal

epithelial cells form a contiguous lining that acts as a barrier

between the moist exterior environment and the remainder

of the host. In addition, these cells, both constitutively and in

response to microbes, together with underlying leukocytes,

secrete many defensive compounds into the mucosal fluid,

including mucins, antibodies, defensins, protegrins, collectins,

cathlecidins, lysozyme, histatins, and nitric oxide. 1 – 3 Together,

these different defensive compounds form a physical barrier and

have direct antimicrobial activity, and the ability to opsonize

microbes to aid clearance. Mucin glycoproteins, however, can

fulfill all of these roles individually.

Mucosal pathogens, almost by definition, have evolved mecha-

nisms to subvert these mucosal defensive measures. The first

barrier the pathogen encounters is the highly hydrated mucus

gel that covers the mucosal surface and protects the epithelial

cells against chemical, enzymatic, microbial, and mechanical

insult. Mucosal surfaces are coated with a layer of viscous mucus

ranging in thickness from 10 � m in the eye 4 and trachea 5 to

300 � m in the stomach and 700 � m in the intestine. 6 – 8 This

mucus layer is not static but moves to clear trapped material. In

the gastrointestinal tract, the outer mucus layer is continually

removed by movement of the luminal contents, whereas in the

respiratory tract cilia drive its movement. Mucin glycoproteins

produced by mucus-producing cells in the epithelium or submu-

cosal glands are the major macromolecular constituent of mucus

and are responsible for the viscous properties of the mucus gel.

In addition to forming a relatively impervious gel, which acts as

a lubricant, a physical barrier, and a trap for microbes, mucus

provides a matrix for a rich array of antimicrobial molecules.

Underneath the mucus layer, the cells present a dense forest

of highly diverse glycoproteins and glycolipids, which form the

glycocalyx. Membrane-anchored cell-surface mucin glyco-

proteins are a major constituent of the glycocalyx in all mucosal

tissues. The glycocalyx is highly variable from tissue to tissue;

for example, the glycocalyx of human intestinal microvilli tips

is thick (100 – 500 nm) in comparison with the glycocalyx of the

lateral microvilli surface (30 – 60 nm). 9,10 The oligosaccharide

moieties of the molecules forming the glycocalyx and the mucus

layer are highly diverse, and the average turnover time of the

human jejunal glycocalyx is 6 – 12 h. 11 Consequently, both the

secreted and adherent mucosal barriers are constantly renewed

and could potentially be rapidly adjusted to changes in the

environment, for example, in response to microbial infection.

MUCIN BIOSYNTHESIS AND STRUCTURE The tremendous energy investment by mucosal tissues in the

production of mucins in the basal state, but particularly in

response to infection, is testimony to the importance of these

Mucins in the mucosal barrier to infection SK Linden 1 , P Sutton 2 , NG Karlsson 3 , V Korolik 4 and MA McGuckin 1

The mucosal tissues of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye present an enormous surface area to the exterior environment. All of these tissues are covered with resident microbial flora, which vary considerably in composition and complexity. Mucosal tissues represent the site of infection or route of access for the majority of viruses, bacteria, yeast, protozoa, and multicellular parasites that cause human disease. Mucin glycoproteins are secreted in large quantities by mucosal epithelia, and cell surface mucins are a prominent feature of the apical glycocalyx of all mucosal epithelia. In this review, we highlight the central role played by mucins in accommodating the resident commensal flora and limiting infectious disease, interplay between underlying innate and adaptive immunity and mucins, and the strategies used by successful mucosal pathogens to subvert or avoid the mucin barrier, with a particular focus on bacteria.

1 Mucosal Diseases Program, Mater Medical Research Institute and The University of Queensland, Level 3 Aubigny Place, Mater Hospitals , South Brisbane , Queensland , Australia . 2 Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne , Melbourne , Victoria , Australia . 3 Centre for BioAnalytical Sciences, Department of Chemistry, National University of Ireland , Galway , Ireland . 4 Institute for Glycomics, Griffith University , Gold Coast , Queensland , Australia . Correspondence: MA McGuckin ( [email protected] )

Received 27 November 2007; accepted 16 January 2008; published online 5 March 2008. doi:10.1038/mi.2008.5

184 VOLUME 1 NUMBER 3 | MAY 2008 | www.nature.com/mi

REVIEW

glycoproteins. Epithelial mucins are a heterogenous family

of large complex glycoproteins containing a dense array of

O -linked carbohydrates typically comprising over 70 % of their

mass. These glycans are concentrated in large peptide domains

of repeating amino-acid sequences rich in serine and threonine.

The size and number of repeats vary between the mucins, and

in many of the genes there are genetic polymorphisms in the

number of repeats (variable number of tandem repeats or VNTR

polymorphisms), which means the size of individual mucins

can differ substantially between individuals. Each mucin is

thought to form a filamentous protein carrying typically 100s

of complex oligosaccharide structures, 12 giving the mucin a

“ bottle-brush ” appearance. To date, at least 16 human mucins have

been included in the family, and the expression profile of mucins

varies between tissues with the gastrointestinal tract showing the

highest and most diverse expression (see Table 1 ).

Mucins can be divided into three distinct subfamilies: (a)

secreted gel-forming mucins, (b) cell-surface mucins, and

(c) secreted non-gel-forming mucins ( Table 1 ). Gel-forming

mucins, which are the major constituent of mucus and con-

fer its viscoelastic properties, are encoded by a cluster of four

highly related genes on chromosome 11 13 – 15 and a similar gene

on chromosome 12. 16 Gel-forming mucins contain N- and

C-terminal cysteine-rich domains that are both involved in

homo-oligomerization mediated by inter-molecular disulfide

bonds. 17,18 The current model for mucin oligomerization is that

dimerization occurs rapidly during biosynthesis in the endoplas-

mic reticulum preceding or concomitant with N -glycosylation

but before O -glycosylation in the Golgi apparatus, which in turn

is followed by multimerization of dimers. 19 Oligomerization is

likely to produce either extended filamentous structures or, more

probably, web-like molecular structures likely to be critical to the

rheological properties of the mucus gel. 20 – 24 The extended con-

formation caused by dense glycosylation enables the molecules

to occupy large volumes, with the secreted oligomeric mucins

occupying volumes equivalent to those of small bacteria. 25 The

secreted non-oligomerizing mucins include the MUC7 salivary

mucin, which can self-aggregate but is not thought to contribute

significantly to mucus properties, and the MUC8 respiratory

mucin, which has not been fully characterized to date.

There are 11 known genes encoding cell-surface mucins

expressed by a wide diversity of mucosal tissues, with substantial

redundancy in many tissues (see Table 1 ). Cell-surface mucins

are present on the apical membrane of all mucosal epithelial

cells and contain large extracellular VNTR domains predicted

to form rigid elongated structures. Together with their high

expression, this indicates that these molecules are likely to be a

prominent, probably dominating, constituent of the glycocalyx

and may provide a barrier that limits access of other cells and

large molecules to the cell surface. During synthesis, most

cell-surface mucins appear to be cleaved into two subunits in

a region known as an SEA module, which is often flanked by

epidermal growth factor-like domains. 26 Structural studies of

the MUC1 SEA module suggest that the cleavage occurs via

autoproteolysis and that the two subunits remain non-covalently

associated throughout biosynthesis. 27 However, importantly, the

extracellular � -subunit can be shed from the cell surface either

mediated via a second distinct cleavage event 28,29 or perhaps

via physical shear forces separating the two domains at the

original cleavage site as suggested by Macao et al . 27 Mutation

Table 1 Tissue distribution of mucins

Mucin Distribution References

Secreted gel forming

MUC2 Small intestine, colon, respiratory tract, eye, middle ear epithelium

231 – 235

MUC5AC Respiratory tract, stomach, cervix, eye, middle ear epithelium

235 – 239

MUC5B Respiratory tract, salivary glands, cervix, gallbladder, seminal fluid, middle ear epithelium

235,236,240 – 244

MUC6 Stomach, duodenum, gallbladder, pancreas, seminal fluid, cervix, middle ear epithelium

235,243,245 – 247

MUC19 Sublingual gland, submandibular gland, respiratory tract, eye, middle ear epithelium

16,235,248

Secreted non-gel forming (monomeric)

MUC7 Salivary glands, respiratory tract, middle ear epithelium

235,249,250

Cell surface

MUC1 Stomach, breast, gallbladder, cervix, pancreas, respiratory tract, duodenum, colon, kidney, eye, B cells, T cells, dendritic cells, middle ear epithelium

235,251 – 256

MUC3A/B Small intestine, colon, gall bladder, duodenum, middle ear epithelium

235,243,257,258

MUC4 Respiratory tract, colon, stomach, cervix, eye, middle ear epithelium

235,255,259 – 261

MUC12 Colon, small intestine, stomach, pancreas, lung, kidney, prostate, uterus

32,262

MUC13 Colon, small intestine, trachea, kidney, appendix, stomach, middle ear epithelium

35,235,262

MUC15 spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, lungs, middle ear epithelium

235,263

MUC16 Peritoneal mesothelium, reproductive tract, respiratory tract, eye, middle ear epithelium

235,264 – 267

MUC17 Small intestine, colon, duodenum, stomach, middle ear epithelium

235,268

MUC20 Kidney, placenta, colon, lung, prostate, liver, middle ear epithelium

235,269


REVIEW

of the cleavage site inhibits MUC1 shedding in transfected

mammary and respiratory epithelial cells without affecting cell

surface expression, indicating the importance of the initial cleav-

age in shedding. 30 Furthermore, most cell-surface mucin genes

appear to undergo alternative splicing and also encode directly

secreted isoforms lacking transmembrane and cytoplasmic

domains. 31 – 34 These isoforms are stored in subapical granules

and in goblet cell thecae and are secreted both constitutively

and following stimuli. Consequently, due to shedding and direct

secretion, cell-surface mucins can also be seen as components

of secreted mucus. 35

MUC1 is the most extensively studied membrane-associ-

ated mucin and is the most ubiquitously expressed across all

mucosal tissues. MUC1 has been estimated to be 200 – 500 nm in

length (depending on the number of tandem repeats), suggest-

ing it will tower above other molecules attached to the plasma

membrane. 36 MUC1 associated with the cell surface is

constantly internalized (0.9 % of the surface fraction min − 1 ) and

recycled. 37 Internalization occurs by clathrin-mediated endo-

cytosis, and alterations in O- glycan structure stimulate endo-

cytosis and intracellular accumulation. 38 During recycling,

sialic acid is added to the premature form of MUC1. 37 Complete

sialylation requires several rounds of recycling, one cycle taking

approximately 2.5 h. 37 Pulse-chase experiments indicate that the

half-life of MUC1 in the plasma membrane is 16 – 24 h, 37,39 sug-

gesting that the average MUC1 molecule recycles up to 10 times

before release. 37 Recycling rates vary between cell lines and pos-

sibly environmental conditions and have not been measured in

non-transformed cells, making it very difficult to extrapolate to

the real rate of recycling and cell-surface half-life in vivo . The

cytoplasmic tail appears to interact with the cytoskeleton and

secondary signaling molecules, 40 – 42 whereas the extracellular

domains of MUC1 and other cell-surface mucins interact with

extracellular matrix components and other cells. 43 – 47

The cytoplasmic domains of the cell surface mucins are com-

plex, often contain known phosphorylation motifs, and are

highly conserved across species, suggesting important intra-

cellular functions. The best-studied mucin in this regard is

MUC1, which has been explored mainly in terms of its role

in cancer cell biology rather than in mucosal defense. We

and others have shown phosphorylation of the MUC1 cyto-

plasmic domain 41,48 – 52 as well as molecular association with

� -catenin, 41,51 linking MUC1 with the Wnt pathway, which

is involved in epithelial growth, migration, and wound repair.

More recently, it has been shown that the cytoplasmic domain

can be cleaved and that the cleaved domain translocates to mito-

chondria and, together with the p53 transcription factor, to the

nucleus, where it modulates the cell cycle and protects against

the apoptotic response to genotoxic stress. 53,54 Some pathogenic

bacteria produce genotoxins, and thus this protective effect, first

identified in cancer cells, may have evolved as part of the natural

epithelial defense against microbial genotoxins. We have recently

shown that in vitro MUC1 protects p53-expressing epithelial

cells from the effects of cytolethal distending toxin, a genotoxin

produced by Campylobacter jejuni . 55,56 In vivo , C. jejuni more

densely colonized the stomachs of Muc1 − / − mice, but this effect

was not seen in isogenic mutants lacking cytolethal distending

toxin, indicating that Muc1 lowers gastric colonization at least

in part via inhibiting the activity of cytolethal distending toxin. 55

Many of the other cell-surface mucins also contain potential

phosphorylation sites and cleavage motifs in the immediate

intracellular region of their cytoplasmic domains and may be

similarly cleaved. Importantly, there is also evidence that inter-

action with bacteria can induce phosphorylation of MUC1

in vitro . 57 Signaling by the cytoplasmic domains of cell-surface

mucins is complex and much remains to be elucidated about

their mode of action. However, the evidence to date suggests

that these domains are involved in cellular programs regulating

growth and apoptosis in mucosal cells perhaps in response to

microbes and / or their toxins.

MUCIN GLYCOSYLATION The carbohydrate structures present on mucosal surfaces vary

according to cell lineage, tissue location, and developmen-

tal stage. Evidence is emerging that mucin glycosylation can

alter in response to mucosal infection and inflammation, and

this may be an important mechanism for unfavorably chang-

ing the niche occupied by mucosal pathogens. The extensive

O- glycosylation of the mucins protects them from proteolytic

enzymes and induces a relatively extended conformation. 25 The

oligosaccharides on secreted mucins are clustered into heavily

glycosylated domains (typically 600 – 1,200 amino acids long)

separated by shorter nonglycosylated regions. 25 The O- linked

glycans contain 1 – 20 residues, which occur both as linear and

branched structures (see Table 2 ). The carbohydrate chain is

initiated with an N- acetylgalactosamine (GalNAc) linked

to serine or threonine and is elongated by the formation of

the so-called core structures followed by the backbone region

(type-1 and type-2 chains). The chains are typically terminated

by fucose (Fuc), galactose (Gal), GalNAc, or sialic acid residues in

the peripheral region, forming histo-blood-group antigens such

as A, B, H, Lewis-a (Le a ), Lewis-b (Le b ), Lewis-x (Le x ), Lewis-y

(Le y ), as well as sialyl-Le a and sialyl-Le x structures. Sulfation

of Gal and N- acetylglucosamine (GlcNAc) residues causes

further diversification. In addition to the O -linked glycans, mucins

contain a smaller number of N -linked oligosaccharides, which

have been implicated in folding, oligomerization (MUC2), or

surface localization (MUC17). 58 – 60

The carbohydrate structures present on mucins are deter-

mined by the expression of specific glycosyl transferases. Thus,

mucin glycosylation is governed by genetics (due to polymor-

phisms in these enzymes), tissue-specific enzyme expression,

and host and environmental factors influencing transferase

expression. As an example of the impact of host genotype, the

H type-1 structure is made by the secretor (Se) gene product, and

the majority (80 % of Caucasians, all South American Indians

and Orientals) carry this structure and are thus referred to as

“ secretors ” . 61 Individuals may also express the Lewis (Le) gene

(90 % of the Caucasian population) and, provided that they are

also secretors, will modify H type-1 into the Le b structure. 61,62

If they are nonsecretors, type-1 chains without its blood group

antigen H will be turned into Le a structures. 61,62 The third


REVIEW

Se phenotype with simultaneous expression of Le a and Le b

antigens has been described as the “ weak-Secretor ” (Se w )

phenotype. 63,64 The dual expression of Le a / Le b is a consequence

of an enzymatically weak Se-transferase in combination with

an intact Le-transferase. 63,64 The terminal structures of mucin

oligosaccharides are highly heterogeneous and vary between /

within species and between and even within tissues. The array

of oligosaccharide structures on individual mucin molecules is

also somewhat determined by stochastic events as the mucin

protein moves through the Golgi apparatus. 65 This structural

diversity may allow the mammalian host to cope with diverse

and rapidly changing pathogens, as reflected by the observation

that susceptibility to specific pathogens differs between peo-

ple with different histo-blood groups, 66 as exemplified by the

observations that individual Se phenotype may determine the

ratio of infection as well as the course and severity of urinary

tract infections, Norwalk virus induced acute gastroenteritis

and Helicobacter pylori -induced gastric diseases. 67 – 69 There is

also a strong correlation between distinct adhesive properties of

H. pylori endemic in specific human populations and the mucin

blood group carbohydrate structures expressed by these popula-

tions. 70 These differences in the external barrier to infection can

be equated with the diversity in underlying innate and adaptive

immunity (e.g., polymorphisms in MHC, cytokines), which is

thought to have evolved for the same reasons.

REGULATION AND MODULATION OF THE MUCIN BARRIER The gel-forming mucins are produced by cells in the epithelial

surface and / or by glands located in the submucosal connective

tissues, and secretion occurs via both constitutive and regulated

pathways. 71 Both gel-forming and cell-surface mucins show

constitutive and inducible gene expression in mucosal epithe-

lial cells. The promoters of the MUC genes have generally not

been fully characterized, although partial promoter characteri-

zation is available for human MUC1 , 72 – 74 MUC2 , 75 – 81 MUC3 , 82

MUC4 , 83 – 85 MUC5AC , 79,86 and MUC5B. 87 Differential regu-

lation of individual mucin genes is evident between different

mucosal tissues and throughout differing regions of the larger

epithelial tracts. For example, differing promoter regions are

involved in the differential regulation of constitutive MUC2

expression in the small and large intestines. 78 Expression of

cell-surface and gel-forming mucins can be upregulated by

inflammatory cytokines such as interleukin (IL)-1 � , IL-4,

IL-6, IL-9, IL-13, interferons, tumor necrosis factor- � , nitric

oxide, and other uncharacterized inflammatory factors. 82,88 – 110

Responsiveness to these cytokines provides a link between

mucins, innate mucosal immunity, and mucosal inflamma-

tory responses. Neutrophils can also stimulate increases in

production of both gel-forming and cell-surface mucins by

mucosal epithelial cells via neutrophil elastase. 111 – 116 Microbial

products can stimulate increased production of mucins by mucosal

epithelial cells. 103,117 – 120 In fact, there is evidence that adherence

of probiotic bacteria upregulates cell-surface mucin expression

in vitro , 121,122 perhaps representing an important part of

the mechanism by which probiotic bacteria limit infection by

pathogens. In contrast, the lipopolysaccharide of the pathogen

H. pylori decreases mucin synthesis in gastric epithelial cells

in vitro via activation of cPLA-2, 123 representing a mechanism by

which a pathogen can favorably modulate the mucus barrier.

Table 2 Common O -linked oligosaccharide structures on mucins

Nomenclature Structure

Core type

Core 1 -Gal � 1-3GalNAc � 1-Ser/Thr

Core 2 -Gal � 1-3(-GlcNAc � 1-6)GalNAc � 1-Ser/Thr

Core 3 -GlcNAc � 1-3GalNAc � 1-Ser/Thr

Core 4 -GlcNAc � 1-3(GlcNAc � 1-6)GalNAc � 1-Ser/Thr

N-Acetyllactosamine elongation type

Type 1 -Gal � 1-3GlcNAc � 1-

Type 2 -Gal � 1-4GlcNAc � 1-

Branching

i-antigen -Gal � 1-4GlcNAc � 1-3Gal � 1- (unbranched)

I-antigen -Gal � 1-4GlcNAc � 1-3(-Gal � 1-4GlcNAc � 1-6)Gal � 1- (branched)

Terminal structures

Blood group H Fuc � 1-2Gal � 1-

Blood group A Fuc � 1-2(GalNAc � 1-3)Gal � 1-

Blood group B Fuc � 1-2(Gal � 1-3)Gal � 1-

Terminal structures (Type 1 based)

Lewis a (Le a ) Gal � 1-3(Fuc � 1-4)GlcNAc � 1-

Lewis b (Le b ) Fuc � 1-2Gal � 1-3(Fuc � 1-4)GlcNAc � 1-(includes H)

Sialyl-Le a NeuAc( � 2-3)Gal � 1-3(Fuc � 1-4)GlcNAc � 1-

Terminal structures (Type 2 based)

Lewis x (Le x ) Gal � 1-4(Fuc � 1-3)GlcNAc � 1-

Lewis y (Le y ) Fuc � 1-2Gal � 1-4(Fuc � 1-3)GlcNAc � 1-(includes H)

Sialyl-Le x NeuAc � 2-3 Gal � 1-4(Fuc � 1-3)GlcNAc � 1-

Sulfation

3 Sulfation HSO 3 -3Gal � 1-

6 Sulfation HSO 3 -6GlcNAc � 1-

Examples of combined epitopes

H-type 1 Fuc � 1-2Gal � 1-3GlcNAc � 1-

Sialylated type 2 NeuAc � 2-3Gal � 1-4GlcNAc � 1-


REVIEW

The constitutive pathway continuously secretes sufficient

mucin to maintain the mucus layer, whereas the regulated path-

way affords a massive discharge as a response to environmen-

tal and / or (patho)physiological stimuli, including cholinergic

stimuli, inflammatory cytokines, prostaglandins, lipopoly-

saccharide, bile salts, nucleotides, nitric oxide, vasoactive intestinal

peptide, and neutrophil elastase. 93,95,103,113,124 – 131 Stimulated

mucin release can occur immediately and is accompanied by

hydration, resulting in approximately a hundredfold expansion

in volume of the secretory granule contents. 132,133 Shedding of

the large extracellular � -subunits of cell-surface mucins from

the cell surface and release of secreted splice variants of cell-

surface mucins are less well understood. The protease ADAM17

(also known as TACE) has been shown to trigger shedding of

MUC1 in endometrial cells in response to tumor necrosis factor-

� , 28,134 and the matrix metalloproteinase-1 also appears to be an

effective MUC1 sheddase. 29

In addition to regulation of their synthesis and release, mucins

are regulated in terms of their glycosylation. Altering mucin carbo-

hydrates may block mechanisms that pathogens use to subvert

the mucin barrier. Tumor necrosis factor- � alters sialylation of

mucins produced by a tracheal cell line 135 and expression of

both fucosyltransferases and � -2,3-sialyltransferases by normal

bronchial mucosal explants. 136 In respiratory epithelial cells,

the Th2 cytokines IL-4 and IL-13 increase expression of core

2 � -1,6- N -acetylglucosaminyltransferase, which forms � -1,6-

branched structures, including core 2, core 4, and blood group

I antigen. 137 In addition, glycosylation changes occur during

infection / inflammation, for example, in individuals with cystic

fibrosis or chronic bronchitis, 138 as well as H. pylori -infected

individuals. 69,139 The inflammation-associated mucin sialylation

shown in patients with H. pylori infection returns to the nor-

mal pattern following successful bacterial clearance with anti-

biotics. 140 In rhesus monkeys that share strong similarities in

mucin glycosylation and the natural history of H. pylori infec-

tion with humans, 141 H. pylori infection induces time-dependent

changes of mucosal glycosylation that alter the H. pylori adhe-

sion targets. 69 Such fine-tuned kinetics of host glycosylation

dynamically modulate host – bacterial interactions, appearing

to balance the impact of infection and thereby may determine

the severity of disease. 69 Another example of dynamic changes

in mucins occurs following infection of rats with the intestinal

parasite Nippostrongylus brasiliensis ; infection induces increased

production and several alterations in the glycosylation of intes-

tinal Muc2 gel-forming mucin, one of which coincides with

expulsion of the parasite. 142 – 147 These alterations in glycosyla-

tion appear to be driven partly by CD4 + T cells, as CD4 but

not CD8 depletion blocks the increase in mucin production,

change in glycosylation and worm expulsion, 148 and also by

T-cell-independent mechanisms. 149 Such changes in mucin

glycosylation need to be considered as a component of innate

and adaptive immune responses to mucosal infection.

MICROBIAL ADHERENCE TO THE EPITHELIUM To colonize mucosal surfaces and invade the host, microbes typi-

cally must first penetrate the secreted mucus barrier and then

either attach to the apical surface of epithelial cells decorated

with the cell-surface mucins or release toxins that disrupt epithe-

lial integrity. Bacterial adhesion to host cells can be mediated by

hydrophobic interactions, cation bridging (i.e., divalent cations

counteracting the repulsion of the negatively charged surfaces

of bacteria and host) and receptor ligand binding. One of the

most extensively studied mechanisms of bacterial adhesion

is via lectins and their corresponding glycosylated receptors.

Binding is usually of low affinity, but clustering of adhesins and

receptors results in multivalent binding. Fimbriae (or pili), outer

membrane proteins, and cell wall components (e.g., lipopoly-

saccharide) may all function as adhesins. Adhesion can affect the

bacteria by stimulation / inhibition of growth as well as induction

of other adhesive structures and proteins required for invasion

such as secretion systems. On the other hand, effects of adhesion

on host cells can include altered morphology, fluid loss, induc-

tion of cytokine release, upregulation of adhesion molecules,

and apoptosis. 150

Many bacterial adhesins bind oligosaccharides present on

mucins. Whether bacterial – mucin binding events favor the

bacteria or the host is a key question. In reality, for some organ-

isms, this may be a truly commensal relationship with benefits

for both the bacteria (by facilitating retention in a favorable

niche and even by providing mucin oligosaccharides for meta-

bolism) and the host (by retaining bacteria in the outer areas

of the mucus barrier where they cannot harm the underlying

epithelium and also limiting the niche available for pathogenic

bacteria). Numerous interactions between microorganisms and

mucins and / or mucin-type carbohydrates have been demon-

strated (see Table 3 ). Bacteria may have multiple adhesins with

different carbohydrate specificities, and modulation of surface

receptor density, kinetic parameters, or topographical distribu-

tions of these receptors on cell membranes regulate adhesion.

As an example, H. pylori binds to mucin oligosaccharides via at

least four adhesins, which differ substantially with anatomical

site along the oro-gastric infection route, mucin type, pH, and

gastric disease status. 139,151 – 153 Thus, for H. pylori , binding to

mucins can have differing consequences during colonization of

the oral-to-gastric niches and during long-term infection.

MUCINS AS DECOYS FOR MICROBIAL ADHESINS Mucus hypersecretion ensuing from infection is testament to the

role of mucus as a component of host defense. Although mucins

are the major macromolecular constituent of mucus and are

largely responsible for formation of the mucus gel, the precise

nature of their role in host defense has not been well demons-

trated empirically. Formation of the mucus gel is important

in itself, as it provides a biophysical barrier as well as a matrix

supporting the retention of a host of antimicrobial molecules.

However, the secreted mucins themselves are likely to function

as decoys for adhesins that have been evolved by pathogens

to engage the cell surface, as the mucins express many of

the oligosaccharide structures found on the cell surface and are

constitutively produced in large amounts, constantly washing

the mucosal surfaces ( Figure 1 ). Some mucins are effective

viral agglutinating agents and exogenously applied mucins


REVIEW

are effective inhibitors of viral infection in in vitro -cultured

cells. 154,155 Streptococcus pyogenes , Tritrichomonas foetus ,

Tritrichomonas mobilensis , influenza viruses, reoviruses,

adenoviruses, enteroviruses, and coronaviruses bind to sialic

acids, which are present both at the epithelial surface and on

mucins. 156 – 165

Despite the accepted dogma that secreted mucins limit infec-

tion, there are few empirical in vivo data demonstrating their

importance. The only secreted mucin for which genetically

deficient animals are available is the intestinal mucin, Muc2.

Muc2 − / − mice develop spontaneous inflammation, presum-

ably due to the absence of the major component of intestinal

mucus, leading to increased exposure to the normal intestinal

microbial flora. 166,167 As yet, there are no reports of controlled

infection experiments in these mice. Further models of secreted

mucin deficiency are required to comprehensively determine

the importance of secreted mucins in preventing and clearing

mucosal infection.

Many pathogens require direct binding to, or penetration of,

mucosal epithelial cells to cause pathology. The widest diversity

of cell-surface mucin expression is in the mucosal tissues most

at risk of infection, such as the gastrointestinal tract, respiratory

tract, and eye; notably, nine of the ten cell-surface mucins are

expressed in the large intestine, which is the most microbe-rich

mucosal environment. Importantly, their ability to be shed from

the cell surface has led us to hypothesize that one of the main

functions of cell-surface mucins is to act as releasable decoy

ligands for microbes attempting to anchor themselves to the

glycocalyx. Cell-surface mucins initiate intracellular signaling

in response to bacteria, suggesting that they have both a bar-

rier and reporting function on the apical surface of all mucosal

epithelial cells. 57 However, until recently, much of the evidence

had been circumstantial or restricted to in vitro analysis. For

example, upregulation of MUC3 expression in colonic cells

has been correlated with decreased binding of enteropatho-

genic E. coli . 121,122 In vitro studies have shown that expression

of MUC1 by transfection inhibits reovirus and adenovirus

infection of MDCK cells by up to tenfold. 168,169 Milk can limit

bacterial and viral infections of the gastrointestinal tract and

this has been attributed in part to the presence of large amounts

of cell-surface mucins, chiefly MUC1 and MUC15, in the milk-

fat globule membrane. 170 – 172 Muc1 − / − mice were reported to

display chronic infection and inflammation of the reproduc-

tive tract, reducing fertility rates. In this latter study, only

normal endogenous bacteria were isolated, suggesting that

these species become opportunistic pathogens in the absence of

Muc1. 173 In addition, Muc1 − / − mice were reported to have

a high frequency of eye inflammation / infection involving

Corynebacteria , Staphylococci and Streptococci , 174 although this

could not be duplicated in a different mouse background held

in alternative housing conditions. 175

We recently demonstrated that the intestinal pathogen

C. jejuni binds to fucosylated mucin oligosaccharides. Controlled

infection experiments demonstrated rapid transit of C. jejuni

across the gastrointestinal barrier and greater intestinal patho-

logy in Muc1 − / − mice. 55 Bone marrow transplantation studies

demonstrated that the increased susceptibility was due to loss

of Muc1 on epithelium rather than on leukocytes (which can

also express Muc1). Loss of Muc1 had no discernable effects

on the abundance or constituency of the intestinal microbial

flora. Muc1 appears to prevent C. jejuni infection both by

protecting cells from the effects of the cytolethal distending

toxin (see above) and by acting as a releasable decoy. 55 We have

also demonstrated that even though H. pylori can bind Muc1,

that primary murine gastric epithelial cells expressing Muc1

bind fewer H. pylori than Muc1 − / − cells. 176 This paradoxical

result is explained by Muc1 acting as a releasable decoy, i.e., the

bacteria bind Muc1 expressed on epithelial cells, which is then

shed by the host. Due to the absence of this decoy molecule,

Muc1 − / − mice develop an approximately fivefold greater coloni-

zation density of H. pylori from the first days following infection

that is maintained for at least 2 months. Consequently, Muc1 − / −

mice develop severe gastritis not found in wild-type mice. 176

Heterozygous mice that have a lower level of gastric Muc1

Table 3 Characterized interactions between mucins and microbes

Tissue derived mucins Mucin Carbohydrate Microbe References

Respiratory mucins MUC1 Sialic acids P. aeruginosa, Haemophilus influenzae, S. aureus, influenza viruses

163,181,270 – 272

Salivary mucins MUC5B MUC7 (DMBT1-Muclin)

Sulfated Le a Sialic acids, Sialyl Le x , Le b

P. aeruginosa, H. pylori, Streptococcus sanguis, Streptococcus gordonii, Actinobacillus actinomycetemcomitans, Streptococcus spp., Candida albicans

273 – 280

Gastric mucins MUC5AC MUC1 A, B, H, Le b H. pylori 139,151,176,281,282

Intestinal mucins MUC2 Enterotoxigenic Escherichia coli, Enteropathogenic E. coli, Salmonella typhimurium, Shigella boydii, Shigella sonnei, Campylobacter upsaliensis, Yersinia enterolitica, C. albicans, reo-viruses

162,283 – 290

In most studies, only the tissue origin of the mucin has been determined. Which mucins and carbohydrates are responsible for the binding was only determined for a small proportion of the interactions. The mucin and carbohydrate columns thus do not indicate that all microbes listed interact via these specifi c structures, but merely that these have been shown to bind to some of the bacteria.


REVIEW

protein expression show intermediate colonization densities,

which suggests that polymorphisms in MUC1 or genes that

regulate its expression could underlie susceptibility to H. pylori -

induced pathology in human populations. In fact, in humans,

individuals with short MUC1 alleles (encoding smaller extra-

cellular mucin domains) have a higher propensity to develop

gastritis following H. pylori infection. 177 – 179 This may be indica-

tive of lower efficacy of smaller MUC1 extracellular mucin

domains allowing increased access of bacteria to the epithelial sur-

face, or these alleles may be surrogate markers of polymorphisms

influencing the level of gastric MUC1 expression. Recently, a

similar protective role has been demonstrated for the extremely

large MUC16 cell-surface mucin in human corneal epithelial

cells. Greater binding of Staphlylococcus aureus occurs to in vitro -

cultured corneal cells when MUC16 is depleted by RNAi. 180

Paradoxically, our demonstrations of Muc1-limiting infec-

tion in the gastrointestinal tract are opposite to that found in a

model of respiratory Pseudomonas aeruginosa infection in the

lung in which Muc1 − / − mice have an increased clearance of

bacteria and a reduced inflammatory response to infection. 181

MUC1 binds the P. aeruginosa flagellin, 182,183 but intriguingly

appears to inhibit flagellin-stimulated TLR5-mediated activation

of NF- � B by an as yet unclear mechanism requiring the MUC1

cytoplasmic domain. 181,184 Whereas an infection-promoting

role of a molecule highly expressed on the apical surface of a

broad array of mucosal epithelia appears counterintuitive, an

anti-inflammatory role for MUC1 in some tissues is consistent

with evolutionary adaptations to clear infection by local defense

without potentially damaging inflammation, where possible.

Further investigations are required with a broad array of patho-

gens in multiple tissues to clearly delineate the participation of

the family of cell-surface mucins in mucosal defense.

OTHER PROTECTIVE ROLES OF MUCINS Mucins have direct and indirect roles in defense from infec-

tion distinct from their ability to form a physical barrier and act

as adhesion decoys. Not only do mucin oligosaccharides bind

microbes, but also, in some cases, they either have direct antimi-

crobial activity or carry other antimicrobial molecules. A mucin

oligosaccharide, � -1-4-linked N -acetyl-glucosamine, which is

expressed by some gastric mucins, has been shown to directly

interfere with synthesis of H. pylori cell wall components. 185

H. pylori must live within gastric mucus to remain protected

from luminal gastric pH and prevent expulsion into the intes-

tine. The antimicrobial mucin oligosaccharide probably acts to

limit H. pylori expansion within gastric mucus. The non-

oligomerizing secreted salivary mucin MUC7 has inher-

ent direct candidacidal activity via a histatin domain at its

N-terminus. 186,187 In addition, there is evidence for direct bind-

ing of antimicrobial molecules such as histatins and statherin

by mucins that would help retain the antimicrobial molecules

in the correct mucosal microenvironment where they can

best protect the host. For example, MUC7 binds statherin and

histatin-1, 188 and the other major mucin in saliva, MUC5B,

binds histatin-1, -3, and -5 and statherin. 189 Secretory IgA

(sIgA) is secreted via mucosal epithelial cells and needs to be

Figure 1 Diagrammatic representation of mucins in the mucosal barrier to infection. ( a ) The normal mucosa is covered with a continuously replenished thick mucus layer retaining host-defensive molecules. Commensal and environmental microbes may live in the outer mucus layer but the layer ensures that contact of microbes with epithelial cells is rare. ( b ) Early in infection, many pathogens actively disrupt the mucus layer and thereby gain access to the epithelial cell surface. In addition, this alters the environment for commensal and environmental microbes and opportunistic pathogenesis may occur. ( c ) Pathogens that break the secreted mucus barrier reach the apical membrane surface, which is decorated with a dense network of large cell-surface mucins. Pathogens bind the cell-surface mucins via lectin interactions and the mucin extracellular domains are shed as releasable decoy molecules. Consequent to contact with microbes and shedding of the extracellular domain, signal transduction by the cytoplasmic domains of the cell-surface mucins modulates cellular response to the presence of microbes. ( d ) In response to infection, there are alterations in mucins that are driven directly by epithelial cells and in response to signals from underlying innate and adaptive immunity. These alterations include goblet cell hyperplasia and increased mucin secretion and altered mucin glycosylation (depicted by the color change) affecting microbial adhesion and the ability of microbes to degrade mucus. These changes in mucins work in concert with other arms of immunity to clear the infection.


REVIEW

retained in the immediate mucosal environment to maximize

exclusion of pathogens. sIgA is retained at high concentrations

in mucus where it can efficiently trap the progress of pathogens,

although the mechanism(s) for retaining sIgA in mucus are not

well understood. Interestingly, secretory component, which is

tightly bound to the Fc region of dimeric-IgA to form sIgA,

carries oligosaccharide structures similar to those on mucins. 190

In the absence of secretory component carbohydrates, sIgA fails

to associate with mucus and fails to prevent infection in a murine

respiratory bacterial infection model, substantiating both

the physiological importance of sIgA – mucin interactions and

the importance of secretory component carbohydrates in main-

taining this interaction. 191 It is also tempting to speculate that

the poly-anionic mucins bind the poly-cationic antimicrobial

defensin peptides that are co-secreted into mucus. Interactions

of mucins with other secreted antimicrobial molecules has not

been fully explored largely due to difficulties in extracting and

purifying mucins in the absence of denaturing agents likely to

disrupt such interactions. The cell-surface mucins are an inte-

gral component of the glycocalyx where they are likely to inter-

act with proteoglycans and other molecules that could retain

host defense molecules in a molecular complex covering the

apical cell surface. 192,193 Therefore, other mucins and mucin

oligosaccharides may yet prove to have direct and indirect

antimicrobial activity. Regardless of whether antimicrobial

molecules are retained in mucus by direct binding with mucins

or by the biophysical properties of mucus, if mucin synthesis

is aberrant or secreted mucins are degraded, the antimicrobial

molecules will have impaired efficacy.

SUBVERSION OF THE MUCIN BARRIER BY MUCOSAL PATHOGENS Perhaps the best evidence for the importance of the mucin bar-

rier to infection is the wide variety of strategies used by microbes

to subvert or avoid this barrier. Mucin barrier subversion strate-

gies used by microbes include the production of enzymes capa-

ble of degrading mucin core proteins and mucin carbohydrates,

and effective motility through mucus gels. Motility is important

for bacterial mucosal pathogens to facilitate breaking through

the physical mucus barrier. In fact, a vast proportion of mucosal

bacterial pathogens are flagellated. 194,195 H. pylori that have

dysfunctional flagella have a greatly reduced ability to infect. 196

H. pylori uses motility for initial colonization and to attain robust

infection. In conjunction with motility, degradative enzymes

such as glycosulfatases, sialidases, sialate O -acetylesterases,

N -acetyl neuraminate lyases and mucinases are produced by

a broad range of bacterial pathogens to destabilize the mucus

gel and remove mucin decoy carbohydrates for adhesins. 197 – 201

The protozoan parasite Entamoeba histolytica cleaves the MUC2

mucin, which is the major structural component of the intes-

tinal mucus, and this cleavage is predicted to depolymerize

the MUC2 polymers. 202 The size of the polymer is important

for the formation of entangled gels and the viscous properties

of mucus; consequently, cleavage of the mucin polymer will

effectively result in a local disintegration of mucus. 203 There is

evidence that these degradative enzymes are critical for micro-

bial pathogenesis. For example, the Vibrio cholerae Hap A,

which has both mucinolytic and cytotoxic activity, is induced by

mucin and required for translocation through mucin-contain-

ing gels. 204 The widespread and critically required expression of

neuraminidases by a wide variety of sialic acid-binding mucosal

viruses underlines the importance of elimination of mucin

carbohydrates for their pathogenicity. 160 Lipopolysaccharide

from H. pylori decreases mucin synthesis, 123 and the mucin

carbohydrate-binding adhesins BabA and SabA undergo phase

variation and change expression during infection, 153,205 which

may allow them to evade this host defense mechanism.

AVOIDANCE OF THE MUCIN BARRIER BY MUCOSAL PATHOGENS Another strategy commonly used by mucosal pathogens is to

avoid the mucin barrier. Intestinal M cells, specifically designed

to capture and present microbes to the underlying lymphoid

tissue, can be regarded as a hole in the mucin barrier. The dome

epithelium in which they lie lacks goblet cells, and therefore

does not produce gel-forming mucins, and their apical cell sur-

face has only sparse microvilli and an apparently thin glycoca-

lyx. 206,207 Although no studies have measured the expression

of individual cell-surface mucins in M cells, there appear to be

differences in the glycocalyx mucins between M cells and adja-

cent intestinal mucosal epithelial cells. In some species, M cells

can be identified by their pattern of lectin binding to specific

cell-surface carbohydrates that differ with other mucosal

epithelial cells. 208,209 Consequently, even though M cells

constitute only a very small percentage of mucosal epithelial

cells, they are the major point of attachment and / or entry used

by a large number of mucosal pathogens including bacteria

(e.g., S. typhimurium , Shigella flexneri , Yersinia enterocolitica ,

and V. cholerae ), viruses (e.g., reovirus, HIV-1, and polio virus)

and parasites (e.g., Cryptosporidia). 206,210,211 Another strategy

used by pathogens to avoid the cell-surface mucin barrier, once

mucus is penetrated or M cells are invaded, is to disrupt the

tight junctions between adjacent mucosal epithelial cells thereby

exposing the vulnerable lateral membranes not protected by the

glycocalyx. Such examples include S. flexneri , 212 enteropatho-

genic E. coli , 213 Porphyromonas gingivalis 214 and H. pylori. 215

MODELS TO INVESTIGATE INTERACTIONS BETWEEN MICROBES AND MUCINS Numerous models, including cancer cell-lines, organ cultures of

gastric biopsies and whole animals have been used to investigate

mucin – microbe interactions. Although they express orthologs

to most human mucins, the most commonly used laboratory

animals such as rats and mice have differing glycosylation of

some of their mucins. In fact, it is tempting to speculate that

differences in mucin glycosylation between mammalian species

may underlie some of the differences in infectivity / pathogenicity

for individual microbial pathogens. Murine knockout models

are only available for Muc1 , 216 Muc2 , 166 and Muc13 (M.A.

McGuckin, unpublished data), and there are also mutants with

aberrant Muc2 assembly. 217 Thus, there is a need for more

models, as mouse knockouts, although limited by the slightly


REVIEW

different glycosylation, still represent an important way to

collect information of the in vivo function of mucins in

infection. Because human pathogens commonly have adhes-

ins for human carbohydrate structures, it is important to select

appropriate models for individual pathogens. For example, the

effects of H. pylori infection on the mouse are mild, and gastric

cancer is not induced even after long-term exposure without

other stimuli or genetic defects, although the mouse may develop

chronic atrophic gastritis. 218,219 H. pylori can colonize the guinea

pig and the Mongolian gerbil and cause a severe inflammatory

response but does not induce cancer in the absence of exogenous

chemical carcinogens. 220 These small animal models are useful

to study some aspects of H. pylori infection and have the advan-

tage of being relatively cheap. In contrast, rhesus monkeys natu-

rally have persistent H. pylori infection leading to loss of mucus,

gastritis, gastric ulcers and even cancer. 221 – 224 In addition, the

anatomy and physiology of the GI tract of the rhesus monkey,

as well as the expression of mucins and mucin glycosylation are

very similar to that in human. 141 However, this model is expen-

sive, the monkeys can have preexisting natural infection, and

primate research has a higher level of ethical considerations.

In vitro microbial – mammalian cocultures are used extensively

to elucidate the mechanisms by which microbes adhere, invade,

and signal to the host, and to examine ensuing mammalian cell

responses. These complex interactions are reliant on appropriate

gene expression and cellular functioning of both the microbial

and mammalian cells. It is therefore critical that appropriate

microbial and mammalian cells are used and that the environ-

ment created experimentally is as similar to the human mucosal

environment as possible. Human cell lines commonly used for

in vitro infection studies have a highly variable expression of

mucins and mucin glycosylation, and generally have very low

production and secretion of gel-forming mucins. 225 Investigators

using these models need to be aware of these limitations and

consider them in interpreting their data. Additional impor-

tant issues to consider are choice of cell line and, depending

on the type of bacteria, oxygen tension. 225,226 With respect to

appropriate mucin production, primary human tracheobron-

chial epithelial cells cultured in an air – liquid interface represent

the most physiological cell cultures in which infection studies

can currently be undertaken. 227 Ex vivo -cultured tissue explants

provide another potential avenue for exploring microbial mucin

interactions in vitro . 228 – 230

CONCLUSIONS The personal repertoire of expression of mucin core proteins

and their glycans, mucin allele length, and transient changes in

mucin expression and glycosylation in response to infection or

stress, as well as variations in environmental conditions may all

affect microbial interaction with host mucins and the patho-

genic consequences of microbial colonization. Rather than a

static barrier, mucins should be considered as a dynamic respon-

sive component of the mucosal barrier that interacts with and

responds to other elements of innate and adaptive immunity.

Difficulties in working with these complex glycoproteins and

the paucity of physiological experimental systems need to be

overcome if we are to fully understand the roles of mucins in

host defense from infection.

DISCLOSURE The authors declared no conflict of interest.

© 2008 Society for Mucosal Immunology

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265 . Hori , Y . , Spurr-Michaud , S . , Russo , C . L . , Argueso , P . & Gipson , I . K . Differential regulation of membrane-associated mucins in the human ocular surface epithelium . Invest. Ophthalmol. Vis. Sci. 45 , 114 – 122 ( 2004 ) .

266 . Andersch-Bjorkman , Y . , Thomsson , K . A . , Holmen Larsson , J . M . , Ekerhovd , E . & Hansson , G . C . Large scale identifi cation of proteins, mucins, and their O -glycosylation in the endocervical mucus during the menstrual cycle . Mol. Cell Proteomics 6 , 708 – 716 ( 2007 ) .

267 . Gipson , I . K . et al. MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence . Biol. Reprod. 78 , 134 – 142 ( 2008 ) .

268 . Gum , J . R . Jr , Crawley , S . C . , Hicks , J . W . , Szymkowski , D . E . & Kim , Y . S . MUC17, a novel membrane-tethered mucin . Biochem. Biophys. Res. Commun. 291 , 466 – 475 ( 2002 ) .

269 . Higuchi , T . et al. Molecular cloning, genomic structure, and expression analysis of MUC20, a novel mucin protein, up-regulated in injured kidney . J. Biol. Chem. 279 , 1968 – 1979 ( 2004 ) .

270 . Kubiet , M . , Ramphal , R . , Weber , A . & Smith , A . Pilus-mediated adherence of Haemophilus infl uenzae to human respiratory mucins . Infect. Immun. 68 , 3362 – 3367 ( 2000 ) .

271 . Scharfman , A . et al. Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth . Infect. Immun. 64 , 5417 – 5420 ( 1996 ) .

272 . Shuter , J . , Hatcher , V . B . & Lowy , F . D . Staphylococcus aureus binding to human nasal mucin . Infect. Immun. 64 , 310 – 318 ( 1996 ) .

273 . Reddy , M . S . Binding of the pili of Pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva . Curr. Microbiol. 37 , 395 – 402 ( 1998 ) .

274 . Hoffman , M . P . & Haidaris , C . G . Analysis of Candida albicans adhesion to salivary mucin . Infect. Immun. 61 , 1940 – 1949 ( 1993 ) .

275 . Veerman , E . C . et al. Sulfated glycans on oral mucin as receptors for Helicobacter pylori . Glycobiology 7 , 737 – 743 ( 1997 ) .

276 . Murray , P . A . , Prakobphol , A . , Lee , T . , Hoover , C . I . & Fisher , S . J . Adherence of oral streptococci to salivary glycoproteins . Infect. Immun. 60 , 31 – 38 ( 1992 ) .

277 . Prakobphol , A . et al. Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria . Biochem. 38 , 6817 – 6825 ( 1999 ) .

278 . Liu , B . et al. The recombinant N-terminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity . Biochem. J. 345 , 557 – 564 ( 2000 ) .

279 . Liu , B . et al. Interaction of human salivary mucin MG2, its recombinant N-terminal region and a synthetic peptide with Actinobacillus actinomycetemcomitans . J. Periodontal. Res. 37 , 416 – 424 ( 2002 ) .

280 . Bosch , J . A . et al. Stress as a determinant of saliva-mediated adherence and coadherence of oral and nonoral microorganisms . Psychosom. Med. 65 , 604 – 612 ( 2003 ) .

281 . Hirmo , S . , Artursson , E . , Puu , G . , Wadstrom , T . & Nilsson , B . Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor . J. Microbiol. Methods 37 , 177 – 182 ( 1999 ) .

282 . Linden , S . et al. Strain- and blood group-dependent binding of Helicobacter pylori to human gastric MUC5AC glycoforms . Gastroenterology 123 , 1923 – 1930 ( 2002 ) .

283 . Sun , R . , Anderson , T . J . , Erickson , A . K . , Nelson , E . A . & Francis , D . H . Inhibition of adhesion of Escherichia coli k88ac fi mbria to its receptor, intestinal mucin-type glycoproteins, by a monoclonal antibody directed against a variable domain of the fi mbria . Infect. Immun. 68 , 3509 – 3515 ( 2000 ) .

284 . Mack , D . R . & Blainnelson , P . L . Disparate in vitro inhibition of adhesion of enteropathogenic Escherichia coli rdec-1 by mucins isolated from various regions of the intestinal tract . Pediatr. Res. 37 , 75 – 80 ( 1995 ) .

285 . Rajkumar , R . , Devaraj , H . & Niranjali , S . Binding of Shigella to rat and human intestinal mucin . Mol. Cell Biochem. 178 , 261 – 268 ( 1998 ) .

286 . Vimal , D . B . , Khullar , M . , Gupta , S . & Ganguly , N . K . Intestinal mucins: the binding sites for Salmonella typhimurium . Mol. Cell Biochem. 204 , 107 – 117 ( 2000 ) .

287 . Jin , L . Z . & Zhao , X . Intestinal receptors for adhesive fi mbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine — a review . Appl. Microbiol. Biotechnol. 54 , 311 – 318 ( 2000 ) .

288 . Sylvester , F . A . , Philpott , D . , Gold , B . , Lastovica , A . & Forstner , J . F . Adherence to lipids and intestinal mucin by a recently recognized human pathogen, Campylobacter upsaliensis . Infect. Immun. 64 , 4060 – 4066 ( 1996 ) .

289 . Mantle , M . & Husar , S . D . Binding of Yersinia enterocolitica to purifi ed, native small intestinal mucins from rabbits and humans involves interactions with the mucin carbohydrate moiety . Infect. Immun. 62 , 1219 – 1227 ( 1994 ) .

290 . de Repentigny , L . , Aumont , F . , Bernard , K . & Belhumeur , P . Characterization of binding of Candida albicans to small intestinal mucin and its role in adherence to mucosal epithelial cells . Infect. Immun. 68 , 3172 – 3179 ( 2000 ) .

Article:Thecancerglycocalyxmechanicallyprimesintegrin-mediatedgrowthandsurvival.Paszeketal,Nature511:319(2014)

ARTICLEdoi:10.1038/nature13535

The cancerglycocalyx mechanically primesintegrin-mediated growth and survivalMatthew J. Paszek1,2,3,4, Christopher C. DuFort1,2, Olivier Rossier5,6, Russell Bainer1,2, Janna K. Mouw1, Kamil Godula7,8{,Jason E. Hudak7, Jonathon N. Lakins1, Amanda C. Wijekoon1,2, Luke Cassereau1,2, Matthew G. Rubashkin1,2, Mark J. Magbanua9,10,Kurt S. Thorn11, Michael W. Davidson12, Hope S. Rugo9,10, John W. Park9,10, Daniel A. Hammer13, Gregory Giannone5,6,Carolyn R. Bertozzi7,14,15 & Valerie M. Weaver1,2,9,16

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx.We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression ofbulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptorspatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyxpromote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulkyglycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state byapplying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteinsare abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is afeature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

The composition of cell surface glycans and glycoproteins changesmarkedly and in tandem with cell fate transitions occurring in embryo-genesis, tissue development, stem-cell differentiation and diseases suchas cancer1–3. Nevertheless, our understanding of the biochemical func-tions of glycans fails to explain fully why broad changes in glycosylationand glycoprotein expression are critical to cell fate specification andin what ways are they linked to disease. It is currently unclear whetherchanges in glycan and glycoprotein expression reflect a global and moregeneral mechanism that directs cell and tissue behaviour.

From a materials perspective, glycan and glycoprotein expressiondictates the bulk physical properties of the glycocalyx—the exterior cellsurface layer across which information flows from the microenviron-ment to signal transduction pathways originating at the plasma mem-brane. Although the biophysical functions of the glycocalyx are largelyuntested, computational models predict that bulky glycoproteins canpromote transmembrane receptor organization, including the clusteringof integrins at adhesion sites4. These models suggest that glycocalyx-mediated integrin clustering would promote the assembly of matureadhesion complexes and collaborate to enhance growth factor signalling5—phenotypes that are associated with cancer6,7. We demonstrate that aglobal modulation of the physical properties of the glycocalyx altersintegrin organization and function, and present evidence for how theglycocalyx can be co-opted in malignancy to support tumour cell growthand survival.

Regulation of integrin assembly by bulky glycoproteinsTo determine whether glycocalyx bulk contributes to a cancer pheno-type, we used gene expression microarray data to relate metastasis toexpression of genes for which protein products contribute to the gly-cocalyx. The likely contribution of gene product to glycocalyx bulk wasestimated based on the protein’s extracellular domain structure andpredicted number of glycosylation sites (Extended Data Fig. 1). Usingthese estimates we obtained evidence for upregulation of transcripts en-coding bulky glycoproteins and some classes of glycosyltransferases, whichcatalyse the glycosylation of cell surface proteins, in primary tumoursof patients with distant metastases relative to those with localized tumourgrowth (P 5 0.032 for bulky transmembrane proteins, Kolmogorov–Smirnov test; Fig. 1a and Extended Data Fig. 1).

To understand whether bulky glycoproteins could promote tumouraggression by regulating integrin adhesions, we developed an integratedbiochemical and mechanical model that incorporates integrins, the extra-cellular matrix (ECM), the cell membrane and the glycocalyx (ExtendedData Fig. 2). The model revealed that the kinetic rates of integrin–ECMinteractions are tightly coupled to the distances between receptor–ligandpairs and, thus, the physical constraints imposed by the glycocalyx. Inthe presence of bulky glycoproteins, the model predicted that integrin–ECM binding is most favourable at sites of pre-existing adhesive con-tact, where the membrane and ECM substrate are in closest proximity(Fig. 1b). Elsewhere, bulky glycoproteins sterically restrict efficient

1Department of Surgery and Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, California 94143, USA. 2Bay Area Physical Sciences-Oncology Program, Universityof California, Berkeley, California 94720, USA. 3School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA. 4Laboratory for Atomic and Solid State Physics and KavliInstitute at Cornell for Nanoscale Science, Cornell University, Ithaca, New York 14853, USA. 5Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, F-33000 Bordeaux, France.6CNRS, Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, F-33000 Bordeaux, France. 7Department of Chemistry, University of California, Berkeley, California 94720, USA.8The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 9Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California94143, USA. 10Division of Hematology/Oncology, University of California, San Francisco, California 94143, USA. 11Department of Biochemistry and Biophysics, University of California, San Francisco,California 94158, USA. 12National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida 32310, USA. 13Departments of Chemical andBiomolecular Engineering and Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 14Department of Molecular Biology, University of California, Berkeley, California 94720,USA. 15Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA. 16Departments of Anatomy and Bioengineering and Therapeutic Sciences and Eli and Edythe BroadCenter for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California 94143, USA. {Present address: Department of Chemistry and Biochemistry, University ofCalifornia, San Diego, California 92093, USA.

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integrin–matrix engagement (Fig. 1b) by increasing the gap betweenthe plasma membrane and ECM. Thus, the model predicted that where-as bulky glycoproteins reduce the overall integrin-binding rate, theyenhance, rather than impede, integrin clustering and focal adhesionassembly by generating a physically based kinetic trap (Fig. 1c).

To test experimentally whether bulky glycoproteins could drive in-tegrin clustering and focal adhesion assembly, we generated a series ofsynthetic mucin glycoprotein mimetics of increasing length that rapidlyintercalate into the plasma membrane and project perpendicularly tothe cell surface8,9. These glycopolymers consisted of a long-chain poly-mer backbone, pendant glycan chains that mimic the structures of nat-ural mucin O-glycans, a phospholipid for membrane insertion, and afluorophore for imaging (Fig. 1d and Extended Data Fig. 3a–c). Wefound that large glycopolymers with lengths of 80 nm, significantlylonger than the reported integrin length of 20 nm10, are consistentlyexcluded from sites of integrin adhesion on the surface of non-malig-nant mammary epithelial cells (MECs; Fig. 1e). Shorter polymers withlengths of 3 or 30 nm were not excluded (Fig. 1e). Because the mimeticspossessed minimal biochemical interactivity with cell surface proteins(Extended Data Fig. 3d), the data suggest a physical interplay betweenbulky glycoproteins and integrin receptors.

To determine how the largest polymer mimetics influence the nano-scale spatial features of the cell–ECM interface, we measured the topo-graphy of the ventral cell membrane using scanning angle interferencemicroscopy (SAIM), a fluorescence-based microscopy technique thatenables imaging with 5–10-nm axial resolution and diffraction-limited(,250 nm) lateral resolution11. Polymers designed to mimic large nativeglycoproteins (,80 nm) expanded the membrane–ECM gap by 19 nm(Fig. 1f). Consistent with computational predictions, the large glycopro-tein mimetics reduced the overall rate of integrin bond formation, butsignificantly enhanced clustering of integrins into focal adhesions (Fig. 1g, h).Shorter glycoprotein mimetics (3 and 30 nm) did not have an impacton integrin clustering, even when incorporated at higher surface densi-ties (Fig. 1h).

We next asked whether cancer-associated glycoproteins could sim-ilarly influence the spatial distribution of integrins and the assemblyof focal adhesions. On the basis of our large-scale gene expression ana-lysis, we determined that the transmembrane mucin glycoprotein, MUC1,which has a highly glycosylated ectodomain that projects out up to200 nm from the cell surface12, was upregulated in metastatic tumours(nominal P 5 0.0028 via one-sided t-test). In agreement with our ana-lysis, we measured high levels of MUC1 on the surface of several breastcancer cell lines, as well as v-Src and HRAS-transformed MECs (Fig. 2a).

To assess the impact of MUC1 on focal adhesion assembly, we ex-pressed MUC1 on the surface of non-malignant MECs, to levels com-parable to those of transformed MECs and breast cancer lines. MUC1expression induced striking membrane topographical features, whichincluded regions of high curvature, and a significant expansion of thecell membrane–ECM gap (Fig. 2b, c and Extended Data Fig. 4a). Expres-sion of an ectodomain-truncated MUC1 construct did not significantlychange the gap compared to control MECs (Fig. 2c and Extended DataFig. 4a). Our model predicted that the membrane topographies weobserved in MUC1-expresing cells would facilitate integrin clusteringthrough the kinetic trap. In agreement with these predictions, express-ion of full-length MUC1 significantly enhanced the size of adhesionclusters and the total adhesion area per cell (Fig. 2d, e and ExtendedData Fig. 5a). The adhesion assembly phenotype did not require theMUC1 cytoplasmic tail, which mediates MUC1’s biochemical activity(Fig. 2e)13, or direct interactions between MUC1 and fibronectin (Ex-tended Data Fig. 5b). Together, these results are consistent with a phys-ically based mechanism of integrin clustering.

To gain additional insight into the coupled dynamics between inte-grins and MUC1, we conducted time-lapse imaging of fluorescentlylabelled MUC1 and the adhesion plaque protein vinculin. We observedthat MUC1 and integrin adhesions spatially segregate on the cell sur-face in a temporally correlated manner (Fig. 2d, Extended Data Fig. 5cand Supplementary Movie 1), suggesting a physical communication bet-ween these molecules. Further evidence for a physical interplay between

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Figure 1 | The cancer glycocalyx drives integrin clustering. a, Violin plotsshowing increased expression of genes encoding bulky transmembraneproteins in primary tumours of patients with distant metastases relative to thosewith local invasion. White dots and thick black lines indicate the medianand interquartile range of the P value distribution of all transcripts within eachclass: all genes, all membrane proteins (Mem.), and bulky transmembraneproteins (Bulky). b, Computed relative rate of integrin–ECM ligand bondformation as a function of distance from a pre-existing adhesion cluster.c, Model of proposed glycocalyx-mediated integrin clustering. Shorterdistances between integrin–ligand pairs result in faster kinetic rates of binding.

d, Cartoon showing structure of glycoprotein mimetics with lipid insertiondomain. e, Fluorescence micrographs of MEC adhesion complexes (vinculin–mCherry) and glycomimetics of the indicated length (scale bar, 3mm). f, SAIMimages of DiI-labelled ventral plasma membrane topography in MECsincorporated with glycomimetics (scale bar, 2.5mm). g, Rate of integrin–substrate adhesion measured using single cell force spectroscopy in MECs withincorporated glycomimetics. h, Quantification of the total adhesion complexarea per cell in MECs with incorporated glycomimetics. All results are themean 6 s.e.m. of three separate experiments. Statistical significance is given by*P , 0.05; **P , 0.01; ***P , 0.001.

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MUC1 and integrins was obtained in mouse embryonic fibroblasts(MEFs) using single-particle tracking photo-activation localization mi-croscopy (sptPALM14,15) to track MUC1 diffusive trajectories. We notedthat whereas MUC1 was mobile in the plasma membrane, it rarely crossedinto integrin adhesion zones (Fig. 2f and Extended Data Fig. 6a).

We next tested our model’s prediction that MUC1 would favour integ-rin clustering by physically impeding integrin–ECM binding outside ofadhesive contacts. We recorded the trajectories of individual b3 integ-rin molecules using sptPALM to determine the location and fraction ofmobile (confined and freely diffusive) and matrix-bound, immobilized

integrin15. Analysis of b3 integrin trajectories after manganese activa-tion in MEFs revealed a significant increase in the total level of immo-bilized integrin at the plasma membrane, both inside and outsideadhesive contacts (Fig. 3a and Extended Data Fig. 6b–e). By contrast,the immobilized b3 integrin in MEFs expressing high MUC1 was re-stricted to sites of adhesion (Fig. 3a–c and Extended Data Fig. 6e). Theseresults are consistent with single-cell force spectroscopy measurements,which indicated that MUC1 expression reduces the net rate of integrin–ECM bond formation (Extended Data Fig. 5d). Mucin expression didnot have a significant impact on the free diffusion of integrins (ExtendedData Fig. 6b–d). Importantly, we observed that integrins frequently dif-fused across the mucin–adhesive zone boundary and could immobilizerapidly once in the adhesive zone (Fig. 3d, Extended Data Fig. 6f, g andSupplementary Movie 2). Together, our results indicate that large gly-coproteins act as physical ‘steric’ barriers that impede integrin immobi-lization and thus funnel integrins into adhesive contacts.

Bulky glycoproteins exert force on integrin bondsIntegrins switch between activity states by undergoing a conformationalchange that is facilitated by tensile force16,17. Given the order of mag-nitude difference in the size of MUC1 (,200 nm12) as compared tointegrins (,20 nm10), and the close proximity of these molecules withinthe cell–ECM interface, we hypothesized that large glycoproteins, suchas MUC1, modify integrin structure and function by applying force tomatrix-bound receptors. Abiding by Newton’s third law, if large gly-coproteins exert a tensile force on integrins, then we should detect areciprocal strain on the glycoproteins. Consistent with this hypothesis,mucins imaged with SAIM appeared compressed or mechanically bentnear integrin adhesive contacts (Fig. 4a and Extended Data Fig. 4a). Fur-thermore, single-cell force spectroscopy revealed that MECs expres-sing high levels of exogenous MUC1 required higher compressive forceapplication at the ECM–substrate interface to promote integrin-mediatedadhesion when compared to control MECs (Fig. 4b).

To test further whether integrin adhesions strain bulky transmem-brane glycoproteins, we generated a genetically encoded construct con-ceptually similar to a strain gauge, consisting of a cysteine-free cyan andyellow fluorescent protein pair (CFP and YFP) separated by an elasticlinker18, which we inserted into the ectodomain of full-length and trun-cated MUC1 proteins (Fig. 4c and Extended Data Fig. 4b). Fluorescenceresonance energy transfer (FRET) served as the readout of distance bet-ween the CFP and YFP pair and, thus, functioned as a reporter of mo-lecular strain. When the full-length reporter was expressed in MECs,we observed high FRET efficiencies in the cell–substrate interface (Fig. 4d, eand Extended Data Fig. 7). FRET efficiency was significantly lower inMECs expressing the ectodomain-truncated construct, indicative oflower molecular strain (Fig. 4d, e). The highest FRET efficiencies cor-related spatially with sites of adhesive contact, consistent with integrinadhesions straining bulky transmembrane glycoproteins and glyco-proteins exerting a reciprocal restoring force on integrins (Fig. 4d andExtended Data Fig. 7e, f).

We next examined whether the bulky glycoprotein MUC1 couldinduce conformational changes that would activate integrins independ-ent of the contractile cytoskeleton. We used a bi-functional crosslinkerthat can specifically link extracellular fibronectin and bounda5b1 integ-rins that are in a tension-dependent conformation17. Inhibition of acto-myosin contractility, using the myosin inhibitor blebbistatin or the Rhokinase inhibitor Y-27632, abrogated most of the fibronectin crosslinkedintegrins in MECs expressing empty vector (Fig. 4f and Extended DataFig. 8a). By contrast, MUC1-expressing MECs formed tensioned bondswith the ECM substrate, even when cells were pre-treated with con-tractility inhibitors before plating (Fig. 4f and Extended Data Fig. 8a).Of note, the myosin-independent integrin clusters observed in the MUC1-expressing MECs recruited activated cytoplasmic adaptors typicallyassociated with mature adhesion structures and nucleated actin (ExtendedData Fig. 8b). These results suggest that large, cancer-associated gly-coproteins not only facilitate integrin clustering but also physically alter

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Figure 2 | The bulky cancer-associated glycoprotein MUC1 drives integrinclustering. a, Cartoon of MUC1 and quantification of MUC1 cell-surfacelevels on control (10A-Cont.), transformed (10A-v-Src, 10A-HRAS) andtumour (MCF7, T47D) cells. b, Topographical SAIM images of representativemCherry–CAAX-labelled ventral plasma membranes in control and MUC1–GFP-expressing (1MUC1) MECs (Scale bar, 5mm; region of interest (ROI)scale bar, 2mm). c, Quantification of mean plasma membrane height in controlMECs and those ectopically expressing ectodomain-truncated MUC1–GFP(1MUC1(DTR)) and wild-type MUC1–GFP (1MUC1). Results are themean 6 s.e.m. of at least 15 cell measurements in duplicate experiments.d, Fluorescence micrographs of MUC1(DTR) or wild-type MUC1 expressed inMECs and their focal adhesions labelled with vinculin–mCherry (scale bar,3mm; ROI scale bar, 1.5mm). e, Quantification of the total adhesion complexarea per cell in control non-malignant MECs (control) and those ectopicallyexpressing MUC1(DTR), wild-type MUC1, or cytoplasmic-tail-deleted MUC1(1MUC1(DCT)). Results are the mean 6 s.e.m. of three separate experiments.f, Left panel: trajectories of individual mEOS2-tagged MUC1 proteins recordedat 50 Hz using sptPALM (green) and focal adhesions visualized withpaxillin–GFP (red) in MEFs (scale bar, 3mm). Right panel: the ROI from the leftpanel with individual MUC1 tracks displayed in multiple colours (scale bar,1mm). Statistical significance is given by *P , 0.05; **P , 0.01; ***P , 0.001.

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integrin state and do so, at least in part, independently of cytoskeletaltension.

Bulky glycoproteins promote growth and survivalTumour metastasis is a multi-step process that depends on the efficientdissemination of primary cancer cells and their subsequent colonization

at distant metastatic sites19. Thus, the ability to survive, particularlywithin unfavourable microenvironments and under minimally adhes-ive conditions, is a prerequisite for efficient tumour cell metastasis19.Given their ability to promote integrin adhesion assembly, we hypoth-esized that bulky glycoproteins could facilitate metastasis by promotingfocal adhesion signalling to enhance tumour cell growth and survival.

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Figure 4 | Integrins are mechanically loaded and re-enforced by bulkyglycoproteins. a, GFP-fluorescence and topographic SAIM images of MUC1–GFP (scale bars, 3mm) and the corresponding focal adhesions visualizedwith vinculin–mCherry. b, Adhesion rate versus force of contact betweencell and substrate (compressive force) measured with single-cell forcespectroscopy for control and MUC1-expressing MECs. Results are themean 6 s.e.m. of at least 10 cell measurements per point. c, Schematic of FRET-based MUC1 compressive strain gauge. d, FRET efficiency maps ofectodomain-truncated (1MUC1(DTR) sensor) and wild-type (1MUC1sensor) strain gauges measured at the ventral cell surface of MECs and the

corresponding vinculin–mCherry-labelled focal adhesions (scale bar, 8mm;ROI scale bar, 1mm). e, Histogram of observed FRET efficiencies of wild-typeMUC1 and MUC1(DTR) strain gauges. f, Quantification of fibronectin-crosslinked a5 integrin in control and MUC1-expressing normal MECs treatedwith solvent alone (DMSO), myosin-II inhibitor (blebbistatin; 50mM), or Rhokinase inhibitor (Y-27632; 10mM) for 1 h followed by detergent-extractionto reveal the fibronectin bound integrin that is under mechanical tension.Results are the mean 6 s.e.m. of three separate experiments. Statisticalsignificance is given by *P , 0.05; **P , 0.01; ***P , 0.001.

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integrins (scale bar, 1 mm). b, Distribution of b3 integrin diffusion coefficientsrecorded before or after Mn21 treatment in control MEFs outside of adhesivecontacts (left), MUC1-transfected MEFs inside MUC1-rich areas (middle),and MUC1-transfected MEFs outside MUC1-rich areas, including adhesivecontacts (right). c, Fraction of immobilized, confined and freely diffusive b3

integrins outside of adhesive contacts in control MEFs (Ctrl) and MUC1-transfected cells (MUC1) before and after Mn21 treatment. Results are themean 6 s.e.m. Statistical significance is given by *P , 0.05; **P , 0.01;***P , 0.001. d, Fluorescence micrograph of MUC1–GFP and an illustrativesingle integrin trajectory in MEFs treated with Mn21 (scale bar, 1mm).

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Consistent with this notion, analysis of human data sets revealed thatpatients with aggressive breast cancers that presented with circulatingtumour cells (CTCs) express disproportionately high amounts of bulkyglycoproteins and have altered glycosyltransferase expression profiles(Fig. 5a and Extended Data Fig. 1d, e). Furthermore, analysis of genesexpressed within CTCs isolated from a cohort of breast cancer patientswith metastatic disease confirmed that several predicted bulky glyco-proteins could be detected in these patient samples (Fig. 5b).

We next examined whether a bulky glycocalyx could promote growthand survival of non-malignant MECs. Using our glycoprotein mimetics,we observed that untreated MECs or MECs incorporated with short(3 nm) or medium (30 nm) length mimetics were not viable 24–48 hafter they were plated on highly compliant hydrogel substrates thatmimic the stiffness of soft sites of colonization, like lung or brain (Young’smodulus, E 5 140 Pa; Fig. 5c). By contrast, MECs incorporated with longglycoprotein mimetics (80 nm) remained viable (Fig. 5c). Analysis ofgene expression profiles and immunofluorescence analysis of freshlyisolated CTCs in our human metastatic breast cancer cohort revealedthat MUC1 could be detected in most of the samples examined (Fig. 5b).Similar to results with the synthetic mimetics, we observed that ectopicexpression of either full-length or a tailless, signalling-defective MUC1in non-malignant MECs permitted their growth and survival even whenplated as single cells on compliant hydrogels (Fig. 5d and Extended DataFig. 9a).

We noted that the CTCs in our cohort also expressed high levels ofCD44, a receptor that binds and retains bulky hyaluronic acid (HA)glycan structures on the cell surface (Extended Data Fig. 10a)20. Sim-ilar to our observations with MUC1 and bulky glycoprotein mimetics,we observed that HA and integrins exhibit an anti-correlated spatial

distribution on the surface of transformed MECs (Extended Data Fig.10b). Inhibition of HA synthesis or HA cell-surface retention sig-nificantly reduced the growth of transformed MECs on complianthydrogels, raising the possibility that bulky cell-surface constituents,in addition to MUC1, could similarly promote tumour aggression (Ex-tended Data Fig. 10b). However, unlike the experiments with taillessMUC1 or the glycoprotein mimetics, which lack signalling capability,we cannot exclude that HA-induced growth and survival phenotypesare not also, at least in part, induced through HA’s direct biochemicalsignalling activity20,21.

We next addressed whether a bulky glycocalyx promotes MEC growthand survival by regulating focal adhesion assembly and crosstalk withgrowth factor signalling pathways5,7. We found that pharmacologicalinhibition of kinases linked to growth factor signalling, including phos-phoinositide 3-kianse (PI(3)K), mitogen-activated kinase, and Src kinase,each independently inhibited the growth and survival of MUC1-expressingMECs on highly compliant substrates (Fig. 5e). We also noted that theMUC1 growth and survival phenotype requires integrin engagementand integrin signalling through focal adhesion kinase (FAK), whichmediates crosstalk between integrin and growth factor signalling path-ways (Fig. 5f and Extended Data Fig. 9b)5,6. Non-malignant MECs ex-pressing the MUC1 ectodomain, but not control MECs, assembleddistinct focal adhesion structures with activated Y397-phosphorylatedFAK on compliant substrates (Extended Data Fig. 8c). Furthermore,MECs expressing wild-type or tailless, signalling defective MUC1, andplated on the compliant substrates showed enhanced Y118-phosphory-lated paxillin, ERK and AKT activation in response to epidermal growthfactor stimulation (Fig. 5g and Extended Data Fig. 8d). This response wasattenuated by FAK inhibition (Fig. 5g, h and Extended Data Fig. 8d).

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Figure 5 | Bulky glycoproteins promote cell survival and are expressed inCTCs. a, Violin plots showing that genes encoding bulky transmembraneproteins are more highly expressed in primary human tumours in patients withcirculating tumour cells (CTCs). White dots and thick black lines indicatethe median and interquartile range of the P-value distribution of transcripts ofall cellular genes (all genes), all transmembrane proteins (membrane), andbulky transmembrane proteins (bulky). b, Heat map quantifying geneexpression of bulky glycoproteins in CTCs isolated from 18 breast cancerpatients (x axis; left), and representative immunofluorescence micrograph ofMUC1 detected on human patient CTCs (right; scale bar, 5 mm). Quantificationof the percentage of CTCs with detectable MUC1 is shown. c, Cell death incontrol non-malignant MECs and those with incorporated glycomimeticsquantified 24 h after plating on a soft (140 Pa) fibronectin-conjugated hydrogelsubstrate. d, Cell death (left graph) and growth (right graph) of control MECsand those expressing cytoplasmic-tail-deleted MUC1 (1MUC1(DCT))

quantified 48 h after plating on a soft hydrogel. e, Quantification of the numberof vehicle (DMSO), PI(3)K inhibitor, MEK inhibitor, or Src inhibitor-treatedcontrol and MUC1(DCT)-expressing MECs per colony 48 h after plating on asoft hydrogel. f, Proliferation of solvent (DMSO) or FAK-inhibitor-treatedMUC1(DCT)-expressing MECs quantified at the indicated day after plating onsoft hydrogels. g, Representative western blots showing phosphorylated andtotal ERK in control and MUC1(DCT)-expressing MECs plated on softhydrogels unstimulated or stimulated with EGF. Cells were treated with solvent(control, 1MUC1(DCT)) or FAK inhibitor (1MUC1(DCT) 1 FAKi) beforestimulation. h, Bar graphs showing quantification of immunoblots probed foractivated AKT in control and MUC1(DCT)-expressing non-malignant MECs24 h after plating on soft versus stiff hydrogels. i, Model summarizingbiophysical regulation of integrin-dependent growth and survival by bulkyglycoproteins. In all bar graphs, results are the mean 6 s.e.m. of at least 2–3separate experiments (*P , 0.05; **P , 0.01; ***P , 0.001).

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Together, these findings indicate that a bulky glycocalyx can promotetumour aggression by enhancing integrin-dependent growth and sur-vival (Fig. 5i).

DiscussionWe present evidence to support a new paradigm for the biological func-tion of cell surface glycans and glycoproteins. Independent of, and inaddition to, their biochemical properties, we demonstrate how bulkyconstituents of the glycocalyx can physically influence receptor organ-ization and activity. Although the current investigation focuses onintegrins, a bulky glycocalyx could, in principle, regulate any transmem-brane receptor that interacts with a tethered ligand. Candidate systemsinclude neurological and immunological synapses22, cell–cell adhesions23,and juxtacrine signalling complexes composed of receptors, like ephrin24.Membrane topographical features imprinted by large glycoproteinscould also directly influence plasma membrane lipid organization, pro-tein sorting and endocytosis25,26. The diversity of these processes sug-gests that the physiological relevance of the glycocalyx may be broad.For example, bulky glycoproteins and glycan structures, such as neu-roligins, neurexins and polysialic acid, have a crucial role in neuronaldevelopment, maintenance and plasticity27,28. Thus, it is plausible thatthe glycocalyx has a prominent role in orchestrating multiple biologicalprocesses occurring at the plasma membrane.

Our observations provide a tractable explanation for why large gly-can structures and glycoproteins, like HA and mucins, as well as reg-ulatory enzymes, are so frequently elevated in many solid tumours13,20.Indeed, the growth and survival advantages afforded by these moleculesmay preferentially select for cancer cells with a prominent glycocalyxand favour tumour cell dissemination and metastasis. Mechanical per-turbations to cell and tissue structure have a causal role in tumour de-velopment and progression29,30, and we now implicate the glycocalyx’simportance in the metastatic mechano-phenotype. Our results suggestthat the glycocalyx and its molecular constituents are attractive targetsfor therapeutic interventions aimed at normalizing transmembranereceptor signalling.

METHODS SUMMARYComplete descriptions of the bioinformatics pipeline, computational model andexpression constructs are presented in Supplementary Notes 1, 2 and 3, respect-ively. Compliant hydrogels were fabricated from soft polyacrylamide (E 5 140 Pa)functionalized with fibronectin31 and plated with single cells for all hydrogel ex-periments. FRET measurements were conducted in living cells on a spinning diskconfocal (photobleaching FRET) or confocal (lifetime imaging) microscope32 (seealso Supplementary Note 4). SptPALM15, SAIM11, single cell force spectroscopy33,integrin crosslinking17, and fibronectin fibrillogenesis34 measurements and assayswere conducted as previously described. Glycoprotein mimetics were synthesizedand characterized as described in Supplementary Note 6 and subsequently incu-bated with suspended cells (2mM for 1 h) to incorporate onto the cell surface imme-diately before experimentation. For gene expression analysis of CTCs, 20 pools ofCTCs were isolated from the blood of 18 metastatic breast cancer patients and quan-tified by qPCR35. Immunofluorescence of CTCs was conducted on samples isolatedfrom three patients35.

Online Content Methods, along with any additional Extended Data display itemsandSourceData, are available in the online version of the paper; references uniqueto these sections appear only in the online paper.

Received 3 August 2013; accepted 23 May 2014.

Published online 25 June 2014.

1. Lanctot, P. M., Gage, F. H. & Varki, A. P. The glycans of stem cells. Curr. Opin. Chem.Biol. 11, 373–380 (2007).

2. Haltiwanger, R. S. & Lowe, J. B. Role of glycosylation in development. Annu. Rev.Biochem. 73, 491–537 (2004).

3. Varki, A., Kannagi, R. & Toole, B. P. in Essentials Glycobiol. (Varki, A. et al.) http://www.ncbi.nlm.nih.gov/books/NBK1963/ (Cold Spring Harbor Laboratory Press,2009).

4. Paszek, M. J., Boettiger, D., Weaver, V. M. & Hammer, D. A. Integrin clustering isdriven by mechanical resistance from the glycocalyx and the substrate. PLOSComput. Biol. 5, e1000604 (2009).

5. Walker, J. L., Fournier, A. K. & Assoian, R. K. Regulation of growth factor signalingand cell cycle progression by cell adhesion and adhesion-dependent changes incellular tension. Cytokine Growth Factor Rev. 16, 395–405 (2005).

6. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications andtherapeutic opportunities. Nature Rev. Cancer 10, 9–22 (2010).

7. Shibue, T. & Weinberg, R. A. Integrin b1-focal adhesion kinase signaling directs theproliferation of metastatic cancer cells disseminated in the lungs. Proc. Natl Acad.Sci. USA 106, 10290–10295 (2009).

8. Godula, K. et al. Control of the molecular orientation of membrane-anchoredbiomimetic glycopolymers. J. Am. Chem. Soc. 131, 10263–10268 (2009).

9. Godula, K., Rabuka, D., Nam, K. T. & Bertozzi, C. R. Synthesis and microcontactprinting of dual end-functionalized mucin-like glycopolymers for microarrayapplications. Angew. Chem. Int. Edn Engl. 48, 4973–4976 (2009).

10. Eng, E. T., Smagghe, B. J., Walz, T. & Springer, T. A. IntactaIIbb3 integrin is extendedafter activation asmeasured by solution X-ray scattering and electron microscopy.J. Biol. Chem. 286, 35218–35226 (2011).

11. Paszek, M. J. et al. Scanning angle interference microscopy reveals cell dynamicsat the nanoscale. Nature Methods 9, 825–827 (2012).

12. Hattrup, C. L. & Gendler, S. J. Structure and function of the cell surface (tethered)mucins. Annu. Rev. Physiol. 70, 431–457 (2008).

13. Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nature Rev. Cancer9, 874–885 (2009).

14. Manley, S. et al. High-density mapping of single-molecule trajectories withphotoactivated localization microscopy. Nature Methods 5, 155–157 (2008).

15. Rossier, O. et al. Integrins b1 and b3 exhibit distinct dynamic nanoscaleorganizations inside focal adhesions. Nature Cell Biol. 14, 1057–1067 (2012).

16. Chen, W., Lou, J., Evans, E. A. & Zhu, C. Observing force-regulated conformationalchanges and ligand dissociation from a single integrin on cells. J. Cell Biol. 199,497–512 (2012).

17. Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically activated integrin switchcontrols alpha5beta1 function. Science 323, 642–644 (2009).

18. Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulationof focal adhesion dynamics. Nature 466, 263–266 (2010).

19. Nguyen, D. X., Bos, P. D. & Massague, J. Metastasis: from dissemination to organ-specific colonization. Nature Rev. Cancer 9, 274–284 (2009).

20. Toole, B. P. Hyaluronan: from extracellular glue to pericellular cue. Nature Rev.Cancer 4, 528–539 (2004).

21. Bono, P., Rubin, K., Higgins, J. M. & Hynes, R. O. Layilin, a novel integral membraneprotein, is a hyaluronan receptor. Mol. Biol. Cell 12, 891–900 (2001).

22. Dustin, M. L. Signaling at neuro/immune synapses. J. Clin. Invest. 122, 1149–1155(2012).

23. Coombs, D., Dembo, M., Wofsy, C. & Goldstein, B. Equilibrium thermodynamics ofcell-cell adhesion mediated by multiple ligand-receptor pairs. Biophys. J. 86,1408–1423 (2004).

24. Salaita, K. et al. Restriction of receptor movement alters cellular response: physicalforce sensing by EphA2. Science 327, 1380–1385 (2010).

25. Groves, J. T. Bending mechanics and molecular organization in biologicalmembranes. Annu. Rev. Phys. Chem. 58, 697–717 (2007).

26. Lundmark,R. & Carlsson, S. R. Driving membrane curvature in clathrin-dependentand clathrin-independent endocytosis. Semin. Cell Dev. Biol. 21, 363–370 (2010).

27. Comoletti, D. et al. Synaptic arrangement of the neuroligin/b-neurexin complexrevealed by X-ray and neutron scattering. Structure 15, 693–705 (2007).

28. Hildebrandt, H., Muhlenhoff, M., Weinhold, B. & Gerardy-Schahn, R. Dissectingpolysialic acid and NCAM functions in brain development. J. Neurochem. 103(Suppl. 1), 56–64 (2007).

29. Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancingintegrin signaling. Cell 139, 891–906 (2009).

30. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. CancerCell 8, 241–254 (2005).

31. Lakins, J. N., Chin, A. R. & Weaver, V. M. Exploring the link between humanembryonic stem cell organization and fate using tension-calibrated extracellularmatrix functionalized polyacrylamide gels. Methods Mol. Biol. 916, 317–350(2012).

32. Bruns, N., Pustelny, K., Bergeron, L. M., Whitehead, T. A. & Clark, D. S. Mechanicalnanosensor based on FRET within a thermosome: damage-reporting polymericmaterials. Angew. Chem. Int. Ed. 48, 5666–5669 (2009).

33. Friedrichs, J., Helenius, J. & Muller, D. J. Quantifying cellular adhesion toextracellular matrix components by single-cell force spectroscopy. NatureProtocols 5, 1353–1361 (2010).

34. Pankov, R. & Momchilova, A. Fluorescent labeling techniques for investigation offibronectin fibrillogenesis (labeling fibronectin fibrillogenesis). Methods Mol. Biol.522, 261–274 (2009).

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Supplementary Information is available in the online version of the paper.

Acknowledgements We thank S. Gendler, J. Goedhart and M. McMahon for cDNAs, asindicated in the Methods section. We thank A. Walker for bioinformatics support,L. Hauranieh for assistance in CTC analysis, H. Aaron for assistance with FLIM,J. B. Sibarita and M. Lagardere for support in sptPALM analysis, B. Hoffman in design ofthe FRET sensor, and T. Wittmann in assistance with pbFRET measurements. Imageacquisition was partly performed at the Nikon Imaging Center and Biological ImagingDevelopment Center at UCSF and the Berkeley Molecular Imaging Center. This workwas supported by the Kavli Institute and UCSF Program for Biomedical Breakthroughpostdoctoral fellowships to M.J.P.; DoD NDSEG Fellowship to M.G.R.; NIH GM59907 toC.R.B.; NIH Pathway to Independence Award K99 EB013446-02 to K.G.; French

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http://www.ncbi.nlm.nih.gov/books/NBK1963

http://www.ncbi.nlm.nih.gov/books/NBK1963


Ministry of Research, CNRS, ANR grant Nanomotility, INSERM, Fondation ARC pour laRecherche sur le Cancer, France BioImaging ANR-10-INBS-04-01, and ConseilRegional Aquitaine to O.R. and G.G.; NIH AI082292-03A1 to D.A.H.; The Breast CancerResearch Foundation to M.J.M., H.S.R. and J.W.P.; NIH 2R01GM059907-13 to C.R.B.and V.M.W.; and BCRP DOD Era of Hope Scholar Expansion grant BC122990, and NIHNCI grants U54CA163155-01, U54CA143836-01, 1U01 ES019458-01, andCA138818-01A1 to V.M.W.

Author Contributions V.M.W. andM.J.P. conceived the project. V.M.W.,M.J.P. and C.R.B.provided project management. M.J.P., A.C.W., M.W.D. and J.N.L. designed andconstructed expression constructs. C.C.D. and M.J.P conducted single cell forcespectroscopy measurements. M.J.P. and K.S.T. designed and implemented theinterference microscope. M.J.P., L.C. and M.G.R. performed fluorescence, FRET andinterferometric imagingand M.J.P. wrote the accompanyinganalysis software. O.R. and

G.G. conducted sptPALM experiments and analysed the results. V.M.W., R.B. and M.J.P.designed the bioinformatics pipeline and R.B. implemented the pipeline andperformed the large-scalegene expression analysis. J.K.M., A.C.W. andM.J.P. fabricatedand conducted experiments on compliant hydrogels. M.J.M., H.S.R. and J.W.P. isolatedCTCs and measured CTC gene and protein expression. K.G., J.E.H. and C.R.B.designed, synthesized and characterized the glycoprotein mimetics. M.J.P andD.A.H. constructed and implemented the computational model. M.J.P. and V.M.Wwrote the manuscript with input from all authors.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondenceand requests for materials should be addressed to V.M.W.([email protected]).

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METHODSBioinformatics. To estimate protein-level contributions to extracellular membranebulkiness, we used TMHMM to identify extracellular domains within each isoformsequence (RefSeq v47) and counted the number of putative extracellular glycosyla-tion sites predicted by NetOGlyc 3.1 and search of N-glycosylation motifs. Gene-wise enrichment of mRNA upregulation among bulky proteins in clinical data (GEOaccessions GSE12276 and GSE31364) was tested by permuting P values quantifyingevidence for upregulation in the appropriate samples. Variance in mRNA upregu-lation explained by membrane bulkiness was estimated by regressing the negative log-transformed P values on the square root of the combined N- and O-glycosylationsites and comparing the residuals with the intercept model. Additional details ofthe analysis and models are provided in Supplementary Note 1.Computational model. A mechanical model of the cell–ECM interface was con-structed as described previously4. A summary of the model is described in Supplemen-tary Note 2 and parameters are detailed in Supplementary Table 1.Antibodies and reagents. Antibodies used include: mouse monoclonal antibody(mAb) vinculin (MAB674; Millipore), mouse mAb talin (8d4; Sigma), rat mAbb1-integrin (AIIBII), rabbit mAb paxillin (Y113; Abcam), rabbit mAb FAK pY397(141-9; Invitrogen), rabbit polyclonal antibody (pAb)a5-integrin (AB1928; Millipore),mouse mAb MUC1 (HMPV; BD Pharminigen), hamster mAb MUC1 (CT2; ThermoScientific), rabbit mAb Src Family pY416 (D49G4; Cell Signaling), mouse mAb FAK(77; BD Transduction Laboratories), rabbit pAb paxillin pY118 (2541; CellSignaling), rabbit mAb pan-AKT (C67E7; Cell Signaling), rabbit pAb AKTpS473 (9271; Cell Signaling); rabbit mAb ERK1/2 pT202/pT204 (197G2; Cell Sig-naling); rabbit pAb ERK1/2 (9102; Cell Signaling); rabbit mAb Gapdh (14C10; CellSignaling); Alexa 488 and Alexa 568 conjugated goat anti-mouse and anti-rabbitmAbs (Invitrogen); FITC conjugated anti-hamster mAbs; Cy5-conjugated goat anti-mouse and rabbit mAbs (Jackson); and HRP conjugated anti-rabbit and anti-mousemAbs. Chemical inhibitors used in these studies include ROCK inhibitor Y-27632(Cayman Chemical), myosin-II inhibitor (-)-blebbistatin (Cayman Chemical), FAKinhibitor FAK inhibitor 14 (Tocris), MEK inhibitor U0126 (Cell Signaling), PI(3)Kinhibitor Wortmannin (Cell Signaling), Src inhibitor Src I1 (Sigma), and DiI (Mo-lecular Probes).Cell culture conditions. All cells were maintained at 37 uC and 5% CO2. MCF10Ahuman MECs (ATCC) were cultured in DMEM F12 (Invitrogen) supplemented with5% donor horse serum (Invitrogen), 20 ng ml21 epidermal growth factor (Peprotech),10mg ml21 insulin (Sigma), 0.5mg ml21 hydrocortisone (Sigma), 0.1mg ml21 choleratoxin (Sigma), and 100 units ml21 penicillin/streptomycin. MCF7 and T47D breasttumour lines (ATCC) were grown in DMEM supplemented with 10% fetal bovineserum (Hyclone) and 100 units ml21 penicillin/streptomycin. 293T cells (ATCC)were maintained in DMEM supplemented with 10% donor horse serum, 2 mML-glutamine, and penicillin/streptomycin. Mouse embryonic fibroblasts (MEFs)were cultured in DMEM with 10% fetal bovine serum. Cell lines were tested rou-tinely for mycoplasma contamination. For transient gene expression in MECs, con-structs in pcDNA3.1 or Clonetech-style vectors were nucleofected with Kit V(Lonza) using program T-024 24 h before experimentation. Transient transfectionin MEFs was conducted 48 h before experimentation using Fugene 6 (Roche) ornucleofection. For stable cell lines harbouring tetracycline inducible transgenes, ex-pression was induced with 0.2 ng ml21 doxycycline 24 h before experimentation.The conditional v-Src oestrogen receptor fusion (v-Src–ER) was activated with 1mM4-hydroxytamoxifen 48 h before experimentation to achieve transformation. ForpERK, pY118paxillin, and pAKT studies, cells were plated on fibronectin-conjugatedpolyacrylamide hydrogels, serum-starved overnight, and stimulated with 20 ng ml21

EGF before collecting protein lysates. Data are reported as the fold increase of phos-phorylated protein relative to total protein, following EGF stimulation.Preparation of cellular substrates. Glass and silicon substrates were prepared byglutaraldehyde activation followed by conjugation with 10mg ml21 (glass) or 20mg ml21

(silicon) fibronectin as described11. Compliant polyacrylamide hydrogel substrates(soft: 2.5% acrylamide, 0.03% Bis-acrylamide; stiff: 10% acrylamide, 0.5% Bis-acrylamide) were prepared as previously described with one modification: func-tionalization with succinimidyl ester was with 0.01% N6, 0.01% Bis-acrylamide,0.025% Irgacure 2959, and 0.002% Di(trimethylolpropane) tetraacrylate (Sigma)31.Following functionalization with succinimidyl ester, hydrogels were conjugated over-night with 20mg ml21 fibronectin at 4 uC and rinsed twice with PBS and DMEMbefore cell plating.Generation of expression constructs. A description of cDNA constructs and theirconstruction is provided in Supplementary Note 3.Generation of stable cell lines. Stable transgene expression was achieved throughretroviral or lentiviral transduction as previously described11,30.Flow cytometry and cell sorting. Cell surface MUC1 was labelled directly withFITC-conjugated mAb MUC1 (clone HMPV). Cytometry and sorting were con-ducted on a FACSAria II (BD Biosciences).

Immunofluorescence and imaging. Cells were fixed and labelled as previouslydescribed and imaged at random on a Zeiss LSM 510 microscope system with a100X Plan Apochromat NA 1.4 objective and 488 nm Argon, 543 nm HeNe, and633 nm HeNe excitation lines30.Live epithelial cell imaging and FRET. Normal growth media was exchanged fora similar formulation lacking phenol red and supplemented with 15 mM HEPESbuffer, pH 7.4. Cells were imaged on a Ti-E Perfect Focus System (Nikon) equippedwith a CSU-X1 spinning disk confocal unit; 454 nm, 488 nm, 515 nm and 561 nmlasers; an Apo TIRF 60X NA 1.49 objective; electronic shutters; a charged-coupleddevice camera (Clara; Andor) and controlled by NIS-Elements software (Nikon).

For measurement of FRET efficiency, the acceptor photobleaching method pbFRETwas implemented with live cells on the spinning disk confocal. Cyan fluorescentprotein (CFP) was first imaged with 454 nm excitation and a 480/20 emission filter,yellow fluorescent protein (YFP) was subsequently bleached using a 100 mW 515 nmlaser for 10 s, and CFP was imaged again following bleaching of YFP. MicroscopeZ-focus was maintained during image acquisition using the Perfect Focus System.Background images were constructed by imaging 10 unique cell-free regions onthe coverslip and averaging the intensity at each pixel. The FRET efficiency wascalculated on a pixel-by-pixel basis according to:

FRET efficiency (%) 5 1{Ipre{Bpre

Ipost{Bpost

� �100%

where Ipre is the CFP intensity before bleaching YFP, Bpre is the CFP-channel back-ground intensity before bleaching YFP, Ipost is the CFP intensity after bleachingYFP, and Bpost is the CFP-channel background intensity after bleaching YFP. Ap-propriate controls were implemented to account for inadvertent CFP photobleach-ing, incomplete YFP photobleaching, and intermolecular FRET (see SupplementaryNote 4).

Time-domain fluorescence lifetime imaging microscopy (FLIM) for additionalFRET sensor characterization was implemented with an inverted Zeiss LSM510Axiovert 200M microscope with a Plan NeoFLUAR 40X/1.3 NA DIC oil-immersionobjective lens, equipped with a TCSPC controller (SPC-830 card; Becker & Hickl,Berlin, Germany) as described previously32. CFP was excited with 440 nm lightgenerated by frequency doubling of 880 nm pulses from a mode-locked Ti:sapphirelaser (Mai-Tai, Spectra-Physics, 120-150 fs pulse width, 80 MHz repetition rate, andFrequency Doubler and Pulse Selector, Spectra-Physics, Model 3980). The emissionlight was passed through a NFT 440 beamsplitter, directed to the fibre-out port of theconfocal scan-head, filtered with a 480BP40 filter (Chroma Technology, Rockingham,VT) and detected by a PMC-100 photomultiplier (Becker & Hickl). The pinhole wasset to give an optical slice of ,4.0mm. Images of 386 3 386 pixels were averaged over,120 s. Data analysis to produce an intensity image and a FLIM image was done off-line using the pixel-based fitting software SPCImage (Becker & Hickl), assumingdouble exponential decay during the first 8.5 ns of the 12.5 ns interval between laserpulses. Images were scaled to 256 3 256 pixels and no binning was used. Lifetimedistributions were calculated for a masked portion of the FLIM image, generatedwith a triangle algorithm threshold of the photo count intensity image.Scanning angle interference microscopy. Cells were plated overnight on reflectivesilicon substrates, fixed or roofed to remove the dorsal membrane (for MUC1–GFP imaging) and then fixed, and imaged randomly as previously described, scan-ning the incident angle of excitation light from 0u to 42uwith a one-degree samplingrate11. Z-positions were localized with custom algorithms previously described andavailable on request11.Single particle tracking photo-activation localization microscopy (sptPALM).sptPALM experiments were performed and analysed as previously described15.Briefly, live MEFs were imaged at 37 uC in a Ludin chamber on a Ti Perfect FocusSystem equipped with a Plan Apo 100X NA 1.45 objective, and an electron multi-plying charge-coupled device (Evolve; Photometrics). For photo-activation local-ization, cells expressing mEOS2-tagged constructs were activated using a 405 nmlaser (Omicron) and the photo-activated fluorophores were excited simultaneouslywith a 561 nm laser (Cobolt Jive). The powers of the activation and excitation laserswere adjusted to keep the number of activated molecules constant and well sepa-rated. GFP fusions of paxillin or MUC1 were imaged in between each sptPALMsequence by imaging the GFP signal above the unconverted mEOS2 background.The acquisition was driven by Metamorph software (Molecular Devices) in stream-ing mode at 50 Hz. For tracking, single-molecules were localized and tracked overtime using a combination of wavelet segmentation and simulated annealing algo-rithms. Trajectories lasting at least 20 frames were selected for further quantifica-tion, including calculation of immobile, confined and free-diffusing fraction (seeSupplementary Note 5)15.Preparation of glycopolymer-coated cell surfaces. Mucin mimetic glycopolymerswith lipid insertion domains were synthesized and characterized as described inSupplementary Note 6. For incorporation into the plasma membrane, cells were

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suspended in DMEM and incubated with 2 mM glycopolymer for 1 h. Cells werepelleted by centrifugation and re-suspended in growth media to remove unincor-porated polymer.Quantification of adhesion complexes. Images of adhesions in fixed, immuno-labelled cells or cells expressing paxillin–mCherry were randomly acquired, smoothedwith a median filter, and background subtracted (12 pixel diameter) in ImageJ.Adhesion sizes and the number of adhesions per cell were subsequently quantifiedin ImageJ with the ‘Analyze Particles’ tool.Integrin crosslinking assay. Cells were incubated in suspension with inhibitor(Y-27632 or Blebbistatin) or control solvent for 1 h before plating on glass substrates.Integrin was crosslinked to fibronectin with 1 mM 3,39-dithiobis(sulfosuccinimi-dylpropionate) (Pierce Chemical) and cells were extracted with SDS buffer as pre-viously described17. Crosslinked a5 integrin was immuno-labelled and imaged atrandom with a Plan Apo VC 60X objective on a Nikon TE2000 epi-fluorescencemicroscope equipped with a charged-coupled device camera (HQ2; Photometrics).Single cell force spectroscopy. Measurements were performed on an Asylum MFP-3D-BIO atomic force microscope as previously described33. Briefly, cells were attachedto a streptavidin-coated, tipless cantilever using biotinylated jacalin (MUC1-expressingcells) or concanavalin A (all other cells) and pressed against the adhesive substratewith a calibrated force and duration before measuring the force required to detachthe cell from the substrate. All measurements were conducted on fibronectin- orBSA-coated glass slides at room temperature. The relative rate of adhesion was cal-culated as the slope of a linear fit of cellular detachment force against contact time.Assessment of fibronectin–fibrillogenesis. Human recombinant fibronectin waslabelled with N-hydroxysuccinimide Alexa568 (Invitrogen) according to manu-facturer’s protocol and dialysed extensively in PBS. Conversion of soluble, fluor-escently labelled fibronectin from the growth media into insoluble fibrils wasimaged according to published protocol34. Briefly, MCF10A complete growth media

was prepared with donor horse serum that was depleted of fibronectin using gelatinSepharose 4B (GE Healthcare). MCF10A cells were plated in the depleted media onfibronectin-conjugated glass coverslips and incubated the next day in 10 mg ml21

labelled fibronectin for one hour. Cells were quickly rinsed in PBS, fixed in 4%paraformaldehyde, and imaged at random on a spinning disk confocal.Isolation and gene expression profiling of CTCs. Twenty CTC samples were iso-lated from the blood of 18 metastatic breast cancer patients as previously described35.Briefly, whole blood was subjected to EpCAM-based immunomagnetic enrich-ment followed by fluorescence-activated cell sorting of CTCs defined as nucleated,EpCAM-positive, CD45-negative cells. CTCs were sorted directly onto lysis buffer(Taqman PreAmp Cells-to-Ct kit, Life Technologies). cDNA of target genes werepre-amplified (14 cycles) and measured via qPCR analysis. The mean Ct for ACTBand GAPDH was used for normalization to calculate relative gene expression (DCt).Studies involving CTCs were approved by the UCSF Committee on Human Re-search. Samples were obtained with IRB approved consent from all patients.Immunofluorescence labelling of CTCs. CTC samples were isolated from theblood of three metastatic breast cancer patients as described for gene expressionprofiling. Isolated CTCs were mounted and fixed on poly-L-lysine-coated slidesand labelled with FITC-conjugated MUC1 mAb (Clone HPMV). As a control,purified white blood cells from the same patients were prepared similarly, andtheir immunofluorescence was compared to CTC samples.Statistics. Statistical significance of experimental data sets was determined byStudent’s t-test after confirming that the data met appropriate assumptions (nor-mality, homogenous variance and independent sampling). Statistical analyses ofmicroarray gene expression data sets are described in detail in Supplementary Note 1.All public microarray data were downloaded from the NCBI Gene Expression Om-nibus website and analysed using custom R scripts (all Perl, PHP and R scripts usedin this work are available on request).

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Extended Data Figure 1 | Large-scale gene expression analysis revealsincreased expression of genes encoding bulky glycoproteins and glycan-modifying enzymes in primary tumours of patients with disseminateddisease. a, Bioinformatics pipeline to estimate the extracellular bulkiness of aprotein from its corresponding amino acid sequence. For each isoformsequence, the transmembrane and extramembrane domains were identifiedusing a hidden Markov model (TMHMM). A combination of motif searchesand neural network prediction then identified likely N- and O-glycosylationsites within each sequence. Isoform-level bulkiness estimates were generated bysumming the number of predicted N- and O-glycosylation sites located withinthe extramembrane regions of the isoform. b, Heat map depicting the pairwisespearman correlation coefficients calculated by comparing all per-geneestimates of the total number of extra-membrane amino acids (AAoutside),N-glycosylation sites (Nglyc), O-glycosylation sites (Oglyc), and the overallbulkiness measure (total sites; for example, the sum of extra-membrane N- andO- glycosylation sites). Correlation coefficients relating the correspondinggene-wise measures are listed in the corresponding cells and depicted on acolour scale, where white corresponds to perfect correlation (rho 5 1), and thedendrograms indicate the overall relationship between the parameters,estimated by Euclidean distance. High correlation coefficients indicate that

gene-wise estimates of the compared parameters are similarly ranked (forexample, genes with high values of X also tend to have high values of Y). Thedata indicate that the number of extracellular N-glycosylation sites andO-glycosylation sites identified within a gene are only weakly correlated, andneither dominates the total number of sites estimated per gene. c, Violin plotscontrasting the distributions of gene-wise one-sided P values (y axis)quantifying evidence for transcriptional upregulation of glycosidases andglycosyltransferases, and subsets of glycosyltransferases (sialyltransferases andN-acetylgalactosaminyltransferases) with the full distribution. White dotsand thick black lines indicate the median and interquartile range of the gene-wise P-value distribution among category members, and the width of the violinalong the y axis indicates the density of the corresponding values. P valuesare derived from comparisons of expression levels in primary tumours ofpatients with or without distant metastases using a t-test. Indicated P valueswere estimated using a one-sided Kolmogorov–Smirnov test. d, Violin plotsquantifying transcriptional upregulation of glycan-modifying enzymes inprimary tumours of patients presenting with circulating tumour cells comparedto tumours without detectable circulating tumour cells. e, Table of bulkyglycoproteins and potential bulk-adding glycosyltransferases whose expressionis upregulated in tumours that present with circulating tumour cells.

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Extended Data Figure 2 | Computational model of the cell–ECM interface.Schematic of an integrated model that describes how the physical properties ofthe glycocalyx influence integrin–ECM interactions. The cell surface ismodelled as a three-dimensional elastic plate; the ECM as a rigid substrateunderneath the cell surface; and the glycocalyx as a repulsive potential betweenthe plate and substrate. To compute stress–strain behaviour, the model isdiscretized using the three-dimensional lattice spring method, the cross-section

of which is depicted above. Integrins are tethered to the cell surface and theirdistance-dependent binding to the ECM–substrate is calculated according tothe Bell model. To calculate integrin-binding rate as a function of lateraldistance from an adhesion cluster, an adhesion cluster is first constructed byassembling a 3 3 3 bond structure. The rates for additional integrin–ECMbonds then are computed at various distances from the cluster.

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Extended Data Figure 3 | Synthesis and characterization of glycoproteinmimetics. a, Scheme for synthesis of lipid-terminated mucin mimetics labelledwith Alexa Fluor 488 (AF488). b, Reagents and yields for the synthesis ofpolymers 3a–c. c, Characteristics of polymers 6a–c based on 1H NMR spectra.Glycoprotien mimetics were engineered to have minimal biochemicalinteractivity with cell surface lectins. d, Flow cytometry results quantifying

incorporation of polymer on the surface of mammary epithelial cells (left) andbinding with recombinant Alexa568-labelled galectin-3 with or withoutcompetitive inhibitor, b-lactose (right). Although a weak affinity betweengalectin-3 and the pendant N-acetylgalactosimes has previously been reported,the results suggest that incorporation of polymer does not significantly changethe affinity of the cell surface for lectins.

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Extended Data Figure 4 | MUC1 expression constructs. a, Schematic ofMUC1 expression constructs. Full-length MUC1 consists of a largeectodomain with 42 mucin-type tandem repeats, a transmembrane domain,and short cytoplasmic tail. The tandem repeats and cytoplasmic tail are deletedin MUC1(DTR) and MUC1(DCT), respectively. For fluorescent proteinfusions, mEmerald (GFP) and mEOS2 are fused to the C terminus of

full-length MUC1 or MUC1(DCT). b, Schematic of MUC1 strain sensorand control constructs. Cysteine-free mTurqoiuse2 (CFP), Venus (YFP), or aFRET module consisting of the fluorescent proteins separated by an elasticlinker (8 repeats of GPGGA) are inserted into the MUC1 ectodomain adjacentto the MUC1 tandem repeats. The mucin tandem repeats are deleted inectodomain-truncated variants (DTR).

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Extended Data Figure 5 | MUC1-mediated adhesion formation.a, Quantification of the average number of large adhesions, greater than 1mm2,per area of cell in control epithelial cells (Control) and those ectopicallyexpressing ectodomain-truncated MUC1 (1MUC1(DTR)), wild-type MUC1(1MUC1), or cytoplasmic-tail-deleted MUC1 (1MUC1(DCT)). Results arethe mean 6 s.e.m. of three separate experiments. b, Fluorescence micrographsshowing immuno-labelled MUC1 and fluorescently labelled fibronectinfibrils in control and MUC1-expressing epithelial cells. Soluble, labelledfibronectin in the growth media was deposited by cells at sites of cell–matrix

adhesion. Binding of soluble fibronectin to MUC1 was not detected. Scale bar,10mm. c, Time lapse images of MUC1–YFP and vinculin–mCherry, showingthe dynamics of adhesion assembly (Vinc.) and MUC1 patterning (MUC1).Scale bar, 1mm. d, Rate of adhesion measured with single cell forcespectroscopy of control (Cont.), a5 integrin-blocked (anti-a5), and MUC1-expressing cells (1MUC1) to fibronectin-coated surfaces and control cells toBSA-coated surfaces (BSA). Results are the mean 6 s.e.m. of at least 15 cellmeasurements. Statistical significance is given by *P , 0.05; **P , 0.01;***P , 0.001.

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Extended Data Figure 6 | b3 integrin mobility in MUC1-expressing cells.a, Molecular diffusivity and adhesion enrichment measured with sptPALM inmouse embryonic fibroblasts (MEFs). Adhesion enrichment is reported as theratio of the number of molecules detected inside focal adhesions per unit area tothe number of molecules detected outside focal adhesions per unit area.b, Mean diffusion coefficients measured for freely diffusive b3 integrin tracksoutside of adhesive contacts in control (Cont.) and MUC1-transfected(1MUC1) MEFs with and without Mn21 to activate b3. c, Mean diffusioncoefficients measured for confined b3 integrin tracks outside of adhesivecontacts in MEFs with and without Mn21. d, Mean radius of confinementmeasured for confined b3 integrin tracks outside of adhesive contacts in MEFswith and without Mn21. e, Fraction of immobilized (Imm.), confined (Conf.),and freely diffusive (Free)b3 integrins inside of adhesive contacts in control andMUC1-transfected MEFs with and without Mn21 treatment. f, From left to

right, panels show GFP-tagged wild-type MUC1 (red) and positions ofindividual b3 integrins (green) in MEFs without Mn21 treatment (left panel)and individual integrin trajectories recorded with sptPALM within MUC1-richregions, outside MUC1-rich regions, and that cross MUC1 boundaries (scalebar, 2 mm). The ratio of integrins crossing out versus crossing in the MUC1boundaries per cell is close to one (1.0 6 0.1, n 5 9 cells, 4,145 trajectories)showing that the flux of free diffusing integrins crossing in or out the mucinregion is the same. g, From left to right, panels show integrin trajectories withinan arbitrary region drawn in a MUC1-rich area (dashed white circles), outsideof the circled region, and that cross the circled region (scale bar, 2mm). Theratio of integrins crossing the MUC1-rich boundaries versus the fictiveboundaries per cell is close to one (1.2 6 0.2, n 5 9 cells, 9,321 trajectories),showing that the MUC1–adhesive zone boundary does not affect the diffusivecrossing of integrins. For all bar graphs, results are the mean 6 s.e.m.

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Extended Data Figure 7 | MUC1 strain gauge. a, Western blot of indicatedconstruct expressed in HEK 293T cells and probed with anti-GFP familyantibody, or full-length MUC1 construct expressed in HEK 293T cells andprobed with an antibody against the MUC1 tandem repeats. b, Pseudo-coloured images showing similar FRET efficiencies measured by thephotobleaching FRET method for mammary epithelial cells (MECs) expressinglow (Low) and high (High) levels of the sensor construct. Scale bar, 5 mm. c, Plotshowing the level of CFP bleaching per CFP imaging cycle in MECs. d, Controlimages showing minimal intermolecular FRET in MECs expressing similarlevels of both MUC1 CFP and MUC1 YFP. e, Micrographs showing the emitted

photons from CFP and their fluorescence lifetimes in MECs expressingectodomain-truncated (MUC1(DTR) sensor) or full-length MUC1 strainsensors (MUC1 sensor). Shorter lifetimes are indicative of higher energytransfer between the CFP donor and YFP acceptor, and thus closer spatialproximity of the donor and acceptor (scale bar, 10mm). f, Representative profileof CFP lifetimes and emitted photons of the full-length MUC1 sensor alongthe red line in panel e. Pixels 0 and 40 correspond to the base and tip of thearrow, respectively. A drop in fluorescence lifetime (Lifetime) is often observedbefore the drop in MUC1 molecular density (Photons) as an adhesive zoneis approached.

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Extended Data Figure 8 | Tension-dependent integrin activation and focaladhesion assembly in MUC1-expressing cells. a, Fluorescence micrographs offibronectin-crosslinked a5 integrin in control and MUC1-expressingmammary epithelial cells (MECs) treated with solvent alone (DMSO), myosin-II inhibitor (blebbistatin; 50mM), or Rho kinase inhibitor (Y-27632; 10mM) for1 h and detergent-extracted following crosslinking. Only fibronectin-boundintegrins under mechanical tension are crosslinked and visualized followingdetergent extraction (scale bar, 15mm). b, Fluorescence micrographs showingformation of myosin-independent adhesion complexes in MUC1-expressingMECs. Cells were pre-treated for 1 h and plated for 2 h in 50mM blebbistatin

(scale bar, 10mm). c, Fluorescence micrographs of paxillin–mCherry andimmuno-labelled activated FAK (pY397) in control and MUC1(DCT)expressing MECs plated on compliant fibronectin-conjugated hydrogels(E 5 140 Pa; scale bar, 3mm; ROI scale bar, 0.5mm). d, Western blots showingphosphorylation of paxillin (pY118) in control and MUC1-expressing MECson compliant substrates (E 5 140 Pa) following overnight serum starvationand stimulation with EGF. MUC1-expressing cells treated with apharmacological inhibitor of focal adhesion kinase (1FAKi) for 1 h before EGFstimulation did not exhibit robust paxillin phosphorylation.

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Extended Data Figure 9 | Cell proliferation on soft ECM. a, Fluorescencemicrographs showing DAPI-stained nuclei of control and MUC1(DCT)-expressing MECs after 24 h of plating on soft, fibronectin-conjugated hydrogels(E 5 140 Pa; scale bar, 250mm). The majority of cells plated as single cells,indicating that multi-cell colonies that formed at later time points were largelyattributed to cell proliferation. b, Quantification of cell proliferation of

MUC1(DCT)-expressing epithelial cells on soft hydrogels conjugated withbovine serum albumin (BSA) or fibronectin (Fn). Cells plated similarly onBSA– and Fn–hydrogels, but cell proliferation was significantly enhanced onFn–hydrogels. Results are the mean 6 s.e.m with statistical significance givenby *P , 0.05; **P , 0.01; ***P , 0.001.

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Extended Data Figure 10 | Hyaluronic acid production by tumour cellspromotes cellular growth. a, Quantification of hyaluronic acid (HA) cellsurface levels on control (10A-Cont.), transformed (10A-v-Src, 10A-HRAS)and malignant (MCF7, T47D) mammary epithelial cells (MECs).b, Fluorescence micrographs of HA and immuno-labelled paxillin on thev-Src transformed MECs (scale bars, 3mm). c, Quantification of the number of

v-Src-transformed MECs per colony 48 h after plating on soft polyacrylamidegels (fibronectin-conjugated) and treated with vehicle (DMSO), hyaluronicacid synthesis inhibitor 4-methylumbelliferone (14MU; 0.3mM), orcompetitive inhibitor HA oligonucleotides (1Oligo; 12-mer averageoligonucleotide size; 100 mg ml21). Results are the mean 6 s.e.m with statisticalsignificance is given by *P , 0.05; **P , 0.01; ***P , 0.001.

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