Mucin Glycoproteins Are Ubiquitous in Mucous Secretions on Cell Surfaces and in Body ﬂuids

Chapter 9 – O-GalNAc Glycans - Essentials of Glycobiology 2nd edition

Intestinal mucosal barrier function in health and disease. Turner, JR Nature Rev. Immunology 9: 799 (2009)

From Mucins to Mucus Thornton DJ and Sheehan JK, Proc Am Thorac Soc., 1:54 (2004)

Mucin structure, aggregation, physiological functions and biomedical applications Bansil R. and Turner BS Curr Opin in Colloid & Interface Sci, 11:164 (2006)

Mucins in the mucosal barrier to infection Linden et al, Nature Immunology 1: 183 (2008)

The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Paszek et al, Nature 511: 319 (2014)

Chapter 9 – O-GalNAc Glycans

Essentials of Glycobiology 2nd edition

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

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

ﬁle:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 1/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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.

file:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 2/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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 file:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 3/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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.

file:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 4/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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. file:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 5/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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- ﬁle:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 6/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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

file:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 7/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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

ﬁle:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 8/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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

ﬁle:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 9/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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).

ﬁle:///Users/tgilstrap/Desktop/READINGS/April%2021-Kamil%20/Chapter%209_2nd%20edition.html 10/12 4/14/2016 O-GalNAc Glycans - Essentials of Glycobiology - NCBI Bookshelf 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-acetylglucosaminyltransferase (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-

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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

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Intestinal mucosal barrier function in health and disease. Turner, JR Nature Rev. Immunology 9: 799 (2009)

REVIEWS

Intestinal mucosal barrier function in health and disease

Jerrold 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.

Mucosa-associated Complex multicellular organisms interface with their as size selectivity) varies with location in the intestine, lymphoid tissue external environments at multiple sites, including as permeability to larger solutes decreases from the (MALT). The collections of mucosae of the airways, oral cavity, digestive tract and crypt to the villus2. In addition to these fixed differences, B cells, T cells, plasma cells, genitourinary tract, and the skin. Although the skin mucosal permeability of many tissues is adaptable and macrophages and other is the most visible site of interface, the combined area may be regulated in response to extracellular stimuli, antigen-presenting cells found in the mucosal linings of organs of the mucosal surfaces is much greater than that of such as nutrients, cytokines and bacteria. including the gastrointestinal the skin. Mucosae are also the primary sites at which the Recent advances have uncovered some of the mech- tract, lungs, salivary glands mucosa-associated lymphoid tissue (MALT) is exposed to anisms by which physiological and immunological and conjuctiva. and interacts with the external environment. The magni- stimuli affect cellular and extracellular components of

Tight junction tude of these interactions is greatest in the gastrointesti- the intestinal barrier. In this article I review our current Also known as the zonula nal tract, which is the largest mucosal surface and is also understanding of the mechanisms that regulate intesti- occludens, this is a site of in continuous contact with dietary antigens and diverse nal barrier integrity, and discuss the hypothesis that the close apposition of adjacent microorganisms. Thus, mucosal surfaces, particularly mucosal barrier can shape pro-inflammatory and immuno- epithelial cell membranes — in the intestines, are crucial sites of innate and adaptive regulatory responses in the context of homeostasis kiss points — that create a barrier against the free immune regulation. and disease. diffusion of water and solutes. A central mediator of interactions between MALT Although they may affect barrier function, den- and the external environment, including the intesti- dritic cells (DCs), which extend dendritic processes nal lumen, is the epithelium that covers the mucosa. across the tight junctions in the distal small intestine3,4, Epithelial cells establish and maintain the barrier, and intraepithelial lymphocytes are not discussed here. which in the skin is a tight, although not impermeant, In addition, the functions of M cells, which are spe- seal. By contrast, most mucosal epithelial cells form cialized epithelial cells that deliver antigens directly leaky barriers. This is necessary to support universal to intra epithelial lymphocytes and to subepithelial functions that include fluid exchange, which occurs lymphoid tissues by transepithelial vesicular transport at nearly all mucosal surfaces, as well as essential from the gut lumen, are reviewed elsewhere5. Finally, tissue-specific functions. Thus, the precise permea- detailed analyses of the interactions between epithelial Department of Pathology, bility characteristics of each barrier type differ based on cells and luminal microorganisms, as well as the intri- The University of Chicago, the functions supported. For example, the ion-selective cacies of mucosal immune regulation, have also been 5841 South Maryland, properties of the barrier vary considerably along the recently reviewed elsewhere6,7. This Review therefore MC 1089, Chicago, length of a nephron to promote or restrict transport of focuses on the roles of the epithelial cell barrier in health Illinois 60637, USA. 1 e-mail: specific ions in each segment . Similarly, the ability and disease, with particular emphasis on the junctional [email protected] of tight junctions to discriminate between and restrict complexes between intestinal epithelial cells, which doi:10.1038/nri2653 passage of solutes based on size (a characteristic known have a crucial role in barrier regulation.

Mucins Anatomy of mucosal barriers absorption by reducing the rate at which nutrients reach A family of heavily glycosylated Extracellular components of the barrier. Most mucosal the transporting protein-rich microvillus brush border, proteins that are secreted as surfaces are covered by a hydrated gel formed by mucins but may also contribute to absorption by limiting the large aggregates by mucous (FIG. 1a). Mucins are secreted by specialized epithelial extent to which small nutrients released by the activities of epithelial cells. cells, such as gastric foveolar mucous cells and intestinal brush border digestive enzymes are lost by diffusion into goblet cells, and create a barrier that prevents large par- the lumen. unstirred layer defects have not been linked Unstirred layer ticles, including most bacteria, from directly contacting to specific diseases. However, it is interesting to note that A thin layer of fluid at epithelial 8 cell surfaces that is separated the epithelial cell layer . The importance of mucus gel increased unstirred layer thickness has been reported in 12 from the mixing forces created hydration is shown by cystic fibrosis, in which the pro- coeliac disease , in which it may contribute to nutrient by luminal flow and, in the duction of hyperviscous mucus contributes to pulmo- malabsorption. intestine, peristalsis. nary, pancreatic and intestinal disease9. Defective mucus production has also been reported in various immune- Cellular components of the mucosal barrier. The pri- Coeliac disease mediated diseases, and spontaneous colitis develops in mary responsibility for mucosal barrier function resides A chronic inflammatory 10 condition of the upper small mice that lack specific mucin genes . with the epithelial cell plasma membrane, which is intestine in humans that is Although small molecules pass through the heavily impermeable to most hydrophilic solutes in the absence caused by immunological glycosylated mucus layer with relative ease, bulk fluid of specific transporters. Accordingly, direct epithelial hypersensitivity to the α-gliadin flow is limited and thereby contributes to the develop- cell damage, such as that induced by mucosal irritants component of wheat gluten. ment of an unstirred layer of fluid at the epithelial cell sur- or cytotoxic agents, including some drugs used for can- It can cause severe villous atrophy, which can lead to face. As the unstirred layer is protected from convective cer chemotherapy, results in a marked loss of barrier malabsorption and mixing forces, the diffusion of ions and small solutes is function. However, in the presence of an intact epithe- malnutrition if slowed. In the stomach, this property of the unstirred layer lial cell layer, the paracellular pathway between cells must gluten-containing foods are not works with epithelial cell bicarbonate secretion to main- be sealed. This function is mediated by the apical junc- removed from the diet. tain a zone of relative alkalinity at the mucosal surface11. tional complex, which is composed of the tight junction The unstirred layer of the small intestine slows nutrient and subjacent adherens junction (FIG. 1b). Both tight and

a b Apical junctional complex Microvilli Mucus Unstirred layer ZO1 Intraepithelial Claudin lymphocyte F-actin Tight junction Epithelial cells Occludin Myosin Goblet cell MLCK α-catenin 1 Basement membrane Adherens E-cadherin junction

β-catenin

Keratin Plasma cell Lamina propria lymphocyte Desmoglein Desmocollin Desmosome Desmoplakin

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.Na Gobletture Re viecells,ws | whichImmunolog y 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.

adherens junctions are supported by a dense perijunc- A second pathway is characterized by small pores that tional ring of actin and myosin that, as discussed below, are thought to be defined by tight junction-associated can regulate barrier function. claudin proteins, which are also primary determinants As implied by the name, the adherens junctions, of charge selectivity18–20. These pores have a radius that along with desmosomes, provide the strong adhesive excludes molecules larger than 4 Å14,15. Expression of spe- bonds that maintain cellular proximity and are also a cific claudins varies between organs and even within dif- site of intercellular communication. Loss of adherens ferent regions of a single organ and, as detailed below, can junctions results in disruption of cell–cell and cell– be modified by external stimuli, such as cytokines. Thus, Paracellular pathway The route of transepithelial matrix contacts, ineffective epithelial cell polariza- tight junctions show both size selectivity and charge selec- 13 transport that involves passive tion and differentiation, and premature apoptosis . tivity, and these properties may be regulated individually movement through the space Adherens junctions are composed of cadherins, a family or jointly by physiological or pathophysiological stimuli. between adjacent cells. of transmembrane proteins that form strong, homotypic interactions with molecules on adjacent cells. The cyto- Interdependence of transport routes Adherens junction Also known as the zonula plasmic tail of the epithelial cadherin, E-cadherin (also Transepithelial transport requires a selectively permeable adherens, this junction is known as cadherin-1), interacts directly with catenin δ1 barrier. The vectorial nature of transcellular transport, immediately subjacent to the (also known as p120 catenin) and β-catenin. In turn, which is required for effective absorption and secre- tight junction and requires β-catenin binds to α-catenin 1, which regulates local tion, generates a transepithelial concentration gradient. the activity of lineage-specific Ca2+-dependent adhesion actin assembly and contributes to development of the Transepithelial transport would, therefore, be ineffective proteins, termed cadherins. perijunctional actomyosin ring. without a tight junction barrier, as diffusion would allow Adherens junctions are required for assembly of the equalization of concentrations on both sides of the epith- Desmosome tight junction, which seals the paracellular space. Tight elium. Thus, active transcellular transport depends on the An adhesive junction that junctions are multi-protein complexes composed of presence of an intact tight junction barrier. The selective connects adjacent epithelial cells. These junctions are transmembrane proteins, peripheral membrane (scaf- permeability of the tight junction barrier also allows trans- composed of multiple protein folding) proteins and regulatory molecules that include epithelial gradients to drive passive paracellular transport subunits and are the points kinases. The most important of the transmembrane of ions and water17,20. For example, claudin-16, which is where keratin filaments attach proteins are members of the claudin family, which define necessary for tight junction cation selectivity, is mutated in to the plasma membrane. several aspects of tight junction permeability, as dis- the human disease familial hypomagnesaemia with hyper- 20 Claudin cussed below. Claudins are expressed in a tissue-specific calciuria and nephro calcinosis . Paracellular absorption of From the Latin claudere, manner, and mutation or deletion of individual family Mg2+ and Ca2+ in the thick ascending limb of Henle requires meaning ‘to close’, members of members can have profound effects on organ function. a cation-selective tight junction barrier, the absence of this family of transmembrane The role of occludin, a transmembrane tight junction which results in urinary loss of Mg2+ and Ca2+ (ReF. 20). proteins are variably expressed + + by specific epithelial cell types protein that interacts directly with claudins and actin, Similarly, apical na –H -exchange protein 3 (nHE3) is + and thereby contribute to the is less well understood. Peripheral membrane proteins, required for transcellular na absorption in the renal unique barrier properties of such as zonula occludens 1 (Zo1) and Zo2, are crucial to tubule and intestine, and also contributes to the trans- different tissues. tight junction assembly and maintenance, partly owing epithelial na+ gradient that drives paracellular water (BOX 1) Occludin to the fact that these proteins include multiple domains absorption . The tight junction barrier to para- + The first transmembrane tight for interaction with other proteins, including claudins, cellular na efflux prevents dissipation of the gradient junction protein identified. The occludin and actin. established by nHE3-mediated transcellular transport. function of occludin remains The tight junction limits solute flux along the para- controversial but it is likely to cellular pathway, which is typically more permeable Tight junction barrier regulation modifies absorption. have roles in barrier regulation transcellular pathway and tumour suppression. It than the . The tight junction is, there- In addition to providing the driving force for paracel- also serves as a cofactor in fore, the rate-limiting step in transepithelial transport lular transport, transcellular transport can activate intra- hepatitis C virus entry. and the principal determinant of mucosal permeability. cellular signalling events that regulate the tight junction Thus, it is important to understand the specific barrier barrier. The best studied physiological example of this Zonula occludens 1 A peripheral membrane, or properties of the tight junction, which can be defined in is the increased intestinal paracellular permeability that is + 21 plaque, protein containing terms of size selectivity and charge selectivity. induced by apical na –nutrient co-transport . This multiple protein interaction At least two routes allow transport across the tight allows passive paracellular absorption of nutrients and domains that, along with the junction, and emerging data suggest that the relative water to amplify transcellular nutrient absorption, partic- related protein zonula contributions of these types of paracellular transport ularly when high luminal nutrient concentrations exceed occludens 2, is required for 2,14,15 + 22 tight junction assembly. may be regulated independently . one route, the the capacity of apical na –nutrient co-transporters . leak pathway, allows paracellular transport of large ultrastructural analyses of intestinal epithelial cells during Transcellular pathway solutes, including limited flux of proteins and bacterial na+–nutrient co-transport revealed condensation of the The route of transepithelial lipopolysaccharides14,15. Although the size at which par- perijunctional actomyosin ring, suggesting that cytoskel- transport that involves active or passive movement across ticles are excluded from the leak pathway has not been etal contraction could be involved in this physiological 21 cell membranes, usually as a precisely defined, it is clear that materials as large as process . Subsequent studies confirmed this hypothesis result of the action of specific whole bacteria cannot pass. As might be expected for and showed that the Ca2+–calmodulin-dependent serine– transport channels. a route that allows large solutes to cross, the leak path- threonine protein kinase myosin light chain kinase (MLCK) way does not show charge selectivity. Flux across the is essential for na+–nutrient co-transport-induced tight Transepithelial transport 23,24 The sum of transport through leak pathway may be increased by cytokines, including junction regulation . Moreover, MLCK activation alone the transcellular and interferon-γ (IFnγ) in vitro and tumour necrosis factor is sufficient to increase tight junction perme ability, both paracellular pathways. (TnF) in vitro and in vivo15–17. in vitro and in vivo25,26. The mechanisms that mediate this

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 a Normal homeostasis b Barrier disruption only Na+ gradient and promotes paracellular H2O H2O water absorption (see the figure, part a). No No diarrhoea diarrhoea The MLCK-dependent increase in tight Na+ Na+ junction permeability enhances paracellular absorption of water and small solutes, such as glucose, that are concentrated in the unstirred layer. This Na+ Na+ 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). c Na+ malabsorption only d Na+ malabsorption and By contrast, tumour necrosis factor barrier disruption activates both protein kinase Cα (PKCα) Mild H2O Copious H2O diarrhoea and MLCK to cause diarrhoea. NHE3 diarrhoea Na+ Na+ 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 Na Na permeability to allow water to flow into the lumen, thereby causing large-volume 17 diarrhoea (see the figure, part d). H2O

Nature Reviews | Immunology MLCK-dependent tight junction regulation have been with TnF-specific antibodies may be largely due to the studied in detail owing to their contributions to nutri- overall reduction in inflammation, it is notable that this ent and water absorption under normal physiological treatment corrects barrier dysfunction in patients with conditions and to the diarrhoea associated with acute Crohn’s disease36. Increased mucosal TnF production barrier loss (BOX 1). may also contribute to increased intestinal permeability and susceptibility to colitis in mice with defective Barrier regulation by immune stimuli mucin biosynthesis10,37. The ability of cytokines, such as TnF and IFnγ, to MLCK has been shown to have a central role in TnF- regulate the function of the tight junction barrier was induced epithelial and endothelial barrier dysregulation, first described 20 years ago27. Since then, increased tight both in vitro and in vivo16,38–41. Similar to na+–nutrient junction protein transcription, vesicular removal of pro- co-transport, TnF-induced MLCK activation seems to teins from the tight junction, tight junction protein deg- increase paracellular flux through the leak pathway16–17,23. radation, kinase activation and cytoskeletal modulation This MLCK activation occurs as a result of increased have all been proposed to mediate cytokine-induced loss enzymatic activity and increased MLCK transcription of tight junction barrier function. Although extensive and translation, both in vitro and in vivo16,40,42. Similarly, Thick ascending limb of apoptosis of epithelial cells may also cause barrier loss, MLCK expression and activity are increased in intesti- Henle the relevance of single-cell apoptosis to barrier dysfunc- nal epithelial cells of patients with inflammatory bowel The portion of the nephron just tion remains controversial owing to differing results in disease43. The degree to which MLCK expression and proximal to the distal tubule. This is a site of active Na+, K+ diverse experimental systems. activity are increased correlates with local disease activ- and Cl– reabsorption, which ity, suggesting that these processes may be regulated by generates an electrochemical Tight junction regulation through the cytoskeleton. local cytokine signalling in these patients43. MLCK is gradient that drives TnF and IFnγ modify tight junction barrier function also a fundamental intermediate in barrier dysfunction paracellular reabsorption of 27,28 29 30 Mg2+ and Ca2+. in intestinal , renal , pulmonary and salivary induced by the TnF family member LIGHT (also known gland31 epithelia as well as between endothelial cells32. as TnFSF14)17,44, interleukin-1β (IL-1β)45, enteropatho- Myosin light chain kinase The effects of TnF on barrier integrity have been best genic Escherichia coli infection39, Helicobacter pylori The Ca2+–calmodulin- studied in the gut, where this cytokine has a central role infection46, giardiasis47, lipopolysaccharide48,49 and the dependent kinase that in many diseases associated with intestinal epith elial ethanol metabolite acetaldehyde50. Thus, MLCK activa- phosphorylates myosin II regulatory light chain at serine barrier dysfunction, including inflammatory bowel tion can be viewed as a common final pathway of acute 33 28,34 19 and threonine 18 to disease , intestinal ischaemia and graft-versus- tight junction regulation in response to a broad range of activate myosin ATPase. host disease35. For example, although the effect of therapy immune and infectious stimuli.

ab 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 cd 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 directly59 and inhibit MLC phosphatase62; and AMP-activated protein kinase52, which is activated during stress and can also directly phosphorylate MLC63. Moreover, Rho kinases Figure 2 | Differential effects of cytokines on tight junction structure and and AMP-activated protein kinase each have diverse function. a | Occludin is normally concentrated at the tight junctionNature Re (arrow)views |in Immunolog jejunal y villus epithelium. The perijunctional actomyosin ring (red) and nuclei (blue) are shown for effects that are separate from myosin function, and it reference. b | Exogenous tumour necrosis factor increases myosin light chain kinase is likely that at least some of these contribute to tight activity, which causes perijunctional myosin II regulatory light chain phosphorylation and junction regulation. triggers occludin (green) endocytosis (arrow). This increases flux across the tight junction leak pathway and enhances paracellular permeability to large solutes. c | Claudin‑2 Tight junction permeability and claudin expression. expression (green) is limited to crypt epithelial cells; it is not expressed by epithelial cells As discussed above, regulation of perijunctional acto- at the mucosal surface in the normal colon. F‑actin (red) and nuclei (blue) are shown for myosin provides a means of rapidly and reversibly reference. d | Interleukin‑13 can stimulate claudin‑2 expression (green) in surface regulating the paracellular leak pathway. By contrast, epithelial cells (arrows). This increases flux across small tight junction pores, thereby enhancing paracellular cation permeability. 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- Although it is clear that MLCK phosphorylates rier regulation, which is consistent with the fact that myosin II regulatory light chain (MLC) within the peri- claudins do not interact with actin directly. Expression junctional actomyosin ring to activate myosin ATPase of specific claudin proteins changes during develop- activity, the subsequent molecular events that cause ment, differentiation and disease and in response to increased permeability are poorly defined. However, stressors — including cytokines — in intestinal64, renal65 recent work suggests that Zo1, which interacts directly and alveolar66 epithelial cells. In addition to affecting with actin, occludin, claudins and other proteins, may barrier function, altered patterns of claudin expression be an essential effector of perijunctional actomyosin may have other consequences. For example, claudin ring-mediated tight junction regulation25,51,52. Endocytic proteins have been associated with control of cell and removal of the transmembrane protein occludin from the organ growth. It may be, therefore, that some changes tight junction is also common in actomyosin-dependent, in claudin protein expression enhance cell proliferation cytokine-mediated tight junction regulation38,53 (FIG. 2). and regeneration, as might be necessary to compensate In vitro studies of tight junction regulation induced for cell loss in colitis. This may explain the increased by LIGHT suggest that occludin endocytosis occurs claudin-1 expression by intestinal epithelial cells of via caveolae and that inhibition of this process can patients with inflammatory bowel disease67. However, prevent loss of barrier integrity despite MLCK activa- claudin-1 has also been shown to enhance neoplastic tion44. Caveolar endocytosis of occludin has also been transformation, tumour growth and metastasis in exper- associated with loss of epithelial and endothelial tight imental models68. Thus, changes in claudin expression junction barrier function in response to actin disrup- may have undesired consequences. tion54 and chemokine signalling55, respectively, in vitro. one common change in claudin expression that However, other mechanisms of occludin removal may directly affects barrier function is the increased claudin-2 also be involved as in vitro studies have shown that expression by intestinal epithelial cells in animal models IFnγ-induced occludin internalization is mediated of colitis (FIG. 2) and patients with inflammatory bowel by myosin ATPase-dependent macropinocytosis53,56, disease64. Consistent with this, IL-13 and IL-17, which and occludin cleavage may even modify the barrier are increased in the mucosa of patients with colitis64,69, to enhance transepithelial migration of inflamma- reduce barrier function and increase claudin-2 expres- tory cells in the lung57. nevertheless, further work sion in cultured intestinal epithelium monolayers64,70.

Caveolae is necessary to define the contributions of occludin In vitro studies have shown that claudin-2 expression Specialized flask-shaped and endocytosis to in vivo tight junction regulation. increases the number of pores that allow paracellular invaginations of the plasma This is particularly important as, despite numerous flux of cations, predominantly na+, and small mole- membrane that contain the in vitro studies demonstrating a role for occludin in cules with radii less than 4 Å14,71, and recent analyses protein caveolin-1 and tight junction function, intestinal barrier function is have provided new insight into the structure of these cholesterol. These proteins 58 72 mediate uptake of some intact in occludin-deficient mice . Future analyses pores . However, it is not clear whether increased extracellular materials and are of the response of occludin-deficient mice to stress, claudin-2 expression contributes to disease progres- involved in cell signalling. with particular reference to intestinal tight junction sion or, as proposed above for claudin-1, is an adaptive

Table 1 | Barrier defects associated with intestinal disease Disease or model Cause of barrier Timing of barrier Effects on tight junction Role of commensal Refs defect defect proteins and cytoskeleton microorganisms Crohn’s disease Unknown, associated Before clinical onset Claudin‑2 upregulation, Antibiotics can be helpful 43,64,109 with a frameshift and before clinical MLCK activation and occludin in maintaining remission insertion at nucleotide relapse downregulation 3020 of NOD2 Ulcerative colitis Unknown Not well studied Claudin‑2 upregulation, Not defined 43,64,109 MLCK activation and occludin downregulation IL‑10‑deficient mice Immune signalling (IL‑10 Before clinical onset Not defined Eradication of 76,97 deficiency) microorganisms prevents disease Coeliac disease Not well studied Not well studied Not defined No demonstrated role 110,111 Systemic T cell MLCK activation Associated with acute MLCK activation and occludin Not defined 38 activation diarrhoea endocytosis CD4+CD45RBhi T cell Cytokine release and With clinical onset MLCK activation, claudin‑2 Eradication of 112–114 adoptive transfer epithelial cell damage upregulation and occludin microorganisms reduces model of colitis endocytosis severity SAMP1/yit mice Unknown Before clinical onset Claudin‑2 upregulation and Not defined 77 occludin downregulation DSS‑induced colitis Epithelial cell damage After DSS treatment Not defined Eradication of 80 but before clinical microorganisms onset exacerbates disease TNBS‑induced colitis Immune signalling Not well studied Claudin‑18 upregulation Probiotics can reduce 92 disease severity Mucin‑2‑deficient Not well studied Not well studied Not defined Not defined 10,37 mice MDR1A‑deficent Unknown, follows After immune cell Reduced occludin Increased epithelial 115,116 mice increased CCL2 activation phosphorylation cell response to LPS production precedes disease JAM‑A‑deficient JAM‑A deficiency Not well studied JAM‑A deficiency Not defined 89,90 mice Clostridium Actomyosin disruption With release of toxin Loss of ZO1 and ZO2 Antibiotics predispose to 117 difficile‑induced and glucosylation of Rho and disease onset disease colitis proteins EPEC infection Type III secretion (of After infection MLCK activation and occludin Not defined 118 bacterial proteins) endocytosis Graft‑versus‑host Associated with After clinical onset Not defined Eradication of 35 disease elevated TNF production microorganisms limits disease 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.

response that promotes homeostasis. nevertheless, the insertion at nucleotide 3020 of the gene encoding the consistency of intestinal epithelial claudin-2 upregula- cytoplasmic sensor nucleotide-binding oligomerization tion in disease suggests that this should be a subject of domain-containing 2 (NOD2)74 (TABLe 1), and the same further study. NOD2 mutation has been associated with decreased IL-10 production by peripheral blood mononuclear Barrier function and immunity cells75. Thus, one explanation for the increased intes- Barrier loss is associated with disease risk. A large body tinal permeability observed in some of these healthy of circumstantial evidence suggests that intestinal bar- relatives is that they have subclinical mucosal immune rier dysfunction is associated with the pathogenesis of activation, perhaps with increased TnF production, that Crohn’s disease. This includes the observation that a sub- leads to barrier dysfunction without overt disease. This SAMP1/yit mice set of first-degree relatives of patients with Crohn’s dis- hypothesis would also explain why IL-10-deficient and An outbred mouse strain that ease has increased intestinal permeability despite being SAMP1/yit mice, which develop spontaneous colitis spontaneously develops a 73 chronic intestinal inflammation completely healthy . Interestingly, genetic analyses have and enteritis, respectively, show increased intestinal per- 76,77 similar to human Crohn’s linked increased intestinal permeability in these healthy meability before disease onset . The suggestion that disease. relatives to the Crohn’s disease-associated frameshift permeability is merely a sensitive indicator of mucosal

immune activation would also explain the observa- epithelial proliferation, migration and apoptosis were tion that, during clinical remission, increased intestinal all similar to that observed in wild-type mice26. This permeability is a predictor of relapse in patients with suggests that the effects of constitutively active MLCK Crohn’s disease78. However, barrier dysfunction must expression in these mice were specific to its effect on also be regarded as a potential contributor to disease the tight junction. Thus, consistent with the presence progression. of barrier dysfunction in healthy relatives of patients The classic experiment showing a role for epithelial with Crohn’s disease, these mice provide evidence to cell function in maintaining mucosal immune homeo- suggest that increased tight junction permeability in stasis used chimeric mice in which dominant-negative the absence of more extensive epithelial cell dysfunc- cadherin was expressed in some intestinal epithelial stem tion is insufficient to cause intestinal disease. Further cells, resulting in disruption of the adherens junctions13. analysis of mice expressing constitutively active MLCK This led to profound epithelial cell defects that included did, however, provide evidence of mucosal immune cell incomplete polarization, brush border and actin cytoskel- activation, including increased numbers of lamina pro- etal disruption, accelerated crypt–villus migration and pria T cells, enhanced mucosal IFnγ, TnF and IL-10 premature apoptosis13. Moreover, the mucus layer over- transcription and repositioning of CD11c+ DCs to the lying epithelial cells expressing dominant-negative cad- superficial lamina propria26. Thus, despite being insuf- herin was disrupted and numerous adherent bacteria ficient to cause disease, chronic increases in intestinal were present at these sites13. Patchy, transmural enteritis permeability as a result of continuous activation of a developed in these mice and was limited to regions where physiological pathway of tight junction regulation does epithelial cells expressed dominant-negative cadherin79. lead to mucosal immune cell activation. In addition, epithelial cell dysplasia was occasionally Because a subset of healthy first-degree relatives of present in these areas79. This study has often been cited patients with Crohn’s disease will ultimately develop the as evidence that tight junction barrier loss is sufficient disease, and because a similar fraction of these healthy to cause inflammatory bowel disease and, although tight relatives have increased intestinal permeability, it has junctions were not specifically examined, they were likely been suggested that tight junction barrier dysfunction to be defective. However, although this important study is one factor that contributes to the development of shows that apical junctional complex disruption causes inflammatory bowel disease84. This hypothesis is consist- enteritis, the broad range of epithelial cell defects induced ent with a case report documenting increased intestinal makes it impossible to draw conclusions regarding the permeability 8 years prior to disease onset in a healthy specific contributions of the tight junction to homeostasis relative of a patient with Crohn’s disease85. However, or disease. Similarly, although dextran-sulphate sodium given that the subject of the case report had two first- (DSS)-induced colitis is associated with increased intes- degree relatives with Crohn’s disease and was, therefore, tinal permeability, this is the result of widespread epithe- at increased risk of developing the disease regardless of lial cell damage80 rather than targeted dysfunction of the intestinal permeability measures, this report does not tight junction barrier. Altered sensitivity of genetically provide evidence that barrier dysfunction itself is related modified mice to DSS must therefore be viewed in the to disease onset. context of epithelial cell injury and repair81–83 and cannot The relationship between barrier dysfunction and be interpreted as a function of disrupted tight junction disease has been assessed using mice with a constitutively permeability alone. active MLCK transgene, which have increases in permeability that are quantitatively and qualitatively similar to Isolated barrier loss is insufficient to cause disease. those in healthy relatives of patients with Crohn’s disease. one study has reported an in vivo model of barrier dys- Recombination-activating gene 1-knockout mice (which function induced by direct activation of endogenous lack mature B and T cells) expressing constitutively tight junction regulatory mechanisms26. Although no active MLCK, as well as littermates that did not express overt developmental defects or disease developed in constitutively active MLCK, received CD4+CD45RBhi mice in which intestinal epithelial cells transgenically naive T cells from wild-type mice (the CD4+CD45RBhi expressed constitutively active MLCK, increased intes- T cell adoptive transfer colitis model). Both groups of mice tinal tight junction permeability was observed26. This developed colitis. However, the disease developed more increase in paracellular permeability was quantitatively rapidly and was more severe, in clinical, biochemical and similar to that induced by activation of na+–glucose histological terms, in the mice that expressed constitu- co-transport and was not an indiscriminate loss of tively active MLCK26. Although one could argue that barrier function26, as occurs with exposure to DSS or this accelerated disease progression was due to effects dominant-negative cadherin expression. In addition, of constitutively active MLCK expression beyond tight CD4+CD45RBhi T cell adoptive transfer colitis MLCK inhibition normalized MLC phosphorylation junction regulation, other epithelial cell defects were not 26 model in epithelial cells and paracellular permeability , sug- apparent in these mice (see above), perhaps because con- A well-characterized model of gesting that compensatory changes in pathways that stitutively active MLCK targets an endogenous signalling chronic colitis induced by regulate MLC phosphorylation or barrier function pathway. Thus, targeted increases in intestinal epithelial + hi transfer of CD4 CD45RB were not induced. Moreover, growth of these mice was tight junction permeability through constitutive activa- (naive) T cells from healthy wild-type mice into normal, and intestinal morphology, enterocyte struc- tion of the pathophysiologically relevant MLCK pathway immunodeficient syngeneic ture, tight junction and adherens junction organiza- are sufficient to accelerate the onset and enhance the recipients. tion, actomyosin and brush border architecture, and severity of immune-mediated colitis.

Conversely, the data above also suggest that inhibi- activity, enhance disease progression and severity and, tion of MLCK, which is sufficient to reverse acute TnF- possibly, be a risk factor for development of disease. induced barrier loss38, may have therapeutic benefit in Finally, although much more investigation is needed, immune-mediated colitis. This notion is supported by early reports indicate that restoration of tight junction a preliminary in vivo report using mice with a knock- barrier function may be effective, either alone or in out of non-muscle MLCK86. However, as non-muscle combination with other agents, in preventing disease MLCK has many functions, including an essential role in at-risk individuals or maintaining remission in in neutrophil transendothelial migration87, the results patients with inflammatory bowel disease. of this report may reflect changes beyond tight junction barrier preservation. Another recent study showed Barrier loss activates immunoregulatory processes. why that a peptide capable of enhancing small-intestinal is tight junction barrier dysfunction alone insufficient barrier function reduced the extent of mucosal inflam- to cause disease? Endoscopic mucosal resection, which mation in IL-10-deficient mice88. However, this result removes the epithelium and mucosa completely and must be interpreted with caution, as the peptide used therefore causes barrier loss far greater than that caused (which is thought to antagonize the putative extra- by targeted tight junction dysfunction, is insufficient cellular pathway signalling molecule zonulin) activates to cause chronic disease. Therefore, immuno regulatory an undefined regulatory pathway and the overall physi- mechanisms must be induced by the host following ology of peptide-treated mice has not been examined barrier loss to prevent inappropriate inflammatory in detail. Thus, although the finding is intriguing, responses, as mucosal damage is a daily occurrence in further investigation is necessary to determine whether the gastrointestinal tract. Moreover, these immuno- barrier preservation, by MLCK inhibition or other means, regulatory mechanisms must be robust because, even in can prevent or reverse intestinal disease. patients with inflammatory bowel disease, endoscopic Two studies have recently reported that junctional mucosal resection is insufficient to initiate a relapse adhesion molecule-A (JAM-A)-deficient mice have to active disease. reduced intestinal barrier function and increased rates The increased transcription of IL-10 in the intestinal of intestinal epithelial cell proliferation and apopto- mucosa of mice with a constitutively active MLCK trans- sis89,90. These mice also have intestinal mucosal immune gene may provide a clue to which immuno regulatory cell activation as defined by increased numbers of processes are triggered by barrier dysfunction. In addi- neutrophils in the intestinal mucosa89,90. In addition, tion, responses to transient barrier loss have been used one study reported an increase in the number of dis- to explore mucosal immunoregulation92. In this model, tal-colonic lymphoid aggregates in JAM-A-deficient intrarectal administration of ethanol caused transient mice90, although neither lymphoid aggregates nor epithelial cell damage, mucosal erosion and barrier enhanced mucosal cytokine expression was detected in loss. The induction of barrier loss was followed by an a subsequent analysis89. However, as JAM-A is widely increase in the numbers of IFnγ- and IL-10-producing expressed by epithelial cells, endothelial cells, platelets, lamina propria mononuclear cells and lamina propria antigen-presenting cells, circulating neutrophils, mono- CD4+CD25+ T cells that express latency-associated peptide cytes, lymphocytes and platelets, and as the mice studied (LAP) on their surface92. Remarkably, such ethanol were not epithelial cell-specific knockouts, it is not clear administration conferred protection from subsequent whether the observed effects were due to altered tight trinitrobenzene sulphonic acid (TnBS)-induced coli- junction integrity, changes in epithelial cell shape91, an tis, and this protection required the presence of LAP+ increased rate of epithelial cell apoptosis90 or trapping T cells92. The induction of these LAP+ T cells was shown of neutrophils within mucosal vessels owing to the loss of to depend on CD11c+ DCs, Toll-like receptor 2 signalling endothelial cell-expressed JAM-A. nevertheless, it is and a normal luminal microorganism population92. intriguing that JAM-A-deficient mice showed increased Although further study is needed to understand sensitivity to DSS-induced colitis89,90. By contrast, the mechanisms of LAP+ T cell induction by transient the sensitivity of mice with an endothelium-specific mucosal damage, it is interesting that in different experi- JAM-A deficiency to DSS was similar to that of wild-type mental systems increased intestinal epithelial cell tight control mice, indicating that the response of mice with junction permeability increased the number of CD11c+ universal JAM-A deficiency was not solely due to loss of DCs in the superficial lamina propria86, and that inter- endothelial JAM-A89. unfortunately, the use of mice with actions with intestinal epithelial cells enhance the abil- a universal JAM-A deficiency and the presence of other ity of bone marrow-derived CD11c+ DCs to induce the epithelial cell abnormalities, particularly increased epi- differentiation of regulatory T cells93. Together, these thelial cell turnover, in the absence of exogenous stimuli observations support the hypothesis that the interactions limits interpretation of these studies. Even so, data from between CD11c+ DCs and luminal materials are regu- JAM-A-deficient mice do support the hypothesis that lated by tight junction permeability and are also central mucosal barrier loss can enhance the severity of colitis. to mucosal immune homeostasis. overall, data from both human subjects and mouse other data also support important roles for luminal Latency-associated peptide experimental models show that defects in tight junc- material, particularly microorganisms and their prod- A small peptide derived from 94 the N-terminal region of the tion barrier function are insufficient to cause disease. ucts, in mucosal immune regulation . For example, TGFβ precursor protein; it can However, several lines of evidence suggest that increased antibiotics can be helpful in the management of Crohn’s modulate TGFβ signalling. paracellular permeability can increase mucosal immune disease95. Although the mechanisms by which antibiotics

Bacterial products example, enhances paracellular permeability in pulmo- and dietary antigens nary and intestinal epithelium48,49. Conversely, epithelial Na+ Small solutes Actomyosin Toll-like receptor 2 activation may restore barrier func- 98 Epithelial cell ring tion . It will therefore be of interest to define the microbial products, dietary components and other factors that influence mucosal immune status following alterations Claudin Claudin-2 in intestinal epithelial tight junction permeability. Occludin MLCK Tight junctions integrate mucosal homeostasis one interpretation of the available data is that the tight junction barrier integrates the relationship between lumi- Na+ Lamina Endosome nal material and mucosal immune function (FIG. 3). In propria Small solutes most individuals, this is a healthy relationship in which TLR TGFβ IFNγ regulated increases in tight junction permeability or IL-13 Retinoic acid APC TNF transient epithelial cell damage trigger the release of pro-inflammatory cytokines, such as TnF and IFnγ, as MHC Antigen well as immunoregulatory responses. Immunoregulatory TCR 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 T 2 cell T cell T 1 cell 99,100 LAP H H differentiation . This precarious balance between pro- inflammatory and immunoregulatory responses can fail if there are exaggerated responses to pro-inflammatory T cell Reg cytokines, as may be associated with mutations in the IL-10 and TGFβ endoplasmic reticulum stress response transcription factor X-box-binding protein 1 (XBP1) (ReF. 101), insuf- Figure 3 | The epithelium and tight junction as integrators of mucosal homeostasis. ficient IL-10 production (as in IL-10-deficient mice Minor barrier defects allow bacterial products and dietary antigensNature to Re crossviews the | Immunolog y and, possibly, in patients with IL10 promoter polymor- epithelium and enter the lamina propria. This can lead to disease or homeostasis. If the phisms102) or inadequate immune tolerance to luminal foreign materials are taken up by antigen‑presenting cells (APCs), such as dendritic cells, antigens and microbial products94 (potentially because that direct the differentiation of T helper 1 (T 1) or T 2 cells, disease can develop. In this H H of NOD2 mutations in patients with inflammatory process, APCs and T 1 cells can release tumour necrosis factor (TNF) and interferon‑ H γ bowel disease103–106). As a result, mucosal immune cell (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 activation may proceed unchecked and the release of the lumen into the lamina propria and amplifying the cycle of inflammation. This may, cytokines, including TnF and IL-13, may enhance bar- ultimately, culminate in established disease. Alternatively, interleukin‑13 (IL‑13) released rier loss that, in turn, allows further leakage of luminal material and perpetuates the pro-inflammatory cycle. by TH2 cells increases flux across small cation‑selective pores, potentially contributing to ongoing disease. Conversely, homeostasis may dominate if APCs promote regulatory This model highlights the roles of a susceptible host and

T (TReg) cell differentiation, which can be enhanced by epithelial cell‑derived transforming defective epithelial cell barrier function as key compo- growth factor‑β (TGFβ) and retinoic acid. The TReg cells display latency‑associated peptide nents of the pathogenesis of intestinal inflammatory (LAP) on their surfaces and may secrete IL‑10 and TGFβ to prevent disease. MLCK, myosin disease and explains the crucial role of the epithelial light chain kinase; TLR, Toll‑like receptor; TCR, T cell receptor. barrier in moulding mucosal immune responses.

Conclusions promote maintenance of remission in patients with Significant progress has been made in understanding Crohn’s disease are not defined, they may be related the processes by which physiological and pathophysio- to the observation that luminal microorganisms are logical stimuli, including cytokines, regulate the tight required in experimental models of inflammatory bowel junction. Early reports suggest that restoration of disease that include IL-10-deficient mice96,97 and adop- tight junction barrier function may have benefit38,86. tive transfer colitis. Microorganisms are also necessary However, the mechanisms of tight junction regulation for the development of increased intestinal permea- will have to be defined in greater detail if they are to be bility before the onset of overt disease in IL-10-deficient viable pharmacological targets. Conversely, recent data mice76. Although this observation is not entirely under- have emphasized the presence of immune mechanisms stood, one interpretation is that the limited flux of that maintain mucosal homeostasis despite barrier microbial products that normally occurs across intact dysfunction, and some data suggest that the epithelium intestinal epithelial tight junctions is sufficient to trig- orchestrates these immunoregulatory events through ger mucosal immune activation in IL-10-deficient mice. direct interactions with innate immune cells. Future This mucosal immune activation could, in turn, result elucidation of the processes that integrate mucosal bar- in the release of cytokines that cause increased intestinal rier function, or dysfunction, and immune regulation to permeability and accelerate disease progression (FIG. 3). prevent or perpetuate disease may lead to novel thera- However, in addition to immune cells, epithelial cells can peutic approaches for diseases associated with increased respond to foreign materials. Lipopolysaccharide, for mucosal permeability.

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From Mucins to Mucus. Thornton DJ and Sheehan JK, Proc Am Thorac Soc., 1:54 (2004)

From Mucins to Mucus Toward 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; and Department 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 chronic play the vital role in determining the physical properties of this airway disease mucus hypersecretion is an important factor in mor- essential barrier in health and disease. However, it should also bidity and mortality. The properties of the mucus gel are dictated be pointed out, especially in pathologic conditions, that other in large part by the oligomeric mucins and, over the past decade, molecules such as proteoglycans and DNA may contribute to we have gained a better understanding of the molecular nature of the physical properties of airways mucus. these complex O-linked glycoproteins. We know now that MUC5AC Over the last decade, great strides have been made in identi- mucins, as well as different glycoforms of the MUC5B mucin, are fying and characterizing the major oligomeric mucins present in the predominant gel-forming glycoproteins in airways mucus. Fur- airways sputa. However, there have been no studies to date that thermore, the amount, molecular size, and morphology of these relate the ensemble of their physical properties (i.e., length, glycoproteins can be altered in disease. From more recent data, it charge density, macromolecular architecture) to a speciﬁc set of has become clear that oligomeric mucins alone do not constitute rheologic parameters for a mucus gel, never mind its optimiza- mucus, and other mucin and nonmucin components must be important contributors to mucus organization and hence airways defense. tion to a speciﬁc function, such as ciliary transport. In this article, Therefore, the challenge over the coming decade will be to investi- we have been given the task of reviewing our own contributions gate how the oligomeric mucins are organized to yield “functional” to this area. We have also suggested further avenues of investiga- mucus. Such studies will provide a clearer perception of airways tion that will be necessary to gain a better understanding of the mucosal protection and may highlight specific components as po- biology and pathobiology of this essential barrier. tential targets for therapeutic strategies for the treatment of hypersecretory disease. MUCINS—WHAT ARE THEY?

Keywords: mucus; sputum; MUC5AC; MUC2; MUC5B Mucins are currently defined as high-Mr glycoproteins that contain at least one and sometimes multiple protein domains that The respiratory mucus gel performs a number of essential func- are sites of extensive O-glycan attachment (mucin-like domains). tions that collectively lead to the protection of the airways. This Thus, the composition of these glycoproteins is dominated by highly hydrated gel, in conjunction with ciliated epithelial cells, carbohydrate, which can total in some cases as much as 80% of forms the mucociliary “escalator,” which, along with cough, is the weight of the molecule. There appear to be two major types essential for the maintenance of sterile and unobstructed air- of mucin, one thought to be monomeric and, primarily, but not ways. By contrast, overproduction of mucus with altered rheo- exclusively, located at the cell surface, and the other oligomeric. logic properties is an important factor in the morbidity and mor- This latter type is secreted and thought to be responsible for tality of chronic airways disease (e.g., asthma, cystic fibrosis [CF], the rheologic properties of mucus. The mucin-like domains are and chronic obstructive pulmonary disease [COPD]) (1–3). As a enriched in the amino acids serine and threonine, which are the result of the change in physical properties of the gel, mucociliary sites for the attachment to the linkage sugar N-acetylgalactos- clearance is impaired or abolished with the attendant problems amine. In addition to N-acetylgalactosamine, mucin oligosaccha- of poor gas exchange and/or inflammation and/or bacterial colo- rides may also contain N-acetylglucosamine, galactose, fucose, nization. Thus, production of mucus with the “correct” proper- sialic acids, and sulfate. The oligosaccharides have remarkable ties for airways protection is paramount for health. structural diversity and all of their specific roles remain to be A variety of factors have been found to trigger mucus hyper- fully understood. However, a consequence of their attachment secretion, including proinflammatory cytokines (e.g., interleu- to the polypeptide is to cause it to stiffen, resulting in a large kin-4, interleukin-13, epidermal growth factor, and tumor necro- expansion of the volume domain of the molecule, which gives sis factor-␣), ATP, bacterial exoproducts, and host proteases mucins their characteristic space-filling, and thus gel-making, (4–9), but the mechanisms underlying mucus biogenesis and properties (10). Furthermore, it has long been known that certain secretion, and its molecular composition and supramolecular or- bacteria bind specific oligosaccharide ligands (for a review see ganization, are still obscure. The major gel-forming components [11]). It is likely that the primary function of the oligosaccharide of the airways mucus gel are the oligomeric mucins, and the diversity is to enhance the possibility that bacteria bind to mucus, hypothesis that drives our research is that these glycoproteins thus facilitating their removal by mucociliary transport. More- over, by providing competing receptors for cell-surface glycoconjugates, mucins may trap bacteria and make them less successful in their attempts to colonize the epithelium. Thus, the array of oligosaccharides expressed on the mucins of an individual may (Received in original form June 19, 2003; accepted in final form September 30, 2003) play a key role in governing the susceptibility to infection. Supported by the Wellcome Trust and the National Asthma Campaign. The studies we shall summarize have been concerned solely Correspondence and requests for reprints should be addressed to David J. Thorn- with the gel-forming mucins. From our studies in collaboration ton, B.Sc., Ph.D., Wellcome Trust Centre for Cell-Matrix Research, School of with Ingemar Carlstedt in the early 1990s, it was clear that the gel- Biological Sciences, University of Manchester, 2.205 Stopford Building, Manches- forming mucins in airways mucus consisted of a heterogeneous ter, M13 9PT, UK. E-Mail: [email protected] mixture of glycoproteins that was physically similar and extremely Proc Am Thorac Soc Vol 1. pp 54–61, 2004 DOI: 10.1513/pats.2306016 large (12–16). The mucus gel could be almost completely solubi- Internet address: www.atsjournals.org 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 mucin, along with a generic model for oligomeric mucin organization and (B) reduced mucin subunits. (A) Transmission electron micrograph of a respiratory mucin preparation showing a single oligomeric mucin chain. Mucins were isolated from sputum by solubilization with 6 M guanidinium chloride (GuCl) followed by purification by two stages of cesium chloride (CsCl) isopynic density gradient centrifugation, first in 4 M GuCl/CsCl and then in 0.2 M GuCl/CsCl (12). Also shown is a cartoon of an expanded view of a section of the mucin chain highlighting the intermolecular, end-to-end, disulfide (S-S)-mediated assembly of the mucin subunits (monomers). The polypeptide consists of alternating stretches of dense O-linked glycosylation (mucin-like domains) and folded, intramolecular disulfide bond stabilized regions (depicted as knots). A single monomer spans the two S-S linkages. (B) Transmission electron micrograph of reduced mucin subunits generated after treatment of oligomeric mucins (prepared as in A) with a reducing agent (e.g., 10 mM dithiothreitol). The reduced subunits are approximately 600 nm in length. Also shown is a cartoon of the reduced mucin subunit highlighting the unfolding of the intramolecular disulfide stabilized domains situated at the ends and interspersed between the mucin-like domains. SH represents reduced cysteine residues.

nium chloride), a process usually achieved overnight but in some MUC5B genes are predominant in sputum (25–29) and that the cases taking days. Thus, entanglement of the long mucin chains MUC2 mucin is barely detectable (see subsequent text). Thus, was considered to be the primary mechanism for gel formation there seems to be a discrepancy between MUC2 messenger RNA and, as a consequence, the size, concentration, and chemical nature levels and the occurrence of the mature MUC2 mucin in sputum. of the mucins are important factors for determining the proper- This may be due to the apparent “insolubility” of this glycopro- ties of the gel. Physical characterization of the mucins isolated tein, as has been reported for MUC2 isolated from small-intesti- from sputum using light scattering and electron microscopy nal mucus (30), causing it to be retained in the airways. While showed them to be polydisperse in both mass (2–40 mD) and size our data do not rule this out, in the single study we have per- (0.5–10 ␮m in length). Furthermore, these studies demonstrated formed, where the mucus was physically removed from an asth- that the component mucin monomers or subunits (2–3 mD) are matic lung postmortem, there was still little evidence for this assembled linearly and held together by disulfide bonds (17). mucin (3, 31). Figure 1 shows electron micrographs of oligomeric respiratory Matching the polypeptides of the different mucins present in mucins before and after treatment with a reducing agent, along mucus to their respective genes has been a long process, which with the model proposed for their structure. It is important to was eventually achieved by fractionation of the mucins (after note that for these studies the mucins were isolated and purified reduction of their disulfide bonds) by anion exchange chroma- in highly chaotropic solvents (e.g., 4–6 M guanidinium chloride) tography and agarose gel electrophoresis, and then by a combina- to preserve their primary structure. Thus, the observed architec- tion of biochemical analyses (i.e., amino acid composition, pep- ture may not reflect their “native” conformation in mucus. A tide sequencing, and peptide fingerprinting) as well as reactivity more realistic view of their native conformation will require char- with mucin-specific antisera (for more details see Figure 2). acterization of the mucins after isolation using nonchaotropic The analysis of multiple sputum samples revealed that the agents. two major mucin gene products, MUC5AC and MUC5B have different chemical properties. The MUC5AC mucin has a more WHICH MUCINS ARE IMPORTANT IN THE AIRWAYS homogeneous charge distribution than MUC5B, which occurs AND WHAT ARE THEY LIKE? in differently charged forms (26). In most samples analyzed, two different MUC5B forms (glycoforms) are found, and while their It is now apparent that these oligomeric glycoproteins are mem- charge density is not identical between individuals it is apparent bers of a larger family of mucins. Of the 13 mucin genes (MUC) that a “high” and “low” charge form occurs in most cases (Fig- currently identified, only 4 (MUC2, MUC5AC, MUC5B, and ure 3). These glycoforms also exhibit different buoyant densities MUC6) contain the cysteine-rich motifs in their C- and N-termi- and, as a result, can be partially separated by isopycnic density nal domains necessary for oligomerization (18–21). Three of centrifugation where there is a direct correlation between their these genes, MUC2, MUC5AC, and MUC5B, are expressed in charge and buoyant densities (17). As far as we can tell, each gly- the airways. A number of important studies on airways mucins coform is found in separate oligomeric mucins and is not part have concentrated on messenger RNA expression, in particular of a heterooligomer. This separation, coupled with the distinctive focusing on MUC2 and MUC5AC, and highlight specific disease- pattern of glycosylation, at least at the level of charge density, related factors that upregulate these two mucins (4–9, 22–24). associated with each mucin species, may reflect their different However, the fact that these macromolecules are synthesized cellular origins (see Where Are Mucins Synthesized?). and then stored awaiting the appropriate stimulus for secretion Studies performed on the intact MUC5AC and MUC5B mu- implies that messenger RNA studies may be misleading for the cins suggest that these mucins have different macromolecular amount of mucin in mucus. This is borne out by biochemical data, characteristics. Sedimentation studies have indicated that both which have demonstrated that the products of the MUC5AC and are polydisperse in size, and this may arise from the oligomeriza-

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Figure 2. Identifying the major oligomeric mucins in sputum. Sputum is a mixture of oligomeric mucins (MUC2, MUC5AC, and MUC5B), which are polydisperse in size and share similar biochemical and biophysical properties. Therefore, purification of the individual mucin species is not possible with intact mucins but can be achieved by working with preparations of reduced mucins. (A) A typical anion exchange chromatogram of a reduced and alkylated respiratory mucin preparation. In this case, the separation matrix was a Mono Q HR 5/5 column, which was eluted with a linear salt gradient of 0–0.25 lithium perchlorate containing 6 M urea (dashed line). The mucin distribution (solid line) was monitored with the periodic acid Schiff’s (PAS) reagent that detects the glycan component of the molecule. In this example, the mucin distribution was pooled into three populations with increasing charge density (I, II, and III), which were further purified by rerunning on the Mono Q column. Fractions from the rerun were pooled on the basis of their electrophoretic mobility on a 1% agarose gel and their reactivity with a number of lectins (data not shown) to yield 3 distinct populations. (B) The 3 resultant pools were subjected to 1% agarose gel electrophoresis and a Western blot of the agarose gel was probed with an antiserum that recognizes oligomeric mucins (64). Other Western blots (not shown) were probed with antisera specific for MUC2, MUC5AC, and MUC5B mucins (25, 26, 44) and the bands that were reactive with these probes are labeled. Two of the bands were reactive with the MUC5B antiserum, and these correspond to populations I and III, which had very different charge densities as assessed by anion exchange chromatography (see Fig. 2A). Therefore, these bands are labeled as high- and low-charge variants of MUC5B. The third band corresponding to the intermediate charge species (population II) on the Mono Q column was reactive with the MUC5AC antiserum. None of the bands were stained with the MUC2 antiserum. The identification of mucins was not based solely on antisera reactivity but also relied on chemical analyses. For example, (C) shows the MALDI-TOF MS peptide fingerprint after trypsin treatment of a reduced and alkylated MUC5B mucin preparation. The four major peptides highlighted (peptides 1–4) were purified by reverse-phase chromatography on a C18 column and their sequence determined by N-terminal sequencing (26). Their sequences and locations within the MUC5B polypeptide are shown in the boxed schematic diagram. This represents the central portion of the MUC5B apoprotein, showing the cysteine-rich domains (cys1–7, black boxes) and the R and R-end domains (hatched boxes) corresponding to the glycosylated mucin-like domains (adapted from [20]). The four major peptides are located in some of the seven cys-domains, which share a similar, repetitive sequence. The 62 amino acid sequence shown is found in three of 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. The multiple occurrences of these four peptides explain their dominance of the MUC5B tryptic fingerprint. Amino acid composition can also be used to identify mucins. Shown in (D) are the predicted contents (calculated from published deduced amino acid sequences) of serine, threonine, and proline in the mucin-like domains of the MUC2, MUC5AC, and MUC5B mucins. This analysis highlights the uniqueness of the MUC2 mucin in this regard. Also shown are the experimentally derived values for these three amino acids generated from amino acid analysis of the highly glycosylated fragments of the mucin (corresponding to the mucin-like domains) obtained after trypsin digestion of population I (MUC5B) and population 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/or glycoprotein, which was taken to indicate that MUC5AC was as a consequence of speciﬁc proteolytic processing and/or degra- smaller than MUC5B (27, 32). However, this is probably not the dation. Moreover, these studies showed that the MUC5AC mucins case, as we have shown that MUC5AC is in fact highly oligomer- exhibited a lower average sedimentation rate than the MUC5B ized and the apparent difference in size may be explained by a

Thornton and Sheehan: From Mucins to Mucus 57

exclusively, from the mucous cells of the submucosal glands (17, 27, 32), and this distribution is consistent with messenger RNA expression as shown by in situ hybridization (35). While antisera to MUC5AC and MUC5B have been used to localize their respective cellular sources, we do not have, and may never have, probes to the polypeptide that are specific for each of the glycoforms of the MUC5B mucin. Thus, we do not know if they have different sites of synthesis. We have previously shown in salivary mucin preparations that the highly charged variant of MUC5B was reactive with a monoclonal antibody F2 that is specific for the a carbohydrate epitope, the sulfo-Lewis antigen (SO3-3Gal␤1– 3GlcNAc) (36). This antibody has been shown to stain a subset of glandular mucous cells in normal human trachea (27). Whereas this suggests different cellular locations for the MUC5B variants, immunolocalization data from carbohydrate-directed probes are not conclusive owing to potential cross-reactivity with other glycoproteins. Thus, polypeptide probes would be desirable and might allow us to conclusively pinpoint their cellular sources; however, unless we can find differences in their polypeptide Figure 3. Agarose gel electrophoresis of reduced sputum samples. moieties, this will not be possible. Sputa, NaCl-induced from healthy airways (lanes 1, 6 and 7) and sponta- It is interesting to speculate that the spatial separation of neous secretions from diseased airways, were solubilized in 6 M GuCl mucin production in the normal airways (i.e., MUC5AC on the and then dialyzed into 6 M urea. The samples were reduced with 10 mM surface and MUC5B in the glands), coupled to the fact that the dithiothreitol, treated with 25 mM iodoacetamide to alkylate-free thiol two sites are controlled by different secretory mechanisms, may groups and then subjected to electrophoresis on a 0.7% agarose gel allow for fine-tuning of the mucus composition to modulate mu- (for more details see Reference [65]). After electrophoresis, gels were cus properties depending on the challenge in the airways. transferred to nitrocellulose and probed with antisera specific for (A) MUC5AC and (B) MUC5B mucins (section from the data presented in On the basis of the immunolocalization and expression data, Reference [39]). These blots were also stained with an antiserum directed it seemed logical at that time to believe that the mucin composi- against MUC2, but no bands were visualized (data not shown). The tion of mucus would inform us on the contribution of the various results show the greater variation in electrophoretic migration of the cellular sources to the secretion and, therefore, might throw up reduced MUC5B mucins compared with MUC5AC; furthermore, with possible cellular targets for therapies aimed at modulating mucin, the exception of one sample (lane 6), all the others exhibit two different and hence mucus production in hypersecretory diseases. How- MUC5B charge forms. These data also demonstrate that there is a larger ever, in hypersecretory disease, this marked spatial separation variation in electrophoretic mobility between low-charge populations of synthesis does not seem to hold. Such diseases are associated than there is between highly charged forms. with significant hyperplasia and metaplasia of mucin secreting cells, and it has been shown that MUC5AC production can also occur in glandular mucous cells, while some MUC5B and MUC2 synthesis can be detected in surface goblet cells (37, 38). Whether difference in physical characteristics of these two mucins (33). these mucins can be coexpressed in the same cell or arise from The MUC5AC polymer has a low mass per unit length, suggesting different cells has not yet been determined. Because we do not small oligosaccharides, and adopts a very stiff extended confor- know the relative change in expression of the mucins at the mation in solution, whereas the MUC5B mucin appears to adopt different locations, we cannot be certain that their occurrence a more compact structure. Further evidence that these two mu- is not a valid indicator of the major source of their production. cins have different macromolecular features is indicated by their Thus, there is an obvious need for further, more in-depth studies behavior on agarose gel electrophoresis. Unreduced MUC5AC on mucin expression in disease. preparations exhibit a ladder-like banding pattern and oligomeric species containing 16 monomers can be discerned (33). ARE AIRWAYS MUCINS ALTERED IN DISEASE? By contrast, unreduced MUC5B mucins barely enter the gel (Thornton and coworkers, unpublished observation). In a series of studies, we have shown that the gel-forming mucins It is clear that MUC5AC and MUC5B are the predominant can be changed in amount, type, and size in airways disease gel-forming mucins in the airways and that they have distinct (3, 12, 13, 31). For example, in the viscid mucus obstructing the chemical and physical features. What effect these differences airways of an individual who died in status asthmaticus, there between the mucins have on the gel properties has yet to be de- were changes in all three parameters. There was an approxi- termined. Moreover, at an even more fundamental level, we do mately seven-fold increase in mucin concentration compared not know whether MUC5AC and MUC5B mucins are blended with healthy secretions. Furthermore, we demonstrated that the together to form the mucus gel or if they form the basis of only mucin type responsible for the aberrant physical properties separate gels. The latter has been suggested in gastric mucus, of this gel was a low-charge glycoform of the MUC5B mucin where laminated layers containing different mucins have been with unusual structural features and extreme size (3, 31). The reported (34). clinical outcome in this case demonstrated the dramatic consequences of alterations in the mucin component of mucus and WHERE ARE MUCINS SYNTHESIZED? highlighted the need to determine the molecular profile of mucins in sputum. Immunolocalization studies on normal airways, using mucin- Therefore, in order to gain a more detailed picture of mucin specific antisera, have shown that MUC5AC mucins are pro- type (gene product and glycoform) and quantity in normal and duced predominantly by goblet cells in the surface epithelium. pathologic respiratory mucus, we developed a quantitative West- MUC5B mucins on the other hand, originate mainly, but not ern blotting assay to measure the levels of MUC2, MUC5AC,

58 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 1 2004 and the different glycoforms of the MUC5B mucins directly from similar macromolecular properties to their counterparts in hu- sputum (39). Data from 44 samples (15 NaCl-induced normal, 10 man airways (44). However, while the MUC5AC mucin exhibits asthma, 10 CF, and 9 COPD) showed, as our previous nonquanti- a similar charge profile to MUC5AC in vivo, by contrast, the tative studies had suggested, that MUC5AC and MUC5B are MUC5B mucin produced is more homogeneous, with evidence the major gel-forming mucins in sputum. By contrast, the MUC2 of only a single (perhaps high-charge) glycoform. Thus, the mu- mucin was present in only trace amounts, confirming our earlier cin phenotype and hence the resultant properties of the secre- observations that MUC2 is only a minor component in spu- tions may not be a truly accurate reflection of in vivo mucus. tum. Moreover, this study showed that airways mucus is not a Nonetheless, such culture systems provide a powerful and manip- single substance and is comprised of variable amounts of MUC5AC ulable experimental system and seem set to form the basis of ever and MUC5B glycoproteins. Somewhat surprisingly, the variation widening studies that should revolutionize our understanding of in content was particularly marked in the “normal” samples. A key the biology and pathobiology of mucus over the next few years. observation to come from this work was that, compared with For example, work from Boucher and collaborators using cul- secretions from normal subjects and individuals with asthma, tures derived from cells with the CF defect has shown that the there was more MUC5B in CF and COPD sputa and, most dynamics of the attached mucus layer are altered and, in these notably, there was a significant increase in the amount of the compared with normal cultures, mucus transport is reduced or low charge form of the MUC5B mucin in the diseased, possibly even abolished (41). infected sputa. This might suggest a link between infection/inflammation and MUC5B production. The functional conse- AIRWAYS MUCUS GEL FORMATION—OLIGOMERIC quences of mucus with different mucin compositions, and in MUCINS ARE NOT THE WHOLE STORY! particular of the different charge forms of the MUC5B mucin, are at present completely obscure. We would suggest the general While size and concentration of the mucins are key factors con- hypothesis that MUC5AC, being primarily the product of goblet trolling gel formation, concentrated solutions of mucins alone cells, may have the major mechanical function of facilitating do not reproduce all the physical properties of the gel (47). This ciliary clearance of mucus, whereas MUC5B emanating from is emphasized in our recent findings where we have shown, using the glands may form the basis of a gel, the primary role of confocal fluorescence recovery after photobleaching, that the which is to help with the clearance of specific pathogens or other network properties of mucus cannot be recapitulated by concen- irritants. The abnormal mucin composition of the gel in CF and trated mucin solutions even at higher than in vivo mucin concen- COPD airways may provide an insight into the altered physical tration (48). Unlike some other methods for looking at mucus nature of these secretions and ultimately suggest potential cellu- properties, confocal fluorescence recovery after photobleaching lar targets for therapy. These observations again highlight the is nondestructive and provides a powerful approach to investigat- need for more studies on mucin expression in disease, and in ing the properties of macromolecules in concentrated solution, particular on MUC5B expression, which has so far been largely including complex mixtures, such as in mucus, under equilibrium ignored. Furthermore, these findings reemphasize the need to conditions, and in the absence of concentration gradients and flow develop reliable probes for the MUC5B mucin glycoforms to and shear forces (48, 49). We have used it to determine lateral establish their cellular origin. tracer diffusion of fluorescent molecular probes (i.e., bovine serum albumin and mucins) and of rigid polystyrene microspheres of GEL-FORMING MUCINS PRODUCED BY AIRWAYS different size in mucus at physiological concentrations. It is important to note that the tracer diffusion measurements alone CELLS IN AIR–LIQUID INTERFACE CULTURE are not a measure of gel formation, which involves polymer cross- Studies of human airways secretions have been, and will continue links and entanglement and occurs in concentrated solutions to be, essential to highlight changes associated with disease. of all flexible polymers. However, they provide a quantitative However, these investigations are fraught with difficulty related comparison of the “network” caused by gel-formation, or entan- to mucus collection, as well as possible changes in the mucins glement, that impedes the free diffusion of other macromolecules being disguised by nonspecific postsecretion processing. Further- in a concentration-dependent way. Our initial confocal fluores- more, the effects of extraneous environmental influences are cence recovery after photobleaching analysis of MUC5B (ex- difficult to control and, for ethical reasons, it is impossible to tracted and purified in the presence of 4–6 M guanidinium chlo- perform in vivo studies of mucin/mucus biogenesis and secretion. ride) showed that in concentrated solutions its properties resulted Thus, to progress our understanding of both normal and diseased from polymer entanglement with no evidence of protein–protein airways, there is an urgent need for in vitro systems that mimic self-association or glycan-mediated interactions (48). A direct com- in vivo mucus production. For this purpose a number of labora- parison of concentrated guanidinium chloride-purified MUC5B tories have developed airways epithelial cell cultures (40–43). mucin solutions with the native saliva from which it was prepared The normal human tracheobronchial air–liquid interface cul- revealed that the MUC5B mucin did not replicate the properties tures instigated by Nettesheim and colleagues are of particular of the parent mucus. At similar MUC5B concentrations to those interest (40). When cultured in the presence of retinoic acid, found in saliva, the guanidinium chloride–purified mucin was normal human tracheobronchial cells grow and differentiate into approximately 20 times more permeable, suggesting that there a mucociliary epithelium. They secrete functional mucus onto was a higher order structure in the native mucus, which may their apical surface (40) that is clearly capable of ciliary transport. involve other components in the secretion (48). Further analyses This culture system appears to mimic in vivo airways mucin pro- have implicated calcium as a key regulator of MUC5B mucin duction in that all three oligomeric airways mucins, MUC5AC, supramolecular organization in salivary mucus (50). This study MUC5B, and MUC2 are expressed. Furthermore, their basal lev- demonstrated a reversible, calcium-dependent interaction be- els of expression can be modulated by physiologically relevant tween MUC5B mucins that was sensitive to denaturation by regulators (e.g., thyroid hormone and epidermal growth factor) chaotropic solvents (e.g., 6 M guanidinium chloride), treatment (44–46). We have investigated the apical secretions from such with reducing agents and the proteinase trypsin (50). This study cultures and have shown that MUC5AC and MUC5B are the also highlighted the need for alternative, nondenaturing solvents predominant mucins stored and secreted by normal human tra- to disassemble mucus gels for investigation of their functions. cheobronchial cells (passage 2). Moreover, these mucins have The fact that gel-forming mucins alone do not constitute mu-

Thornton and Sheehan: From Mucins to Mucus 59 cus may not be totally surprising because it is a complex mixture (61–63). There is little biochemical characterization of these of ions, mucins, glycoproteins, proteins, and lipids and, like the mucins, and their amounts in mucus are unknown. Furthermore, mucins, the total amount of these other components is increased their role in mucus has yet to be defined and there is clearly an in hypersecretory disease (51–54). Some of these components urgent need to understand their function in mucus. contribute to airways protection by targeting pathogens (e.g., secretory IgA, proline-rich proteins, defensins, lysozyme, and Conclusions transferrin), and others may act to modulate the organization As far as the oligomeric mucins are concerned, an extensive and hence the properties of the gel. For example, in the gastro- array of mucin-specific probes are available, the methodologies intestinal tract the mitogenic/reparative trefoil peptides are inti- necessary for their separation and characterization have been mately associated with mucus and have been suggested to bind developed, and the air–liquid cultures offer a more physiologi- to oligomeric mucins and increase mucus viscosity (55). Work cally relevant in vitro system for their study. Thus, we are well from our laboratory has demonstrated that a large glycoprotein, placed to perform new studies of the cell and structural biology gp-340, is found in complexes with MUC5B in airways mucus of the mucins and mucus, which is essential for a more coherent (56). It is interesting to speculate on the consequences of an picture of how this essential barrier functions to protect the interaction of gp-340 with the mucin network. Gp-340 clearly airways. Finally, there are currently few effective therapies to has an important antibacterial role in mucus, either via its direct alleviate mucus hypersecretion, and these studies may highlight binding to bacteria (57) or by its association with the bacterial- specific components as potential targets for future therapeutic binding collectin surfactant protein-D (58). Thus, binding of strategies for the treatment of hypersecretory conditions. gp-340, and likely other protective factors, to the gel network increases the functionality of the mucus by facilitating clearance Acknowledgment : The authors acknowledge the substantial contributions to the research reviewed here by Ingemar Carlstedt (University of Lund, Sweden), Paul of sequestered bacteria from the respiratory tract via the muco- Richardson (St. Georges Hospital, London, UK), Paul Nettesheim, Tom Gray, and ciliary escalator or cough. Peter Koo (NIEHS, U.S.A.), and Sara Kirkham, Marj Howard, Nagma Khan, David It is clear from these two examples that nonmucin glycopro- Knight, Bertrand Raynal, and Tim Hardingham (University of Manchester, UK). teins and proteins play key roles in mucosal protection and mucus organization; but while there have been many studies on References the molecular components of mucus, we do not yet know their 1. Phillips GJ, James SL, Lethem MI. Rheological properties and hydration full complement or which of these are associated with the mucins of airway mucus. In Rogers DF and Lethem MI, editors. Airway and what function(s) they perform. These other components may mucus: basic mechanisms and clinical perspectives. Basel: Birkhauser be over- or under-expressed in disease conditions, which would Verlag; 1997. pp. 117–147. 2. Puchelle E, Zahm JM, de Bentzmann S, Gaillard D. In Rogers DF and have an effect on the hosts ability to prevent colonization or Lethem MI, editors. 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Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, water availability of the environment into which they are se- Elias JA. Pulmonary expression of interleukin-13 causes inflammation, creted. This will in turn be affected by the other biomolecules al- mucus hypersecretion, subepithelial fibrosis, physiologic abnormali- ready present in that environment. Aberrant mucin hydration ties, and eotaxin production. J Clin Invest 1999;103:779–788. 8. Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. may be of particular importance in the airways of patients with Neutrophil elastase increases MUC5AC mRNA and protein expres- CF. In CF, the competition for water on the airways epithelium sion in respiratory epithelial cells. Am J Physiol 1999;276:L835–L843. appears to be more severe, and the water imbalance in the 9. Nadel J. 2001. 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16. Sheehan JK, Thornton DJ, Somerville M, Carlstedt I. The structure and 37. Davies JR, Carlstedt I. Respiratory tract mucins. In Salathe M, editor. heterogeneity of respiratory mucus glycoproteins. Am Rev Respir Dis Cilia and mucus. New York: Marcel Dekker; 2001. pp. 167–178. 1991;144:S4–S9. 38. Groneberg DA, Eynott PR, Oates T, Lim S, Wu R, Carlstedt I, Nicholson 17. Thornton DJ, Davies JR, Carlstedt I, Sheehan JK. Structure and bio- AG, Chung KF. Expression of MUC5AC and MUC5B mucins in chemistry of human respiratory mucins. In Rogers DF and Lethem normal and cystic fibrosis lung. Respir Med 2002;96:81–86. MI, editors. Airway mucus: basic mechanisms and clinical perspectives. 39. Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. Hetero- Basel: Birkhauser Verlag; 1997. pp. 19–39. geneity of airways mucus: variations in the amounts and glycoforms 18. Gum JR, Hicks JW, Toribara MW, Siddiki B, Kim YS. Molecular cloning of the major oligomeric mucins MUC5AC and MUC5B. 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Epidermal growth factor system regulates mucin pro- by cultured normal human tracheobronchial epithelial cells. Am J duction in airways. Proc Natl Acad Sci USA 1999;96:3081–3086. Physiol 2000;278:L1118–L1128. 23. Takeyama K, Jung B, Shim JJ, Burgel PR, Pick TD, Ueki IF, Protin U, 45. Gray T, Nettesheim P, Basbaum C, Koo JS. Regulation of mucin gene Kroschel P, Nadel JA. Activation of epidermal growth factor receptors expression in human tracheobronchial epithelial cells by thyroid hor- is responsible for mucin synthesis induced by cigarette smoke. Am J mone. Biochem J 2001;353:727–734. Physiol Lung Cell Mol Physiol. 2001;280:L165–L172. 46. Gray T, Koo PS, Nettesheim P. Regulation of mucous differentiation and 24. Shim JJ, Dabbagh K, Ueki IF, Pick TD, Burgel PR, Takeyama K, Tam mucin gene expression in the tracheobronchial epithelium. Toxicology DCW, Nadel JA. IL-13 induces mucin production by stimulating epi- 2001;160:35–46. dermal growth factor receptors and by activating neutrophils. Am J 47. Sellers A, Allen A. Gastrointestinal mucus rheology. In Chantler E and Physiol Lung Cell Mol Physiol 2001;280:L134–L140. Ratcliffe NA, editors. Mucus and related topics. Cambridge: The Com- 25. Thornton DJ, Carlstedt I, Howard M, Devine PL, Price MR, Sheehan pany of Biologists Limited; 1989. pp. 65–71. JK. Respiratory mucins: identification of core proteins and glycoforms. 48. Raynal BD, Hardingham TE, Thornton DJ, Sheehan JK. Concentrated Biochem J 1996;316:967–975. solutions of salivary MUC5B mucin do not replicate the gel-forming 26. Thornton DJ, Howard M, Khan N, Sheehan JK. Identification of two properties of saliva. Biochem J 2002;362:289–296. 49. Gribbon P, Hardingham TE. Macromolecular diffusion of biological glycoforms of the MUC5B mucin in human respiratory mucus. 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In cal characterization of a low-charge glycoform of the MUC5B mucin Rogers DF and Lethem MI, editors. Airway mucus: basic mechanisms comprising the gel-phase of an asthmatic respiratory mucous plug. and clinical perspectives. Basel: Birkhauser Verlag; 1997. pp. 1–18 Biochem J 1999;338:507–513. 55. Thim L, Madsen F, Poulsen SS. Effect of trefoil factors on the viscoelastic 32. Hovenberg HW, Davies JR, Carlstedt I. Different mucins are produced properties of mucus gels. Eur J Clin Invest 2002;32:519–527. by the surface epithelium and the submucosa in human trachea: identi- 56. Thornton DJ, Davies JR, Kirkham S, Gautrey A, Khan N, Richardson fication of MUC5AC as a major mucin from goblet cells. Biochem J PS, Sheehan JK. Identification of a nonmucin glycoprotein (gp-340) 1996;318:319–324. from a purified respiratory mucin preparation: evidence for an associa- 33. Sheehan JK, Brazeau C, Kutay S, Pigeon H, Kirkham S, Howard M, tion involving the MUC5B mucin. Glycobiology 2001;11:969–977. Thornton DJ. Physical characterization of the MUC5AC mucin: a 57. Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, highly oligomeric glycoprotein whether isolated from cell culture or Frangsmyr L, Holmskov U, Leffler H, Nilsson C, et al. Salivary aggluti- in vivo from respiratory mucous secretions. Biochem J 2000;347:37–44. nin, which binds Streptococcus mutans and Helicobacter pylori,isthe 34. Ota H, Katasuyama T. Alternating laminated array of two types of mucin lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 2000; in the human gastric surface mucous layer. Histochem J 1992;24:86–92. 275:39860–39866. 35. Copin MC, Devisme L, Buisine MP, Marquette CH, Wurtz A, Aubert JP, 58. Holmskov U, Lawson P, Teisner B, Tornoe I, Willis AC, Morgan C, Gosselin B, Porchet N. From normal respiratory mucosa to epidermoid Kock C, Reid BM. Isolation and characterisation of a new member carcinoma: expression of human mucin genes. Int J Cancer 2000;86: of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as 162–168. a lung surfactant protein-D binding molecule. J Biol Chem 1997;272: 36. Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer 13743–13749. NH, Sheehan JK. Salivary mucin MG1 is comprised almost entirely 59. Joo NS, Irokawa T, Wu JV, Robbins RC, Whyte RI, Wine JJ. Absent of different glycosylated forms of the MUC5B gene product. Glyco- secretion to vasoactive intestinal peptide in cystic fibrosis airway biology 1999;9:293–302. glands. J Biol Chem 2002;277:50710–50715.

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60. Ballard ST, Trout L, Mehta A, Inglis SK. Liquid secretion inhibitors worth MA, Engelhardt JF. MUC5B and MUC7 are differentially ex- reduce mucociliary transport in glandular airways. Am J Physiol Lung pressed in mucous and serous cells of submucosal glands in human Cell Mol Physiol 2002;283:L329–L335. bronchial airways. Am J Respir Cell Mol Biol 1998;19:30–37. 61. Moniaux N, Escande F, Batra SK, Porchet N, Laine A, Aubert JP. 64. Sheehan JK, Boot-Handford RP, Chantler E, Carlstedt I, Thornton DJ. Alternative splicing generates a family of putative secreted and mem- Evidence for shared epitopes within the ’naked’ protein domains of brane-associated MUC4 mucins. Eur J Biochem 2000;267:4536–4544. human mucus glycoproteins. Biochem J 1991;274:293–296. 62. Williams SJ, Wreschner DH, Tran M, Eyre HJ, Sutherland GR, McGuckin 65. Thornton DJ, Khan N, Sheehan JK. Identiﬁcation and separation of MA. MUC13, a Novel human cell surface mucin expressed by epithe- mucins and their glycoforms. In Corﬁeld AP, editor. Mucin methods lial and hemopoietic cells. J Biol Chem 2001;276:18327–18336. and protocols; methods in molecular biology series. Totowa, NJ: Hu- 63. Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollings- mana; 2000. pp. 77–86.

Mucin structure, aggregation, physiological functions and biomedical applications. Bansil R. and Turner BS Curr Opin in Colloid & Interface Sci, 11:164 (2006)

Current Opinion in Colloid & Interface Science 11 (2006) 164 – 170 www.elsevier.com/locate/cocis

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 States b 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 2. Molecular biology and biochemical structure of mucins

Mucus is a complex viscous adherent secretion synthesized 2.1. Composition of mucus by specialized goblet cells in the columnar epithelium that lines all of the organs that are exposed to the external environment. Mucus is composed primarily of water (¨95%), but also This includes the respiratory tract, the gastrointestinal tract, the contains salts, lipids such as fatty acids, phospholipids and reproductive tract, and the oculo-rhino-otolaryngeal tracts. It cholesterol [1], proteins which serve a defensive purpose such serves many functions in those locations, among which are as lysozyme, immunoglobulins, defensins, growth factors and lubrication for the passage of objects, maintenance of a hydrated trefoil factors. However, the main component that is respon- layer over the epithelium, a barrier to pathogens and noxious sible for its viscous and elastic gel-like properties is the substances and as a permeable gel layer for the exchange of glycoprotein mucin. gases and nutrients with the underlying epithelium [1,2]. In addition to its protective functions, mucus is also 2.2. Mucins involved in many disease processes. Mucus also is the first barrier with which nutrients and enteric drugs must interact and Mucins are large, extracellular glycoproteins with molecular diffuse through, in order to be absorbed and gain access to the weights ranging from 0.5 to 20 MDa. Both membrane bound circulatory system and their target end organs. mucins, and secreted mucins [3&] share many common features. They are both highly glycosylated consisting of ¨80% carbohydrates primarily N-acetylgalactosamine, N-acetylglucosamine, fucose, galactose, and sialic acid (N-acetylneu- * Corresponding author. Tel.: +1 617 353 2969. E-mail addresses: [email protected](R.Bansil), raminic acid) and traces of mannose and sulfate. The [email protected](B.S.Turner). oligosaccharide chains consisting of 5–15 monomers, exhibit 1 Tel.: +1 617 667 1215. moderate branching and are attached to the protein core by

1359-0294/$ - see front matter D 2005 Published by Elsevier Ltd. doi: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 cluster within ¨500 kb on the short arm of chromosome 11 threonines and arranged in a ‘‘bottle brush’’ configuration about (11p15) [9]. It is thought that they represent homologs that the protein core. diverged from a common ancestor gene on chromosome 11 The protein core, making up the remaining 20% of the [10]. The STP-repeat sequences for each MUC gene are unique molecular mass (¨200–500 kDa), is arranged into distinct to each species, whereas the cys-rich regions share a large regions. First, a central glycosylated region, which is com- degree of similarity [5&]. Different collections of mucin genes prised of a large number of tandem repeats that are rich in are expressed in different tissues. Such repeats and super serine, threonine and proline (STP repeats), which can make up repeats are relatively uncommon in normal proteins. greater than 60% of the amino acids. Second, located at the amino and carboxy terminals, and sometimes interspersed 3. Physical properties of mucin in dilute solution between the STP-repeats, are regions with an amino acid composition more representative of globular proteins, relatively Mucin has been difficult to characterize, owing to its large little O-glycosylation and a few N-glycosylation sites [4] and a molecular weight, polydispersity and high degree of glycosyl- high proportion of cysteine (>10%). These cysteine rich ation. Earlier biophysical studies, reviewed by Bansil et al. [11&] regions contain domains that possess sequence similarity to and Harding [12&] primarily using light scattering methods von Willebrand factor (vWF) C and D domains, and C-terminal showed that mucin was a somewhat stiffened random coil with a cystine knot domains [5&,6,7], and have been shown to be radius of gyration around 100 nm. NMR studies of MUC1 involved in dimerization via disulfide bond formation, and [13,14&] revealed very little alpha helix, a small amount of beta subsequent polymerization of the dimers to form multimers [8] and mostly random coil. The large size of mucin has, on the (Fig. 1). other hand, turned out to be of great advantage in imaging the molecule directly. Earlier transmission electron micrographic 2.3. Mucin genes studies by Fiebrig et al. [15] revealed long fibers approximately 400 nm long in pig gastric mucin (PGM). More recent Atomic Currently, approximately 19 mucin (designated MUC) Force Microscopy (AFM) studies of ocular mucin [16&] show genes have been identified cloned and partially sequenced in individual fibers with a broad distribution of contour lengths. the human, and homologs to many of them have been While most of the fibers are between 200–600 nm long, the tail identified in the mouse and rat [5&]. Only three MUC of the distribution extends to 1500 nm. They also estimated a (MUC1, MUC2 and MUC5B) genes have been totally persistence length of about 36 nm from these images, which sequenced due to the large size of the central tandem repeats, confirms the extended nature of the polymer. In another AFM which are difficult to accurately assemble. Many of the smaller study of ocular mucin, Brayshaw et al. [17] demonstrated the membrane bound mucins are not considered ‘‘true’’ mucins multimeric nature of mucin, by observing in situ depolymer- since they only share the STP repeats and glycosylation with ization on treatment with DTT (dithiothreitol). Longer fibers, up other mucins [3&]. Of the remaining ‘‘true’’ mucins (MUC1–7), to 2 Am in length, were observed in PGM by Deacon et al. [18]. the secreted mucins (MUC 2, 5AC, 5B and 6) are located in a McMaster et al. [19] examined ocular mucin using tapping

Fig. 1. (a) A schematic drawing of the pig gastric mucin (PGM) monomer consisting of glycosylated regions flanked by regions with relatively little glycosylation. (b) & The symbols indicate the different domains in the sketch in (a). (This representation is based in part on Figs. 1 and 2 of Dekker et al. [3 ]). At the bottom of the figure we show (c) a dimer formed by two monomeric subunits linked via disulfide bonds in the non-glycosylated regions and in (d) dimers that are further disulfide linked to form higher multimers. This gives rise to the high molecular weight and polydispersity of secretory mucins. Polymers>16-mers have been described in MUC5AC from human airway secretions by Sheehan et al. [8]. (The bottom part of the figure is adapted from Fig. 8 of Ref. [8].) Reprinted with permission from [21]. 166 R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170 mode AFM under a buffer and observed regular variations in the protein core of PGM as indicated by its increased binding of height along the length of the fiber which they interpret as the fluorescent hydrophobic dye 1-anilinonaphthyl-8-sulfonic glycosylated regions of the mucin molecules. Round et al. [20] acid (ANS). Atomic force microscopy images confirm that correlated the conformations with differing amounts of glyco- while PGM exists as single molecules at pH 6 (about 400 nm in sylation by imaging different fractions obtained on a CsCl length), it forms aggregates below pH 4 [21,28]. The viscosity gradient. However the sugar chains are too mobile in their increase and gelation are not observed if PGM is broken into its solvated state to be seen fully extended in an AFM image, subunits (by treating with enzymes such as pronase or disulfide which requires a high degree of immobilization on the substrate, reduction with DTT) or salt concentration is increased. the AFM tip might have penetrated the brush without sensing it. Commercial preparations of PGM available from Sigma AFM images of PGM which was about 50% deglycosylated chemicals (which include protease treatment during purifica- showed that the deglycosylated portions re-folded forming tion) also do not gel [28]. compact globular structures [21]. Thus the sugars are important for maintaining the extended conformation of mucin. 4.2. Model for pH induced gelation of PGM The conformation of mucin also depends on factors such as pH and ionic strength. For example, dynamic light scattering This accumulated data showing a sharp increase in the (DLS) studies [22] have shown that as the pH is lowered in observed changes at pH 4 and the lack of gelation in PGM dilute solutions (<5 mg/ml) PGM undergoes a conformational which is broken into the subunits has led us to suggest that change from an isotropic random coil (with end to end length PGM gelation involves interactions of side chains of amino of 390 nm and persistence length of 8–10 nm) at pH 7 to an acids with pKs around 4, such as Asp or Glu from the non- anisotropic extended random coil (with end-to-end length of glycosylated regions of the molecule. At neutral pH of 6–7 490 nm, persistence length 43 nm) at pH 2. This is paralleled these non-glycosylated Cys-rich regions of PGM are in by an increase in the sedimentation coefficient from 11S at pH conformations with hydrophobic domains hidden in folds of 7 to greater than 31S at pH 3 [23]. In a recent paper the stabilized by salt bridges between negatively charged carbox- conformation of commercially available PGM from Sigma in ylates and positively charged amino groups. At acidic pH 2 the dilute solution examined by viscosity and circular dichroism carboxylates of the salt bridges are protonated, breaking the salt measurements reveals similar changes as function of pH which bridges and allowing the unfolding and exposure of hydro- are attributed to the unfolding of hydrophobic domains at low phobic regions of protein (Fig. 2). Circular dichroism studies pH [24]. These authors also measured the zeta (1) potential of reported by Lee et al. [24] on pig gastric mucin from Sigma mucin and found that its isoelectric point lies between 2 and 3, also confirm the conformational transition at pH 4 and below. suggesting that the charge on the molecule varies as pH is The hydrophobic domains on adjacent molecules are then able lowered. to associate acting as the crosslinks of a gel. Hydrophobic interactions among protein domains were also shown to be 4. Colloidal properties of mucin involved in aggregation of human tracheobronchial mucin [25]. The gelation of mucin is a complex problem, involving the 4.1. Aggregation and gelation interplay of electrostatic and hydrophobic interactions, somewhat reminiscent of the association of multiblock copolymers Mucins exhibit a tendency to aggregate and form gels. in solvents that have differential solubility to the component Taylor et al. [23] used rheological techniques to investigate the blocks. The entanglement of the sugar side chains further structure and formation of the pig gastric mucus gel and contributes to the high viscosity of mucin solutions (Fig. 2). showed that both transient and non-transient interactions are responsible in maintaining the gel matrix. Purified mucin also 4.3. Liquid crystalline properties of mucin has been shown to form gels. Human tracheobronchial mucin forms a gel below 30 -C at concentrations above 14 mg/mL Mucin glycoprotein has also been shown to exhibit liquid [25]. Gelation was also observed in canine submaxillary mucin crystalline order. Viney, et al. [30] had shown that slug mucin in the chaotropic (denaturing) solvent guanidine HCl [26]. forms nematic liquid crystals. A detailed study of the These authors suggest that since non-covalent interactions are temperature and concentration dependence of the liquid destabilized by guanidine HCl, gelation in these high molecular crystalline behavior of commercially available mucin from weight fractions involves the interpenetration of the carbohy- Sigma was reported by Davis et al. [31] who suggest that the drate side chains [26]. interactions of interdigitated sugar side chains are responsible Our laboratory has focused on gelation of gastric mucin at for the nematic behavior. Waigh et al. [32] confirmed these low pH, which has implications for the physiologically relevant observations by neutron scattering, comparing Sigma mucin at question of how the stomach is prevented from being digested high concentrations in the presence and absence of an external by the acidic gastric juice it secretes. Bhaskar et al. [27] showed magnetic field to produce orientation. Purified PGM, on the a reversible increase in viscosity of aqueous solutions of pig other hand, did not show this liquid crystalline orientation, gastric mucin (PGM) at pH 2. DLS studies [22] show that at suggesting that the Sigma mucin, which consists of a single concentrations above 10 mg/ml gelation occurs below pH 4. At mucin monomer, is more rigid and easier to orient than the low pHs we also observed an increase in the hydrophobicity of multimeric form of native PGM in which the non-glycosylated R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170 167

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 coiltoa 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, design of drugs which have to diffuse through the mucus layer, monomeric domains. or kept from entering it. While many small molecules diffuse readily through mucus, the diffusion of larger particles depends 4.4. Adhesive properties of mucin both on size and muco-adhesive interactions. The diffusion of latex particles and lipid vesicles in purified gall bladder mucin The well know tendency of other substances to adhere to has been examined using DLS [38] and fluorescence recovery mucin, known as mucoadhesivity, is not surprising given that after photobleaching [39] methods. Olmsted et al. [40] showed this glycoprotein exhibits electrostatic, hydrophobic, and H- that while many small viruses (20–200 nm) could diffuse bonding interactions [33]. By the same token the molecule can through cervical mucus, others such as the Herpes Simplex be anti-adhesive, for example to negatively charged species. virus got stuck to the mucus. Celli et al. [41] have exploited the Thus mucin coated AFM tips adhered to mica in presence of movement of latex particles through mucin solutions to divalent cations [34] but did not adhere to mica coated with measure its rheological properties as a function of pH using mucin, presumably due to the polyelectrolytic charge repul- microscope based DLS. sion. Bovine submaxillary mucin from Sigma could be Finally, it is worth noting that the transport of water and adsorbed to hydrophobic polystyrene surface rendering it other fluids through mucus and purified mucin solutions is a hydrophilic [35]. Mucin lipid interactions are well known, very interesting problem in fluid dynamics. Due to its high reflecting the presence of hydrophobic domains [36]. In this viscosity, water and other low viscosity fluids injected under a context, it is worth noting that since mucin is negatively pressure gradient across an unstirred mucin solution do not charged it binds to positive ions. For example, Chromium III simply diffuse, but are transported by a viscous fingering binding has been shown to cause significant conformational mechanism [42,43]. This transport of HCl through ‘‘channels’’ changes and it can lead to aggregation of mucin [37]. produced by the viscous fingering mechanism has also been demonstrated in vivo in rats [44]. The mucus in the 4.5. Diffusion of macromolecules and particles and fluid flow immediate vicinity of the surface of the acid channel will through mucin gel, thereby confining the acid in a ‘‘tube’’. Similarly the mucus layer on the luminal surface of the stomach will gel as In view of the protective function of mucus studying the the acid is emptied into the lumen. The negatively charged diffusion of other molecules through it is of considerable mucin gel prevents back diffusion of H+ via the mechanism physiological interest. It is also an important factor in the of Donnan equilibrium. 168 R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170

5. Biomedical function and applications polymeric viewpoint mucin can be considered as a multiblock copolymer with alternating polyelectrolytic domains having a 5.1. Mucus and disease grafted sugar brush, connected by flexible regions with less glycosylation and the tendency to form multimers. The The physical state of the mucus, change in the concentra- molecule has both hydrophobic and hydrophilic regions with tion of secreted mucin, and the strong dependence of its the ability to form H-bonds, and electrostatic interactions. This physicochemical properties on environmental factors such as wide range of interactions causes it to aggregate gel and form ionic strength and pH play an important role in many diseases. mucoadhesive interactions with other substances. These For example, besides serving as a barrier to bacteria, many physical properties are of direct relevance to the physiological bacteria reside within the mucus and possess specific adhesins functions of mucus in normal and diseased states. An that specifically bind to it [45]. This includes pathogenic understanding these properties is of considerable current strains of Pseudomonas, Streptococcus and Pneumococcus. interest because of the many biomedical applications. Helicobacter pylori particularly resides in the mucus layer of [Note: Topics that are not covered in this review such as the stomach, and is a common cause of ulcers [46].Some mucin glycosylation [58], signal transduction [59],and parasitic organisms also produce their own layers of mucus to secretion [60], can be found in the indicated references.] evade the immune system [47]. Second, overproduction of mucus is involved in cystic fibrosis [48&], bronchitis, asthma Acknowledgements [49] and in middle ear infections, and mucus gels serve as the matrix in which gallstones are nucleated and grow [50]. The authors would like to thank Dr. Nezam H. Afdhal, Dr. Thirdly, mucus underproduction is present in dry eye K. Ramakrishnan Bhaskar, Dr. XingXiang Cao, Prof. Bernard syndromes [51&] and in some forms of ulcer disease. Finally, Chasan, and Dr. Zhenning Hong for our long-standing mucus expression and composition is altered in cancers of collaboration on studies of mucin. Our research on mucin has epithelial origin [52&&]. been supported by grants from NIH (P.I. Dr. N.H. Afdhal) and NSF (P.I. R.B.). 5.2. Mucus and pharmacology References and recommended readings Mucus is the first barrier with which nutrients and enteric [1] Allen A. Structure and function of gastrointestinal mucus. In: Johnson L, drugs must interact and diffuse through, in order to be absorbed editor. Physiology of the gastroenterology tract, 1st edn. New York, NY’ and gain access to the circulatory system and their target end Raven Press; 1981. p. 617–39. organs. There is great interest in methods to optimize these so- [2] Neutra M, Forstner J. Gastrointestinal mucus: synthesis, secretion, and called muco-adhesive interactions for improved drug delivery. function. In: Johnson L, editor. Physiology of the gastrointestinal tract, ’ Various molecular interactions have been exploited to enhance 2nd edn. New York, NY Raven Press; 1987. Chapter 34. [3] Dekker J, Rossen J, Bu¨ller H, Einerhand A. The MUC family: an obituary. & mucoadhesion, including, polyelectrolytic interactions (chito- Trends Biochem Sci 2002;27(3):126–31. sans, poly-acrylic acid, etc.) hydrogen bonds (hydrogels), [33] Review article discussing the controversies concerning the defining char- and disulfide binding (thiomers) [53]. Efforts are underway to acteristics of the MUC family. Contains clear schematic illustrations of the design pH sensitive drug carriers such as gels which will not two major groups of mucins and their structural domains. release the drug in the acidic environment of the stomach but [4] Bell S, Xu G, Khatri I, Wang R, Rahman S, Forstner J. N-linked oligosaccharides play a role in disulphide-dependent dimerization of will do so in the basic environment of the intestine and colon. intestinal mucin Muc2. Biochem J 2003;373(Pt 3):893–900. In this context, we emphasize recent studies [54] which show [5] Perez-Vilar J, Hill R. The structure and assembly of secreted mucins. & that the resting stomach has a pH close to 4 which drops to 2 J Biol Chem 1999;274(45):31751–4. Review article which contains during active acid secretion. Mucin can be used as a high numerous sequence alignments between the various homologous regions molecular weight additive to improve the adherence of artificial of the different mucin genes. [6] Turner B, Bhaskar K, Hadzopoulou-Cladaras M, LaMont J. Cysteine-rich tear drops in treating dry eye syndrome [55]. Efforts to develop regions of pig gastric mucin contain von Willebrand factor and cystine nanoparticles for mucosal DNA vaccines and gene therapy are knot domains at the carboxyl terminal. Biochim Biophys Acta 1999; also being considered [56]. The abnormality of the sugars in 1447(1):77–92. cell membrane bound mucins from cancerous cells has also [7] Bell S, Xu G, Forstner J. Role of the cystine-knot motif at the C-terminus been targeted as a potential for cancer vaccine development. of rat mucin protein Muc2 in dimer formation and secretion. Biochem J 2001;357(Pt1):203–9. Excellent reviews of oral mucosal drug delivery are collected in & [8] Sheehan J, Kirkham S, Howard M, Woodman P, Kutay S, Brazeau C, et al. Ref. [57 ]. Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin. J Biol Chem 2004;279(15):15698–705. 6. Conclusions [9] Pigny P, Guyonnet-Duperat V, Hill A, Pratt W, Galiegue-Zouitina S, D’Hooge M, et al. Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes. Genomics 1996; We have reviewed recent, as well as some pertinent older 38(3):340–52. 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 & Of special interest. organization into domains with different structures. From the && Of outstanding interest. R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170 169

[10] Desseyn J, Aubert J, Porchet N, Laine A. Evolution of the large secreted [32] Waigh T, Papagiannopoulos A, Voice A, Bansil R, Unwin A, Dewhurst C, gel-forming mucins. Mol Biol Evol 2000;17(8):1175–84. et al. Entanglement coupling in porcine stomach mucin. Langmuir [11] Bansil R, Stanley E, LaMont J. Mucin biophysics. Annu Rev Physiol 2002;18(19):7188–95. & 1995;57:635–657. [33] Harding S, Davis S, Deacon M, Fiebrig I. Biopolymer mucoadhesives. This review describes the various biophysical methods that have been used to Biotechnol Genet Eng Rev 1999;16:41–86. elucidate mucin structure and function. [34] Berry M, McMaster T, Corfield A, Miles M. Exploring the molecular [12] Harding SE. The macrostructure of mucus glycoproteins in solution. Adv adhesion of ocular mucins. Biomacromolecules 2001;2(2):498–503. & Carbohydr Chem Biochem 1989;47:345–81. Excellent review of the [35] Shi L, Caldwell K. Mucin adsorption to hydrophobic surfaces. J Colloid solution properties of mucins. Interface Sci 2000;224(2):372–81. [13] Fontenot J, Tjandra N, Bu D, Ho C, Montelaro R, Finn O. Biophysical [36] Rogunova M, Blackwell J, Jamieson A, Pasumar-Thy M, Gerken T. characterization of one-, two-, and three-tandem repeats of human mucin Effects of lipids on the structure and rheology of gels formed by canine (muc-1) protein core. Cancer Res 1993;53(22):5386–94. submaxillary mucin. Biorheology 1997;34(4–5):295–308. [14] Otvos L, Cudic M. Conformation of glycopeptides. Mini Rev Med Chem [37] Shrivastava H, Nair B. Structural modification and aggregation of & 2003;3(7):703–711. mucin by chromium (III) complexes. J Biomol Struct Dyn A review article that describes the biophysical and computational 2003;20(4):575–87. techniques that have been employed to determine the structures of the [38] Cao X, Bansil R, Gantz D, Moore EW, Niu N, Afdhal N. Diffusion glycosylated regions of mucins and other glycoproteins. behavior of lipid vesicles in entangled polymer solutions. Biophys J 1997; [15] Fiebrig I, Harding S, Rowe A, Hyman S, Davis S. Transmission electron 73(4):1932–9. microscopy studies on pig gastric mucin and its interactions with chitosan. [39] Afdhal NH, Cao X, Bansil R, Hong Z, Thompson C, Brown B, et al. Carbohydr Polym 1995;28(3):239–44. Interaction of mucin with cholesterol enriched vesicles: role of mucin [16] Round A, Berry M, McMaster T, Stoll S, Gowers D, Corfield A, et al. structural domains. Biomacromolecules 2004;5(2):269–75. & Heterogeneity and persistence length in human ocular mucins. Biophys J [40] Olmsted S, Padgett J, Yudin A, Whaley K, Moench T, Cone R. Diffusion 2002;83(3):1661–70. of macromolecules and virus-like particles in human cervical mucus. Research article which uses atomic force microscopy in order to obtain a Biophys J 2001;81(4):1930–7. measure of the sizes and size distributions of mucin molecules. [41] Celli J, Gregor B, Turner B, Afdhal N, Bansil R, Erramilli S. Viscoelastic [17] Brayshaw D, Berry M, McMaster T. Reducing a polymer to its properties and dynamics of porcine gastric mucin. Biomacromolecules subunits as an aid to molecular mapping. Nanotechnology 2004;15(11): 2005;6(3):1329–33. 1391–6. [42] Bhaskar K, Garik P, Turner B, Bradley J, Bansil R, Stanley H, et al. [18] Deacon M, McGurk S, Roberts C, Williams P, Tendler S, Davies M, et al. Viscous fingering of HCl through gastric mucin. Nature 1992;360(6403): Atomic force microscopy of gastric mucin and chitosan mucoadhesive 458–61. systems. Biochem J 2000;348(Pt 3):557–63. [43] Fujita T, Ohara S, Sugaya T, Saigenji K, Hotta K. Effects of rabbit [19] McMaster T, Berry M, Corfield A, Miles M. Atomic force microscopy of gastrointestinal mucins and dextran on hydrochloride diffusion in vitro. the submolecular architecture of hydrated ocular mucins. Biophys J Comp Biochem Physiol B 2000;126(3):353–9. 1999;77(1):533–41. [44] Johansson M, Synnerstad I, Holm L. Acid transport through channels in [20] Round A, Berry M, McMaster T, Corfield A, Miles M. Glycopolymer the mucous layer of rat stomach. Gastroenterology 2000;119:1297–304. charge density determines conformation in human ocular mucin gene [45] Scharfman A, Lamblin G, Roussel P. Interactions between human products: an atomic force microscope study. J Struct Biol 2004;145(3): respiratory mucins and pathogens. Biochem Soc Trans 1995;23(4):836–9. 246–53. [46] Slomiany B, Slomiany A. Role of mucus in gastric mucosal protection. [21] Hong Z, Chasan B, Bansil R, Turner B, Bhaskar K, Afdhal N. Atomic J Physiol Pharmacol 1991;42(2):147–61. force microscopy reveals aggregation of gastric mucin at low pH. [47] Jain M, Karan D, Batra S, Varshney G. Mucins in protozoan parasites. Biomacromolecules 2005;6(6):3458–66. Front Biosci 2001;6:D1276–83. [22] Cao X, Bansil R, Bhaskar K, Turner B, LaMont J, Niu N, et al. pH- [48] Boucher R. New concepts of the pathogenesis of cystic fibrosis lung & dependent conformational change of gastric mucin leads to sol–gel disease. Eur Respir J 2004;23(1):146–58. transition. Biophys J 1999;76(3):1250–8. Review article of the role of many factors such as ions, bacterial infection, [23] Taylor C, Allen A, Dettmar P, Pearson J. The gel matrix of gastric mucus fluid dynamics and their relationship to and effect on mucus in cystic is maintained by a complex interplay of transient and nontransient fibrosis. associations. Biomacromolecules 2003;4(4):922–7. [49] Rose M. Mucins: structure, function and role in pulmonary diseases. Am J [24] Lee S, Mu¨ller M, Rezwan K, Spencer N. Porcine Gastric Mucin (PGM) at Physiol 1992;263(4 Pt 1):L413–29. the Water/Poly(dimethylsiloxane) (PDMS) Interface: influence of pH and [50] Smith B, LaMont J: The Pathogenesis of Gallstones. Hospital Practice. ionic strength on its conformation, adsorption, and aqueous lubrication 1984. 19(12): 93–99, 103–104. properties. Langmuir 2005;21(18):8344–53. [51] Argu¨eso P, Gipson I. Epithelial mucins of the ocular surface: structure, & [25] Bromberg L, Barr D. Self-association of mucin. Biomacromolecules biosynthesis and function. Exp Eye Res 2001;73(3):281–9. 2000;1(3):325–34. Review of the basic elements of mucin structure and regulation with special [26] McCullagh C, Gupta R, Jamieson A, Blackwell J. Gelation of fractionated reference to the surface of the eye in the normal and disease states. canine submaxillary mucin in a chaotropic solvent. Int J Biol Macromol [52] Hollingsworth M, Swanson B. Mucins in cancer: protection and control && 1996;18(4):247–53. of the cell surface. Nat Rev Cancer 2004;4(1):45–60. [27] Bhaskar K, Gong D, Bansil R, Pajevic S, Hamilton J, Turner B, et al. An excellent comprehensive review of both secreted and membrane mucins, Profound increase in viscosity and aggregation of pig gastric mucin at low their possible functions in signal transduction. It also compares the roles of pH. Am J Physiol 261(5 Pt 1):G827–32. mucins in normal and cancerous states and also contains very informative [28] Koe`evar-Nared J, Kristl J, Sˇmid-Korbar J. Comparative rheological illustrations. investigation of crude gastric mucin and natural gastric mucus. Biomater- [53] Leitner V, Walker G, Bernkop-Schnurch A. Thiolated polymers: evidence ials 1997;18(9):677–81. for the formation of disulphide bonds with mucus glycoproteins. Eur J [29] Cao X. Aggregation and gelation of mucin and its interactions with lipid Pharm Biopharm 2003;56(2):207–14. vesicles. Ph. D Dissertation, Boston University, Boston MA 1997. [54] Baumgartner H, Montrose M. Regulated alkali secretion acts in tandem [30] Viney C, Huber A, Verdugo P. Liquid-crystalline order in mucus. with unstirred layers to regulate mouse gastric surface pH. Gastroenter- Macromolecules 1993;26(4):852–5. ology 2004;126(3):774–83. [31] Davies J, Viney C. Water-mucin phases: conditions for mucus liquid [55] Khanvilkar K, Donovan M, Flanagan D. Drug transfer through mucus. crystallinity. Thermochim Acta 1998;315(1):39–49. Adv Drug Deliv Rev 2001;48:173–93. 170 R. Bansil, B.S. Turner / Current Opinion in Colloid & Interface Science 11 (2006) 164–170

[56] Dawson M, Krauland E, Wirtz D, Hanes J. Transport of polymeric [59] Basbaum C, Lemjabbar H, Longphre M, Li D, Gensch E, McNamara N. nanoparticle gene carriers in gastric mucus. Biotechnol Prog 2004;20(3): Control of mucin transcription by diverse injury-induced signaling 851–7. pathways. Am J Respir Crit Care Med 1999;160:544–9. [57] Rathbone MJ, editor. Oral mucosal drug delivery. Marcel Dekker Inc; [60] Verdugo P. Goblet cells secretion and mucogenesis. Annu Rev Physiol & 1993. 1990;52:157–76. This book has excellent contributions by many authors and provides a comprehensive review of the issues related to mucus in oral drug delivery. [58] Van den Steen P, Rudd P, Dwek R, Opdenakker G. Concepts and priciples of O-linked glycosylation. Crit Rev Biochem Mol Biol 1998; 33(3):151–208.

Mucins in the mucosal barrier to infection. Linden et al, Nature Immunology 1: 183 (2008)

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Mucins in the mucosal barrier to infection

S K L i n d e n 1, P S u t t o n 2, N G K a r l s s o n 3, V K o r o l i k 4 a n d M A M c G u c k i n 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.

MUCINS — AN INTEGRAL PART OF THE MUCOSAL BARRIER respiratory tract cilia drive its movement. Mucin glycoproteins Mucosal epithelial tissues have evolved multiple mechanisms produced by mucus-producing cells in the epithelium or submu- of defense in response to their vulnerability to microbial attack cosal glands are the major macromolecular constituent of mucus due to their exposure to the external environment. The mucosal and are responsible for the viscous properties of the mucus gel. epithelial cells form a contiguous lining that acts as a barrier In addition to forming a relatively impervious gel, which acts as between the moist exterior environment and the remainder a lubricant, a physical barrier, and a trap for microbes, mucus of the host. In addition, these cells, both constitutively and in provides a matrix for a rich array of antimicrobial molecules. response to microbes, together with underlying leukocytes, Underneath the mucus layer, the cells present a dense forest secrete many defensive compounds into the mucosal fluid, of highly diverse glycoproteins and glycolipids, which form the including mucins, antibodies, defensins, protegrins, collectins, glycocalyx. Membrane-anchored cell-surface mucin glyco- cathlecidins, lysozyme, histatins, and nitric oxide. 1 – 3 Together, proteins are a major constituent of the glycocalyx in all mucosal these different defensive compounds form a physical barrier and tissues. The glycocalyx is highly variable from tissue to tissue; have direct antimicrobial activity, and the ability to opsonize for example, the glycocalyx of human intestinal microvilli tips microbes to aid clearance. Mucin glycoproteins, however, can is thick (100 – 500 nm) in comparison with the glycocalyx of the fulfill all of these roles individually. lateral microvilli surface (30– 60 nm). 9,10 The oligosaccharide Mucosal pathogens, almost by definition, have evolved mecha- moieties of the molecules forming the glycocalyx and the mucus nisms to subvert these mucosal defensive measures. The first layer are highly diverse, and the average turnover time of the barrier the pathogen encounters is the highly hydrated mucus human jejunal glycocalyx is 6– 12 h. 11 Consequently, both the gel that covers the mucosal surface and protects the epithelial secreted and adherent mucosal barriers are constantly renewed cells against chemical, enzymatic, microbial, and mechanical and could potentially be rapidly adjusted to changes in the insult. Mucosal surfaces are coated with a layer of viscous mucus environment, for example, in response to microbial infection. ranging in thickness from 10 m in the eye 4 and trachea 5 t o 300 m in the stomach and 700 m in the intestine.6 – 8 This MUCIN BIOSYNTHESIS AND STRUCTURE mucus layer is not static but moves to clear trapped material. In The tremendous energy investment by mucosal tissues in the the gastrointestinal tract, the outer mucus layer is continually production of mucins in the basal state, but particularly in removed by movement of the luminal contents, whereas in the response to infection, is testimony to the importance of these

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

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glycoproteins. Epithelial mucins are a heterogenous family Table 1 Tissue distribution of mucins of large complex glycoproteins containing a dense array of O-linked carbohydrates typically comprising over 70% of their Mucin Distribution References mass. These glycans are concentrated in large peptide domains Secreted gel forming of repeating amino-acid sequences rich in serine and threonine. MUC2 Small intestine, colon, respiratory 231 – 235 The size and number of repeats vary between the mucins, and tract, eye, middle ear epithelium in many of the genes there are genetic polymorphisms in the MUC5AC Respiratory tract, stomach, cervix, 235 – 239 number of repeats (variable number of tandem repeats or VNTR eye, middle ear epithelium polymorphisms), which means the size of individual mucins MUC5B Respiratory tract, salivary glands, 235,236, cervix, gallbladder, seminal fluid, 240 – 244 can differ substantially between individuals. Each mucin is middle ear epithelium thought to form a filamentous protein carrying typically 100s 12 MUC6 Stomach, duodenum, gallbladder, 235,243, of complex oligosaccharide structures, giving the mucin a pancreas, seminal fluid, cervix, 245 – 247 “ bottle-brush ” appearance. To date, at least 16 human mucins have middle ear epithelium been included in the family, and the expression profile of mucins MUC19 Sublingual gland, submandibular 16,235,248 varies between tissues with the gastrointestinal tract showing the gland, respiratory tract, eye, highest and most diverse expression (see Table 1 ) . middle ear epithelium Mucins can be divided into three distinct subfamilies: (a) secreted gel-forming mucins, (b) cell-surface mucins, and Secreted non-gel forming (monomeric) (c) secreted non-gel-forming mucins (Table 1 ). Gel-forming MUC7 Salivary glands, respiratory tract, 235,249,250 mucins, which are the major constituent of mucus and con- middle ear epithelium fer its viscoelastic properties, are encoded by a cluster of four 13– 15 highly related genes on chromosome 11 and a similar gene Cell surface on chromosome 12.16 Gel-forming mucins contain N- and MUC1 Stomach, breast, gallbladder, 235,251 – C-terminal cysteine-rich domains that are both involved in cervix, pancreas, respiratory tract, 256 homo-oligomerization mediated by inter-molecular disulfide duodenum, colon, kidney, eye, bonds.17,18 The current model for mucin oligomerization is that B cells, T cells, dendritic cells, dimerization occurs rapidly during biosynthesis in the endoplas- middle ear epithelium mic reticulum preceding or concomitant with N -glycosylation MUC3A/B Small intestine, colon, gall 235,243, bladder, duodenum, middle ear 257,258 but before O-glycosylation in the Golgi apparatus, which in turn epithelium is followed by multimerization of dimers.19 Oligomerization is MUC4 Respiratory tract, colon, stomach, 235,255, likely to produce either extended filamentous structures or, more cervix, eye, middle ear epithelium 259 – 261 probably, web-like molecular structures likely to be critical to the 20– 24 MUC12 Colon, small intestine, stomach, 32,262 rheological properties of the mucus gel. The extended con- pancreas, lung, kidney, prostate, formation caused by dense glycosylation enables the molecules uterus to occupy large volumes, with the secreted oligomeric mucins MUC13 Colon, small intestine, trachea, 35,235,262 occupying volumes equivalent to those of small bacteria.25 The kidney, appendix, stomach, secreted non-oligomerizing mucins include the MUC7 salivary middle ear epithelium mucin, which can self-aggregate but is not thought to contribute MUC15 spleen, thymus, prostate, testis, 235,263 significantly to mucus properties, and the MUC8 respiratory ovary, small intestine, colon, peripheral blood leukocyte, bone mucin, which has not been fully characterized to date. marrow, lymph node, tonsil, There are 11 known genes encoding cell-surface mucins breast, fetal liver, lungs, middle expressed by a wide diversity of mucosal tissues, with substantial ear epithelium redundancy in many tissues (see Table 1 ). Cell-surface mucins MUC16 Peritoneal mesothelium, 235,264 – are present on the apical membrane of all mucosal epithelial reproductive tract, respiratory 267 cells and contain large extracellular VNTR domains predicted tract, eye, middle ear epithelium to form rigid elongated structures. Together with their high MUC17 Small intestine, colon, duodenum, 235,268 stomach, middle ear epithelium expression, this indicates that these molecules are likely to be a prominent, probably dominating, constituent of the glycocalyx MUC20 Kidney, placenta, colon, lung, 235,269 prostate, liver, middle ear and may provide a barrier that limits access of other cells and epithelium 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 extracellular -subunit can be shed from the cell surface either the MUC1 SEA module suggest that the cleavage occurs via mediated via a second distinct cleavage event 28,29 o r p e r h a p s autoproteolysis and that the two subunits remain non-covalently via physical shear forces separating the two domains at the associated throughout biosynthesis.27 However, importantly, the original cleavage site as suggested by Macao et al . 27 M u t a t i o n

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of the cleavage site inhibits MUC1 shedding in transfected was not seen in isogenic mutants lacking cytolethal distending mammary and respiratory epithelial cells without affecting cell toxin, indicating that Muc1 lowers gastric colonization at least surface expression, indicating the importance of the initial cleav- in part via inhibiting the activity of cytolethal distending toxin. 55 age in shedding. 30 Furthermore, most cell-surface mucin genes Many of the other cell-surface mucins also contain potential appear to undergo alternative splicing and also encode directly phosphorylation sites and cleavage motifs in the immediate secreted isoforms lacking transmembrane and cytoplasmic intracellular region of their cytoplasmic domains and may be domains. 31– 34 These isoforms are stored in subapical granules similarly cleaved. Importantly, there is also evidence that inter- and in goblet cell thecae and are secreted both constitutively action with bacteria can induce phosphorylation of MUC1 and following stimuli. Consequently, due to shedding and direct in vitro .57 Signaling by the cytoplasmic domains of cell-surface secretion, cell-surface mucins can also be seen as components mucins is complex and much remains to be elucidated about of secreted mucus. 35 their mode of action. However, the evidence to date suggests MUC1 is the most extensively studied membrane-associ- that these domains are involved in cellular programs regulating ated mucin and is the most ubiquitously expressed across all growth and apoptosis in mucosal cells perhaps in response to mucosal tissues. MUC1 has been estimated to be 200– 500 nm in microbes and / or their toxins. length (depending on the number of tandem repeats), suggesting it will tower above other molecules attached to the plasma MUCIN GLYCOSYLATION membrane.36 MUC1 associated with the cell surface is The carbohydrate structures present on mucosal surfaces vary constantly internalized (0.9 % of the surface fraction min − 1 ) a n d according to cell lineage, tissue location, and developmen- recycled. 37 Internalization occurs by clathrin-mediated endo- tal stage. Evidence is emerging that mucin glycosylation can cytosis, and alterations in O- glycan structure stimulate endo- alter in response to mucosal infection and inflammation, and cytosis and intracellular accumulation. 38 During recycling, this may be an important mechanism for unfavorably chang- sialic acid is added to the premature form of MUC1. 37 Complete ing the niche occupied by mucosal pathogens. The extensive sialylation requires several rounds of recycling, one cycle taking O- glycosylation of the mucins protects them from proteolytic approximately 2.5 h. 37 Pulse-chase experiments indicate that the enzymes and induces a relatively extended conformation. 25 T h e half-life of MUC1 in the plasma membrane is 16 – 24 h,37,39 s u g - oligosaccharides on secreted mucins are clustered into heavily gesting that the average MUC1 molecule recycles up to 10 times glycosylated domains (typically 600– 1,200 amino acids long) before release. 37 Recycling rates vary between cell lines and pos- separated by shorter nonglycosylated regions.25 The O- linked sibly environmental conditions and have not been measured in glycans contain 1 – 20 residues, which occur both as linear and non-transformed cells, making it very difficult to extrapolate to branched structures (see Table 2 ). The carbohydrate chain is the real rate of recycling and cell-surface half-life in vivo . T h e initiated with an N- acetylgalactosamine (GalNAc) linked cytoplasmic tail appears to interact with the cytoskeleton and to serine or threonine and is elongated by the formation of secondary signaling molecules,40 – 42 whereas the extracellular the so-called core structures followed by the backbone region domains of MUC1 and other cell-surface mucins interact with (type-1 and type-2 chains). The chains are typically terminated extracellular matrix components and other cells.43 – 47 by fucose (Fuc), galactose (Gal), GalNAc, or sialic acid residues in The cytoplasmic domains of the cell surface mucins are com- the peripheral region, forming histo-blood-group antigens such plex, often contain known phosphorylation motifs, and are as A, B, H, Lewis-a (Lea ), Lewis-b (Le b ), Lewis-x (Lex ), Lewis-y highly conserved across species, suggesting important intra- (Ley ), as well as sialyl-Lea and sialyl-Le x structures. Sulfation cellular functions. The best-studied mucin in this regard is of Gal and N- acetylglucosamine (GlcNAc) residues causes MUC1, which has been explored mainly in terms of its role further diversification. In addition to the O -linked glycans, mucins in cancer cell biology rather than in mucosal defense. We contain a smaller number of N -linked oligosaccharides, which and others have shown phosphorylation of the MUC1 cyto- have been implicated in folding, oligomerization (MUC2), or plasmic domain 41,48– 52 as well as molecular association with surface localization (MUC17). 58 – 60 -catenin, 41,51 linking MUC1 with the Wnt pathway, which The carbohydrate structures present on mucins are deter- is involved in epithelial growth, migration, and wound repair. mined by the expression of specific glycosyl transferases. Thus, More recently, it has been shown that the cytoplasmic domain mucin glycosylation is governed by genetics (due to polymor- can be cleaved and that the cleaved domain translocates to mito- phisms in these enzymes), tissue-specific enzyme expression, chondria and, together with the p53 transcription factor, to the and host and environmental factors influencing transferase nucleus, where it modulates the cell cycle and protects against expression. As an example of the impact of host genotype, the the apoptotic response to genotoxic stress.53,54 Some pathogenic H type-1 structure is made by the secretor (Se) gene product, and bacteria produce genotoxins, and thus this protective effect, first the majority (80 % of Caucasians, all South American Indians identified in cancer cells, may have evolved as part of the natural and Orientals) carry this structure and are thus referred to as epithelial defense against microbial genotoxins. We have recently “ s e c r e t o r s ” . 61 Individuals may also express the Lewis (Le) gene shown that in vitro MUC1 protects p53-expressing epithelial (90 % of the Caucasian population) and, provided that they are cells from the effects of cytolethal distending toxin, a genotoxin also secretors, will modify H type-1 into the Le b s t r u c t u r e .61,62 produced by Campylobacter jejuni .55,56 In vivo , C. jejuni m o r e If they are nonsecretors, type-1 chains without its blood group densely colonized the stomachs of Muc1 − / − mice, but this effect antigen H will be turned into Le a structures.61,62 The third

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Table 2 Common O -linked oligosaccharide structures on of an enzymatically weak Se-transferase in combination with mucins an intact Le-transferase. 63,64 The terminal structures of mucin oligosaccharides are highly heterogeneous and vary between / Nomenclature Structure within species and between and even within tissues. The array Core type of oligosaccharide structures on individual mucin molecules is Core 1 -Gal 1-3GalNAc 1-Ser/Thr also somewhat determined by stochastic events as the mucin Core 2 -Gal 1-3(-GlcNAc 1-6)GalNAc 1- protein moves through the Golgi apparatus. 65 This structural Ser/Thr diversity may allow the mammalian host to cope with diverse Core 3 -GlcNAc 1-3GalNAc 1-Ser/Thr and rapidly changing pathogens, as reflected by the observation Core 4 -GlcNAc 1-3(GlcNAc 1-6)GalNAc 1- that susceptibility to specific pathogens differs between peo- Ser/Thr ple with different histo-blood groups,66 as exemplified by the observations that individual Se phenotype may determine the N-Acetyllactosamine elongation type ratio of infection as well as the course and severity of urinary Type 1 -Gal 1-3GlcNAc 1- tract infections, Norwalk virus induced acute gastroenteritis 67 – 69 Type 2 -Gal 1-4GlcNAc 1- and Helicobacter pylori -induced gastric diseases. There is also a strong correlation between distinct adhesive properties of H. pylori endemic in specific human populations and the mucin Branching blood group carbohydrate structures expressed by these popula- i-antigen -Gal 1-4GlcNAc 1-3Gal 1- tions. 70 These differences in the external barrier to infection can (unbranched) be equated with the diversity in underlying innate and adaptive I-antigen -Gal 1-4GlcNAc 1-3(-Gal 1- 4GlcNAc 1-6)Gal 1- (branched) immunity (e.g., polymorphisms in MHC, cytokines), which is thought to have evolved for the same reasons.

Terminal structures REGULATION AND MODULATION OF THE MUCIN BARRIER Blood group H Fuc 1-2Gal 1- The gel-forming mucins are produced by cells in the epithelial Blood group A Fuc 1-2(GalNAc 1-3)Gal 1- surface and /or by glands located in the submucosal connective Blood group B Fuc 1-2(Gal 1-3)Gal 1- tissues, and secretion occurs via both constitutive and regulated 71 Terminal structures pathways. Both gel-forming and cell-surface mucins show (Type 1 based) constitutive and inducible gene expression in mucosal epithe- Lewis a (Le a ) Gal 1-3(Fuc 1-4)GlcNAc 1- lial cells. The promoters of the MUC genes have generally not Lewis b (Le b ) Fuc 1-2Gal 1-3(Fuc 1-4)GlcNAc 1- been fully characterized, although partial promoter characteri- 72 – 74 75– 81 82 (includes H) zation is available for human MUC1 , MUC2 , MUC3 , 83 – 85 79,86 87 Sialyl-Le a NeuAc( 2-3)Gal 1-3(Fuc 1-4) MUC4 , MUC5AC , a n d MUC5B. Differential regu- GlcNAc 1- lation of individual mucin genes is evident between different Terminal structures mucosal tissues and throughout differing regions of the larger (Type 2 based) epithelial tracts. For example, differing promoter regions are Lewis x (Le x ) Gal 1-4(Fuc 1-3)GlcNAc 1- involved in the differential regulation of constitutive MUC2 78 Lewis y (Le y ) Fuc 1-2Gal 1-4(Fuc 1-3)GlcNAc 1- expression in the small and large intestines. Expression of (includes H) cell-surface and gel-forming mucins can be upregulated by Sialyl-Le x NeuAc 2-3 Gal 1-4(Fuc 1-3) inflammatory cytokines such as interleukin (IL)-1 , IL-4, GlcNAc 1- IL-6, IL-9, IL-13, interferons, tumor necrosis factor- , n i t r i c 82,88 – 110 oxide, and other uncharacterized inflammatory factors. Sulfation Responsiveness to these cytokines provides a link between mucins, innate mucosal immunity, and mucosal inflamma- 3 Sulfation HSO -3Gal 1- 3 tory responses. Neutrophils can also stimulate increases in 6 Sulfation HSO -6GlcNAc 1- 3 production of both gel-forming and cell-surface mucins by mucosal epithelial cells via neutrophil elastase. 111 – 116 M i c r o b i a l Examples of combined epitopes products can stimulate increased production of mucins by mucosal H-type 1 Fuc 1-2Gal 1-3GlcNAc 1- epithelial cells.103,117 – 120 In fact, there is evidence that adherence Sialylated type 2 NeuAc 2-3Gal 1-4GlcNAc 1- 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 Se phenotype with simultaneous expression of Lea a n d L e b H. pylori decreases mucin synthesis in gastric epithelial cells antigens has been described as the “ weak-Secretor ” (Se w ) in vitro via activation of cPLA-2, 123 representing a mechanism by phenotype. 63,64 The dual expression of Lea / L e b i s a c o n s e q u e n c e which a pathogen can favorably modulate the mucus barrier.

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The constitutive pathway continuously secretes sufficient either attach to the apical surface of epithelial cells decorated mucin to maintain the mucus layer, whereas the regulated path- with the cell-surface mucins or release toxins that disrupt epithe- way affords a massive discharge as a response to environmen- lial integrity. Bacterial adhesion to host cells can be mediated by tal and / or (patho)physiological stimuli, including cholinergic hydrophobic interactions, cation bridging (i.e., divalent cations stimuli, inflammatory cytokines, prostaglandins, lipopoly- counteracting the repulsion of the negatively charged surfaces saccharide, bile salts, nucleotides, nitric oxide, vasoactive intestinal of bacteria and host) and receptor ligand binding. One of the peptide, and neutrophil elastase. 93,95,103,113,124 – 131 S t i m u l a t e d most extensively studied mechanisms of bacterial adhesion mucin release can occur immediately and is accompanied by is via lectins and their corresponding glycosylated receptors. hydration, resulting in approximately a hundredfold expansion Binding is usually of low affinity, but clustering of adhesins and in volume of the secretory granule contents.132,133 Shedding of receptors results in multivalent binding. Fimbriae (or pili), outer the large extracellular -subunits of cell-surface mucins from membrane proteins, and cell wall components (e.g., lipopoly- the cell surface and release of secreted splice variants of cell- saccharide) may all function as adhesins. Adhesion can affect the surface mucins are less well understood. The protease ADAM17 bacteria by stimulation / inhibition of growth as well as induction (also known as TACE) has been shown to trigger shedding of of other adhesive structures and proteins required for invasion MUC1 in endometrial cells in response to tumor necrosis factor- such as secretion systems. On the other hand, effects of adhesion ,28,134 and the matrix metalloproteinase-1 also appears to be an on host cells can include altered morphology, fluid loss, induc- effective MUC1 sheddase.29 tion of cytokine release, upregulation of adhesion molecules, In addition to regulation of their synthesis and release, mucins and apoptosis.150 are regulated in terms of their glycosylation. Altering mucin carbo- Many bacterial adhesins bind oligosaccharides present on hydrates may block mechanisms that pathogens use to subvert mucins. Whether bacterial– mucin binding events favor the the mucin barrier. Tumor necrosis factor- alters sialylation of bacteria or the host is a key question. In reality, for some organ- mucins produced by a tracheal cell line 135 and expression of isms, this may be a truly commensal relationship with benefits both fucosyltransferases and -2,3-sialyltransferases by normal for both the bacteria (by facilitating retention in a favorable bronchial mucosal explants. 136 In respiratory epithelial cells, niche and even by providing mucin oligosaccharides for meta- the Th2 cytokines IL-4 and IL-13 increase expression of core bolism) and the host (by retaining bacteria in the outer areas 2 -1,6- N -acetylglucosaminyltransferase, which forms -1,6- of the mucus barrier where they cannot harm the underlying branched structures, including core 2, core 4, and blood group epithelium and also limiting the niche available for pathogenic I antigen. 137 In addition, glycosylation changes occur during bacteria). Numerous interactions between microorganisms and infection/ inflammation, for example, in individuals with cystic mucins and/ or mucin-type carbohydrates have been demon- fibrosis or chronic bronchitis,138 a s w e l l a s H. pylori -infected strated (see Table 3). Bacteria may have multiple adhesins with individuals.69,139 The inflammation-associated mucin sialylation different carbohydrate specificities, and modulation of surface shown in patients with H. pylori infection returns to the nor- receptor density, kinetic parameters, or topographical distribu- mal pattern following successful bacterial clearance with anti- tions of these receptors on cell membranes regulate adhesion. biotics.140 In rhesus monkeys that share strong similarities in As an example, H. pylori binds to mucin oligosaccharides via at mucin glycosylation and the natural history of H. pylori infec- least four adhesins, which differ substantially with anatomical tion with humans, 141 H. pylori infection induces time-dependent site along the oro-gastric infection route, mucin type, pH, and changes of mucosal glycosylation that alter the H. pylori a d h e - gastric disease status. 139,151– 153 Thus, for H. pylori , binding to sion targets.69 Such fine-tuned kinetics of host glycosylation mucins can have differing consequences during colonization of dynamically modulate host– bacterial interactions, appearing the oral-to-gastric niches and during long-term infection. to balance the impact of infection and thereby may determine the severity of disease.69 Another example of dynamic changes MUCINS AS DECOYS FOR MICROBIAL ADHESINS in mucins occurs following infection of rats with the intestinal Mucus hypersecretion ensuing from infection is testament to the parasite Nippostrongylus brasiliensis; infection induces increased role of mucus as a component of host defense. Although mucins production and several alterations in the glycosylation of intes- are the major macromolecular constituent of mucus and are tinal Muc2 gel-forming mucin, one of which coincides with largely responsible for formation of the mucus gel, the precise expulsion of the parasite.142 – 147 These alterations in glycosyla- nature of their role in host defense has not been well demons- tion appear to be driven partly by CD4 + T cells, as CD4 but trated empirically. Formation of the mucus gel is important not CD8 depletion blocks the increase in mucin production, in itself, as it provides a biophysical barrier as well as a matrix change in glycosylation and worm expulsion,148 and also by supporting the retention of a host of antimicrobial molecules. T-cell-independent mechanisms.149 Such changes in mucin However, the secreted mucins themselves are likely to function glycosylation need to be considered as a component of innate as decoys for adhesins that have been evolved by pathogens and adaptive immune responses to mucosal infection. to engage the cell surface, as the mucins express many of the oligosaccharide structures found on the cell surface and are MICROBIAL ADHERENCE TO THE EPITHELIUM constitutively produced in large amounts, constantly washing To colonize mucosal surfaces and invade the host, microbes typi- the mucosal surfaces ( Figure 1 ). Some mucins are effective cally must first penetrate the secreted mucus barrier and then viral agglutinating agents and exogenously applied mucins

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Table 3 Characterized interactions between mucins and microbes

Tissue derived mucins Mucin Carbohydrate Microbe References Respiratory mucins MUC1 Sialic acids P. aeruginosa, Haemophilus influenzae, 163,181,270 – 272 S. aureus, influenza viruses Salivary mucins MUC5B MUC7 Sulfated Le a Sialic acids, P. aeruginosa, H. pylori, Streptococcus 273 – 280 (DMBT1-Muclin) Sialyl Le x , Leb sanguis, Streptococcus gordonii, Actinobacillus actinomycetemcomitans, Streptococcus spp., Candida albicans Gastric mucins MUC5AC MUC1 A, B, H, Leb H. pylori 139,151,176,281,282 Intestinal mucins MUC2 Enterotoxigenic Escherichia coli, 162,283 – 290 Enteropathogenic E. coli, Salmonella typhimurium, Shigella boydii, Shigella sonnei, Campylobacter upsaliensis, Yersinia enterolitica, C. albicans, reoviruses 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.

are effective inhibitors of viral infection in in vitro - c u l t u r e d infection of MDCK cells by up to tenfold.168,169 Milk can limit cells. 154,155 Streptococcus pyogenes , Tritrichomonas foetus , bacterial and viral infections of the gastrointestinal tract and Tritrichomonas mobilensis, influenza viruses, reoviruses, this has been attributed in part to the presence of large amounts adenoviruses, enteroviruses, and coronaviruses bind to sialic of cell-surface mucins, chiefly MUC1 and MUC15, in the milk- acids, which are present both at the epithelial surface and on fat globule membrane. 170– 172 Muc1 − / − mice were reported to mucins.156 – 165 display chronic infection and inflammation of the reproduc- Despite the accepted dogma that secreted mucins limit infec- tive tract, reducing fertility rates. In this latter study, only tion, there are few empirical in vivo data demonstrating their normal endogenous bacteria were isolated, suggesting that importance. The only secreted mucin for which genetically these species become opportunistic pathogens in the absence of deficient animals are available is the intestinal mucin, Muc2. Muc1.173 In addition, Muc1 − / − mice were reported to have Muc2 − / − mice develop spontaneous inflammation, presum- a high frequency of eye inflammation/ infection involving ably due to the absence of the major component of intestinal Corynebacteria , Staphylococci a n d Streptococci , 174 a l t h o u g h t h i s mucus, leading to increased exposure to the normal intestinal could not be duplicated in a different mouse background held microbial flora. 166,167 As yet, there are no reports of controlled in alternative housing conditions.175 infection experiments in these mice. Further models of secreted We recently demonstrated that the intestinal pathogen mucin deficiency are required to comprehensively determine C. jejuni binds to fucosylated mucin oligosaccharides. Controlled the importance of secreted mucins in preventing and clearing infection experiments demonstrated rapid transit of C. jejuni mucosal infection. across the gastrointestinal barrier and greater intestinal patho- Many pathogens require direct binding to, or penetration of, logy in Muc1 − / − mice. 55 Bone marrow transplantation studies mucosal epithelial cells to cause pathology. The widest diversity demonstrated that the increased susceptibility was due to loss of cell-surface mucin expression is in the mucosal tissues most of Muc1 on epithelium rather than on leukocytes (which can at risk of infection, such as the gastrointestinal tract, respiratory also express Muc1). Loss of Muc1 had no discernable effects tract, and eye; notably, nine of the ten cell-surface mucins are on the abundance or constituency of the intestinal microbial expressed in the large intestine, which is the most microbe-rich flora. Muc1 appears to prevent C. jejuni infection both by mucosal environment. Importantly, their ability to be shed from protecting cells from the effects of the cytolethal distending the cell surface has led us to hypothesize that one of the main toxin (see above) and by acting as a releasable decoy. 55 We h a v e functions of cell-surface mucins is to act as releasable decoy also demonstrated that even though H. pylori can bind Muc1, ligands for microbes attempting to anchor themselves to the that primary murine gastric epithelial cells expressing Muc1 glycocalyx. Cell-surface mucins initiate intracellular signaling bind fewer H. pylori t h a n Muc1 − / − cells. 176 This paradoxical in response to bacteria, suggesting that they have both a bar- result is explained by Muc1 acting as a releasable decoy, i.e., the rier and reporting function on the apical surface of all mucosal bacteria bind Muc1 expressed on epithelial cells, which is then epithelial cells.57 However, until recently, much of the evidence shed by the host. Due to the absence of this decoy molecule, had been circumstantial or restricted to in vitro a n a l y s i s . F o r Muc1 − / − mice develop an approximately fivefold greater coloni- example, upregulation of MUC3 expression in colonic cells zation density of H. pylori from the first days following infection has been correlated with decreased binding of enteropatho- that is maintained for at least 2 months. Consequently, Muc1 − / − genic E. coli . 121,122 In vitro studies have shown that expression mice develop severe gastritis not found in wild-type mice. 176 of MUC1 by transfection inhibits reovirus and adenovirus Heterozygous mice that have a lower level of gastric Muc1

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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.

protein expression show intermediate colonization densities, without potentially damaging inflammation, where possible. which suggests that polymorphisms in MUC1 or genes that Further investigations are required with a broad array of patho- regulate its expression could underlie susceptibility to H. pylori - gens in multiple tissues to clearly delineate the participation of induced pathology in human populations. In fact, in humans, the family of cell-surface mucins in mucosal defense. individuals with short MUC1 alleles (encoding smaller extracellular mucin domains) have a higher propensity to develop OTHER PROTECTIVE ROLES OF MUCINS gastritis following H. pylori infection. 177 – 179 This may be indica- Mucins have direct and indirect roles in defense from infec- tive of lower efficacy of smaller MUC1 extracellular mucin tion distinct from their ability to form a physical barrier and act domains allowing increased access of bacteria to the epithelial sur- as adhesion decoys. Not only do mucin oligosaccharides bind face, or these alleles may be surrogate markers of polymorphisms microbes, but also, in some cases, they either have direct antimi- influencing the level of gastric MUC1 expression. Recently, a crobial activity or carry other antimicrobial molecules. A mucin similar protective role has been demonstrated for the extremely oligosaccharide, -1-4-linked N-acetyl-glucosamine, which is large MUC16 cell-surface mucin in human corneal epithelial expressed by some gastric mucins, has been shown to directly cells. Greater binding of Staphlylococcus aureus o c c u r s t o in vitro - interfere with synthesis of H. pylori cell wall components. 185 cultured corneal cells when MUC16 is depleted by RNAi. 180 H. pylori must live within gastric mucus to remain protected Paradoxically, our demonstrations of Muc1-limiting infec- from luminal gastric pH and prevent expulsion into the intes- tion in the gastrointestinal tract are opposite to that found in a tine. The antimicrobial mucin oligosaccharide probably acts to model of respiratory Pseudomonas aeruginosa infection in the limit H. pylori expansion within gastric mucus. The non- lung in which Muc1 − / − mice have an increased clearance of oligomerizing secreted salivary mucin MUC7 has inher- bacteria and a reduced inflammatory response to infection.181 ent direct candidacidal activity via a histatin domain at its MUC1 binds the P. ae r ug ino s a flagellin, 182,183 but intriguingly N-terminus.186,187 In addition, there is evidence for direct bind- appears to inhibit flagellin-stimulated TLR5-mediated activation ing of antimicrobial molecules such as histatins and statherin of NF- B by an as yet unclear mechanism requiring the MUC1 by mucins that would help retain the antimicrobial molecules cytoplasmic domain. 181,184 Whereas an infection-promoting in the correct mucosal microenvironment where they can role of a molecule highly expressed on the apical surface of a best protect the host. For example, MUC7 binds statherin and broad array of mucosal epithelia appears counterintuitive, an histatin-1,188 and the other major mucin in saliva, MUC5B, anti-inflammatory role for MUC1 in some tissues is consistent binds histatin-1, -3, and -5 and statherin.189 S e c r e t o r y I g A with evolutionary adaptations to clear infection by local defense (sIgA) is secreted via mucosal epithelial cells and needs to be

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retained in the immediate mucosal environment to maximize bial pathogenesis. For example, the Vibrio cholerae H a p A , exclusion of pathogens. sIgA is retained at high concentrations which has both mucinolytic and cytotoxic activity, is induced by in mucus where it can efficiently trap the progress of pathogens, mucin and required for translocation through mucin-contain- although the mechanism(s) for retaining sIgA in mucus are not ing gels.204 The widespread and critically required expression of well understood. Interestingly, secretory component, which is neuraminidases by a wide variety of sialic acid-binding mucosal tightly bound to the Fc region of dimeric-IgA to form sIgA, viruses underlines the importance of elimination of mucin carries oligosaccharide structures similar to those on mucins. 190 carbohydrates for their pathogenicity.160 Lipopolysaccharide In the absence of secretory component carbohydrates, sIgA fails from H. pylori decreases mucin synthesis,123 and the mucin to associate with mucus and fails to prevent infection in a murine carbohydrate-binding adhesins BabA and SabA undergo phase respiratory bacterial infection model, substantiating both variation and change expression during infection, 153,205 w h i c h the physiological importance of sIgA– mucin interactions and may allow them to evade this host defense mechanism. the importance of secretory component carbohydrates in maintaining this interaction. 191 It is also tempting to speculate that AVOIDANCE OF THE MUCIN BARRIER BY MUCOSAL the poly-anionic mucins bind the poly-cationic antimicrobial PATHOGENS defensin peptides that are co-secreted into mucus. Interactions Another strategy commonly used by mucosal pathogens is to of mucins with other secreted antimicrobial molecules has not avoid the mucin barrier. Intestinal M cells, specifically designed been fully explored largely due to difficulties in extracting and to capture and present microbes to the underlying lymphoid purifying mucins in the absence of denaturing agents likely to tissue, can be regarded as a hole in the mucin barrier. The dome disrupt such interactions. The cell-surface mucins are an inte- epithelium in which they lie lacks goblet cells, and therefore gral component of the glycocalyx where they are likely to inter- does not produce gel-forming mucins, and their apical cell sur- act with proteoglycans and other molecules that could retain face has only sparse microvilli and an apparently thin glycoca- host defense molecules in a molecular complex covering the lyx.206,207 Although no studies have measured the expression apical cell surface. 192,193 Therefore, other mucins and mucin of individual cell-surface mucins in M cells, there appear to be oligosaccharides may yet prove to have direct and indirect differences in the glycocalyx mucins between M cells and adja- antimicrobial activity. Regardless of whether antimicrobial cent intestinal mucosal epithelial cells. In some species, M cells molecules are retained in mucus by direct binding with mucins can be identified by their pattern of lectin binding to specific or by the biophysical properties of mucus, if mucin synthesis cell-surface carbohydrates that differ with other mucosal is aberrant or secreted mucins are degraded, the antimicrobial epithelial cells.208,209 Consequently, even though M cells molecules will have impaired efficacy. constitute only a very small percentage of mucosal epithelial cells, they are the major point of attachment and / or entry used SUBVERSION OF THE MUCIN BARRIER BY MUCOSAL by a large number of mucosal pathogens including bacteria PATHOGENS (e.g., S. typhimurium , Shigella flexneri , Yersinia enterocolitica , Perhaps the best evidence for the importance of the mucin bar- and V. cholerae ), viruses (e.g., reovirus, HIV-1, and polio virus) rier to infection is the wide variety of strategies used by microbes and parasites (e.g., Cryptosporidia). 206,210,211 Another strategy to subvert or avoid this barrier. Mucin barrier subversion strate- used by pathogens to avoid the cell-surface mucin barrier, once gies used by microbes include the production of enzymes capa- mucus is penetrated or M cells are invaded, is to disrupt the ble of degrading mucin core proteins and mucin carbohydrates, tight junctions between adjacent mucosal epithelial cells thereby and effective motility through mucus gels. Motility is important exposing the vulnerable lateral membranes not protected by the for bacterial mucosal pathogens to facilitate breaking through glycocalyx. Such examples include S. flexneri , 212 enteropatho- the physical mucus barrier. In fact, a vast proportion of mucosal genic E. coli ,213 Porphyromonas gingivalis 214 and H. pylori. 215 bacterial pathogens are flagellated. 194,195 H. pylori t h a t h a v e dysfunctional flagella have a greatly reduced ability to infect. 196 MODELS TO INVESTIGATE INTERACTIONS BETWEEN H. pylori uses motility for initial colonization and to attain robust MICROBES AND MUCINS infection. In conjunction with motility, degradative enzymes Numerous models, including cancer cell-lines, organ cultures of such as glycosulfatases, sialidases, sialate O-acetylesterases, gastric biopsies and whole animals have been used to investigate N -acetyl neuraminate lyases and mucinases are produced by mucin – microbe interactions. Although they express orthologs a broad range of bacterial pathogens to destabilize the mucus to most human mucins, the most commonly used laboratory gel and remove mucin decoy carbohydrates for adhesins. 197 – 201 animals such as rats and mice have differing glycosylation of The protozoan parasite Entamoeba histolytica cleaves the MUC2 some of their mucins. In fact, it is tempting to speculate that mucin, which is the major structural component of the intes- differences in mucin glycosylation between mammalian species tinal mucus, and this cleavage is predicted to depolymerize may underlie some of the differences in infectivity/ pathogenicity the MUC2 polymers.202 The size of the polymer is important for individual microbial pathogens. Murine knockout models for the formation of entangled gels and the viscous properties are only available for Muc1 ,216 Muc2 ,166 a n d Muc13 (M.A. of mucus; consequently, cleavage of the mucin polymer will McGuckin, unpublished data), and there are also mutants with effectively result in a local disintegration of mucus. 203 There is aberrant Muc2 assembly.217 Thus, there is a need for more evidence that these degradative enzymes are critical for micro- models, as mouse knockouts, although limited by the slightly

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different glycosylation, still represent an important way to overcome if we are to fully understand the roles of mucins in collect information of the in vivo function of mucins in h o s t d e f e n s e f r o m i n f e c t i o n . infection. Because human pathogens commonly have adhesins for human carbohydrate structures, it is important to select DISCLOSURE appropriate models for individual pathogens. For example, the The authors declared no conflict of interest. effects of H. pylori infection on the mouse are mild, and gastric cancer is not induced even after long-term exposure without © 2008 Society for Mucosal Immunology other stimuli or genetic defects, although the mouse may develop 218,219 REFERENCES chronic atrophic gastritis. H. pylori can colonize the guinea 1 . Kagnoff , M .F . & Eckmann , L. Epithelial cells as sensors for microbial pig and the Mongolian gerbil and cause a severe inflammatory infection . J. Clin. 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MucosalImmunology | VOLUME 1 NUMBER 3 | MAY 2008 197

Article:

The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Paszek et al, Nature 511: 319 (2014)

ARTICLE doi:10.1038/nature13535

The cancer glycocalyx mechanically primes integrin-mediated growth and survival

Matthew 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, Gre´gory 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 of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying 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 glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

The composition of cell surface glycans and glycoproteins changes Regulation of integrin assembly by bulky glycoproteins markedly and in tandem with cell fate transitions occurring in embryo- To determine whether glycocalyx bulk contributes to a cancer pheno- genesis, tissue development, stem-cell differentiation and diseases such type, we used gene expression microarray data to relate metastasis to as cancer1–3. Nevertheless, our understanding of the biochemical func- expression of genes for which protein products contribute to the gly- tions of glycans fails to explain fully why broad changes in glycosylation cocalyx. The likely contribution of gene product to glycocalyx bulk was and glycoprotein expression are critical to cell fate specification and estimated based on the protein’s extracellular domain structure and in what ways are they linked to disease. It is currently unclear whether predicted number of glycosylation sites (Extended Data Fig. 1). Using changes in glycan and glycoprotein expression reflect a global and more these estimates we obtained evidence for upregulation of transcripts en- general mechanism that directs cell and tissue behaviour. coding bulky glycoproteins and some classes of glycosyltransferases, which From a materials perspective, glycan and glycoprotein expression catalyse the glycosylation of cell surface proteins, in primary tumours dictates the bulk physical properties of the glycocalyx—the exterior cell of patients with distant metastases relative to those with localized tumour surface layer across which information flows from the microenviron- growth (P 5 0.032 for bulky transmembrane proteins, Kolmogorov– ment to signal transduction pathways originating at the plasma mem- Smirnov test; Fig. 1a and Extended Data Fig. 1). brane. Although the biophysical functions of the glycocalyx are largely To understand whether bulky glycoproteins could promote tumour untested, computational models predict that bulky glycoproteins can aggression by regulating integrin adhesions, we developed an integrated promote transmembrane receptor organization, including the clustering biochemical and mechanical model that incorporates integrins, the extra- of integrins at adhesion sites4. These models suggest that glycocalyx- cellular matrix (ECM), the cell membrane and the glycocalyx (Extended mediated integrin clustering would promote the assembly of mature Data Fig. 2). The model revealed that the kinetic rates of integrin–ECM adhesion complexes and collaborate to enhance growth factor signalling5— interactionsare tightlycoupled to thedistances between receptor–ligand phenotypes that are associated with cancer6,7. We demonstrate that a pairs and, thus, the physical constraints imposed by the glycocalyx. In global modulation of the physical properties of the glycocalyx alters the presence of bulky glycoproteins, the model predicted that integrin– integrin organization and function, and present evidence for how the ECM binding is most favourable at sites of pre-existing adhesive con- glycocalyx canbe co-opted inmalignancy to supporttumour cellgrowth tact, where the membrane and ECM substrate are in closest proximity and survival. (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, University of 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 Kavli Institute 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, California 94143, 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 and Biomolecular 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 Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California 94143, USA. {Present address: Department of Chemistry and Biochemistry, University of California, San Diego, California 92093, USA.

a Upregulation with b Computational c Slow Fast Slow d Glycoprotein mimetics metastasis model 12 1.0 10 Integrin HO OH 1010 50 O 0.8 Kinetic HO 8 45 trap AcHN 10 O OH 0.6 40 N HO 6 AcHN 10 35 O OH 0.4 ✯ ✯ Glycocalyx HO OH

4 thickness (nm) ✯ 10 30 Glyo- O N O One-sided P 0.2 2 protein HO Relative bond rate 10 AcHN d O 0 1 N 0 100 200 300 400 500 600 Bulky Extracellular matrix Lipid tail All genes Distance from adhesion (nm) Membrane

e Mimetic Vinculin Merge f Short (3 nm) Long (80 nm) g h 120 Mimetic Mimetic ) Membrane height (nm) ) –1 50 110 2 100 100 40 *** *** 90 30

Short (3 nm) 80 80 20 70 60 60 10 50 Adhesion area ( μ m Adhesion rate (pN s 0 40 40 None3 nm None3 nm Long (80 nm) Mean: 75 nm Mean: 94 nm 80 nm 30 nm80 nm

Figure 1 | The cancer glycocalyx drives integrin clustering. a, Violin plots d, Cartoon showing structure of glycoprotein mimetics with lipid insertion showing increased expression of genes encoding bulky transmembrane domain. e, Fluorescence micrographs of MEC adhesion complexes (vinculin– proteins in primary tumours of patients with distant metastases relative to those mCherry) and glycomimetics of the indicated length (scale bar, 3 mm). f,SAIM with local invasion. White dots and thick black lines indicate the median images of DiI-labelled ventral plasma membrane topography in MECs and interquartile range of the P value distribution of all transcripts within each incorporated with glycomimetics (scale bar, 2.5 mm). g, Rate of integrin– class: all genes, all membrane proteins (Mem.), and bulky transmembrane substrate adhesion measured using single cell force spectroscopy in MECs with proteins (Bulky). b, Computed relative rate of integrin–ECM ligand bond incorporated glycomimetics. h, Quantification of the total adhesion complex formation as a function of distance from a pre-existing adhesion cluster. area per cell in MECs with incorporated glycomimetics. All results are the c, Model of proposed glycocalyx-mediated integrin clustering. Shorter mean 6 s.e.m. of three separate experiments. Statistical significance is given by distances between integrin–ligand pairs result in faster kinetic rates of binding. *P , 0.05; **P , 0.01; ***P , 0.001. integrin–matrix engagement (Fig. 1b) by increasing the gap between We next asked whether cancer-associated glycoproteins could sim- the plasma membrane and ECM.Thus, the model predicted that where- ilarly influence the spatial distribution of integrins and the assembly as bulky glycoproteins reduce the overall integrin-binding rate, they of focal adhesions. On the basis of our large-scale gene expression ana- enhance, rather than impede, integrin clustering and focal adhesion lysis, we determined that the transmembrane mucin glycoprotein, MUC1, assembly by generating a physically based kinetic trap (Fig. 1c). which has a highly glycosylated ectodomain that projects out up to To test experimentally whether bulky glycoproteins could drive in- 200 nm from the cell surface12, was upregulated in metastatic tumours tegrin clustering and focal adhesion assembly, we generated a series of (nominal P 5 0.0028 via one-sided t-test). In agreement with our ana- synthetic mucin glycoprotein mimetics of increasing length that rapidly lysis, we measured high levels of MUC1 on the surface of several breast intercalate into the plasma membrane and project perpendicularly to cancer celllines, as well as v-Src and HRAS-transformed MECs (Fig. 2a). the cell surface8,9. These glycopolymers consisted of a long-chain poly- To assess the impact of MUC1 on focal adhesion assembly, we ex- mer backbone, pendant glycan chains that mimic the structures of nat- pressed MUC1 on the surface of non-malignant MECs, to levels com- ural mucin O-glycans, a phospholipid for membrane insertion, and a parable to those of transformed MECs and breast cancer lines. MUC1 fluorophore for imaging (Fig. 1d and Extended Data Fig. 3a–c). We expression induced striking membrane topographical features, which found that large glycopolymers with lengths of 80 nm, significantly included regions of high curvature, and a significant expansion of the longer than the reported integrin length of 20 nm10, are consistently cell membrane–ECM gap (Fig. 2b, c and Extended Data Fig. 4a). Expres- excluded from sites of integrin adhesion on the surface of non-malig- sion of an ectodomain-truncated MUC1 construct did not significantly nant mammary epithelial cells (MECs; Fig. 1e). Shorter polymers with change the gap compared to control MECs (Fig. 2c and Extended Data lengths of 3 or 30 nm were not excluded (Fig. 1e). Because the mimetics Fig. 4a). Our model predicted that the membrane topographies we possessed minimal biochemical interactivity with cell surface proteins observed in MUC1-expresing cells would facilitate integrin clustering (Extended Data Fig. 3d), the data suggest a physical interplay between through the kinetic trap. In agreement with these predictions, express- bulky glycoproteins and integrin receptors. ion of full-length MUC1 significantly enhanced the size of adhesion To determine how the largest polymer mimetics influence the nano- clusters and the total adhesion area per cell (Fig. 2d, e and Extended scale spatial features of the cell–ECM interface, we measured the topo- Data Fig. 5a). The adhesion assembly phenotype did not require the graphy of the ventral cell membrane using scanning angle interference MUC1 cytoplasmic tail, which mediates MUC1’s biochemical activity microscopy (SAIM), a fluorescence-based microscopy technique that (Fig. 2e)13, or direct interactions between MUC1 and fibronectin (Ex- enables imaging with 5–10-nm axial resolution and diffraction-limited tended Data Fig. 5b). Together, these results are consistent with a phys- (,250 nm) lateral resolution11. Polymers designed tomimic largenative ically based mechanism of integrin clustering. glycoproteins (,80 nm) expanded the membrane–ECM gap by 19 nm To gain additional insight into the coupled dynamics between inte- (Fig. 1f). Consistent with computational predictions, the large glycopro- grins and MUC1, we conducted time-lapse imaging of fluorescently tein mimetics reduced the overall rate of integrin bond formation, but labelled MUC1 and the adhesion plaque protein vinculin. We observed significantly enhanced clustering of integrins into focal adhesions (Fig. 1g, h). that MUC1 and integrin adhesions spatially segregate on the cell sur- Shorter glycoprotein mimetics (3 and 30 nm) did not have an impact face in a temporally correlated manner (Fig. 2d, Extended Data Fig. 5c on integrin clustering, even when incorporated at higher surface densi- and Supplementary Movie 1), suggesting a physical communication bet- ties (Fig. 1h). ween these molecules. Further evidence for a physical interplay between

15 a b Membrane Membrane (ROI) integrin . Analysis of b3 integrin trajectories after manganese activa- 10A-Cont. tion in MEFs revealed a significant increase in the total level of immo- 10A-v-Src bilized integrin at the plasma membrane, both inside and outside 10A-HRAS adhesive contacts (Fig. 3a and Extended Data Fig. 6b–e). By contrast, MUC1

MCF7 Control the immobilized b3 integrin in MEFs expressing high MUC1 was re- T47D stricted to sites of adhesion (Fig. 3a–c and Extended Data Fig. 6e). These results are consistent with single-cell force spectroscopy measurements, 1.0 which indicated that MUC1 expression reduces the net rate of integrin– 0.8 ECM bond formation (Extended Data Fig. 5d). Mucin expression did not have a significant impact on the free diffusion of integrins (Extended 0.6

+MUC1 Data Fig. 6b–d). Importantly, we observed that integrins frequently dif- 0.4 fused across the mucin–adhesive zone boundary and could immobilize 0.2 Normalized counts rapidly once in the adhesive zone (Fig. 3d, Extended Data Fig. 6f, g and 0 1 2 3 4 5 6 50 74 99 Supplementary Movie 2). Together, our results indicate that large gly- 10 10 10 10 10 10 123 147 171 196 220 Intensity (AU) Membrane height (nm) coproteins act as physical ‘steric’ barriers that impede integrin immobilization and thus funnel integrins into adhesive contacts. c d MUC1 Vinculin MUC1/Vinc. (ROI) 200 Bulky glycoproteins exert force on integrin bonds ** 160 Integrins switch between activity states by undergoing a conformational 120 change that is facilitated by tensile force16,17. Given the order of mag- +MUC1( Δ TR) 12 80 nitude difference in the size of MUC1 (,200 nm ) as compared to , 10 40 integrins ( 20 nm ), and the close proximity of these molecules within the cell–ECM interface, we hypothesized that large glycoproteins, such Membrane height (nm) 0 as MUC1, modify integrin structure and function by applying force to +MUC1 Control +MUC1 matrix-bound receptors. Abiding by Newton’s third law, if large gly-

+MUC1(ΔTR) coproteins exert a tensile force on integrins, then we should detect a e

) 250 f MUC1 tracks/paxillin Tracks/paxillin (ROI) reciprocal strain on the glycoproteins. Consistent with this hypothesis, 2 200 *** mucins imaged with SAIM appeared compressed or mechanically bent 150 near integrin adhesivecontacts(Fig. 4a and Extended Data Fig. 4a). Fur- ** 100 thermore, single-cell force spectroscopy revealed that MECs expres- 50 sing high levels of exogenous MUC1 required higher compressive force

Adhesion area ( μ m 0 application at the ECM–substrate interface to promote integrin-mediated

ΔCT) adhesion when compared to control MECs (Fig. 4b). Control +MUC1 To test further whether integrin adhesions strain bulky transmem- +MUC1(ΔTR)+MUC1( brane glycoproteins, we generated a genetically encoded construct con- Figure 2 | The bulky cancer-associated glycoprotein MUC1 drives integrin ceptually similar to a strain gauge, consisting of a cysteine-free cyan and clustering. a, Cartoon of MUC1 and quantification of MUC1 cell-surface yellow fluorescent protein pair (CFP and YFP) separated by an elastic levels on control (10A-Cont.), transformed (10A-v-Src, 10A-HRAS) and linker18, which we inserted into the ectodomain of full-length and trun- tumour (MCF7, T47D) cells. b, Topographical SAIM images of representative cated MUC1 proteins (Fig. 4c and Extended Data Fig. 4b). Fluorescence mCherry–CAAX-labelled ventral plasma membranes in control and MUC1– resonance energy transfer (FRET) served as the readout of distance bet- GFP-expressing (1MUC1) MECs (Scale bar, 5 mm; region of interest (ROI) ween the CFP and YFP pair and, thus, functioned as a reporter of mo- scale bar, 2 mm). c, Quantification of mean plasma membrane height in control lecular strain. When the full-length reporter was expressed in MECs, MECs and those ectopically expressing ectodomain-truncated MUC1–GFP (1MUC1(DTR)) and wild-type MUC1–GFP (1MUC1). Results are the we observed high FRET efficiencies in the cell–substrate interface (Fig. 4d, e mean 6 s.e.m. of at least 15 cell measurements in duplicate experiments. and Extended Data Fig. 7). FRET efficiency was significantly lower in d, Fluorescence micrographs of MUC1(DTR) or wild-type MUC1 expressed in MECs expressing the ectodomain-truncated construct, indicative of MECs and their focal adhesions labelled with vinculin–mCherry (scale bar, lower molecular strain (Fig. 4d, e). The highest FRET efficiencies cor- 3 mm; ROI scale bar, 1.5 mm). e, Quantification of the total adhesion complex related spatially with sites of adhesive contact, consistent with integrin area per cell in control non-malignant MECs (control) and those ectopically adhesions straining bulky transmembrane glycoproteins and glyco- expressing MUC1(DTR), wild-type MUC1, or cytoplasmic-tail-deleted MUC1 proteins exerting a reciprocal restoring force on integrins (Fig. 4d and (1MUC1(DCT)). Results are the mean 6 s.e.m. of three separate experiments. Extended Data Fig. 7e, f). f, Left panel: trajectories of individual mEOS2-tagged MUC1 proteins recorded We next examined whether the bulky glycoprotein MUC1 could at 50 Hz using sptPALM (green) and focal adhesions visualized with paxillin–GFP (red) in MEFs (scale bar, 3 mm). Right panel: the ROI from the left induce conformational changes that would activate integrins independ- panel with individual MUC1 tracks displayed in multiple colours (scale bar, ent of the contractile cytoskeleton. We used a bi-functional crosslinker 1 mm). Statistical significance is given by *P , 0.05; **P , 0.01; ***P , 0.001. that can specifically link extracellular fibronectin and bound a5b1 integrins that are in a tension-dependent conformation17. Inhibition of actomyosin contractility, using the myosin inhibitor blebbistatin or the Rho MUC1 and integrins was obtained in mouse embryonic fibroblasts kinase inhibitor Y-27632, abrogated most of the fibronectin crosslinked (MEFs) using single-particle tracking photo-activation localization mi- integrins in MECs expressing empty vector (Fig. 4f and Extended Data croscopy (sptPALM14,15) to track MUC1 diffusivetrajectories. We noted Fig. 8a). By contrast, MUC1-expressing MECs formed tensioned bonds that whereas MUC1 was mobile in the plasma membrane, it rarely crossed with the ECM substrate, even when cells were pre-treated with con- into integrin adhesion zones (Fig. 2f and Extended Data Fig. 6a). tractility inhibitors before plating (Fig. 4f and Extended Data Fig. 8a). We next tested our model’s prediction that MUC1 would favour integ- Of note, the myosin-independent integrin clusters observed in the MUC1- rin clustering by physically impeding integrin–ECM binding outside of expressing MECs recruited activated cytoplasmic adaptors typically adhesive contacts. We recorded the trajectories of individual b3 integ- associated with mature adhesion structures and nucleated actin (Extended rin molecules using sptPALM to determine the location and fraction of Data Fig. 8b). These results suggest that large, cancer-associated gly- mobile (confined and freely diffusive) and matrix-bound, immobilized coproteins not only facilitate integrin clustering but also physically alter

a Control +MUC1 b Control - outside Paxillin/β integrin Paxillin/β tracks (ROI) MUC1/β integrin MUC1/β tracks (ROI) +MUC1 - inside + MUC1: inside 3 3 3 3 adh. contacts MUC1-rich areas adh. contacts 25 25 15 Imm. Mobile Imm. Mobile Imm. Mobile

Mobile (free) 20 β 20 β β 3 3 3 β + Mn2+ β 2+ 10 β 2+ 15 3 15 3 + Mn 3 + Mn 2+ 10 10 5 5

No Mn 5 Occurence (%) 0 0 0 –5 –4 –3 –2 –1 0 –5 –4 –3 –2 –1 0 –5 –4 –3 –2 –1 0 Mobile (confined) log[D (μm2 s–1)]

c Cont. +MUC1 d Free 100 *** MUC1-rich 80 Free Confined 60

40 Immobile activation Immobile 2+ contacts (%) 20 Immobile Outside adhesive Mn 0 2+ 2+ 2+ 2+ Adhesion zone – Mn+ Mn – Mn+ Mn

Figure 3 | Bulky glycoproteins spatially regulate immobilization of integrins (scale bar, 1 mm). b, Distribution of b3 integrin diffusion coefficients activated integrins. a, Left panels: fluorescence micrographs displaying recorded before or after Mn21 treatment in control MEFs outside of adhesive paxillin–GFP-labelled focal adhesions in control cells or MUC1-rich regions in contacts (left), MUC1-transfected MEFs inside MUC1-rich areas (middle), MUC1–GFP-expressing MEFs, and positions of individual mEOS2-fused b3 and MUC1-transfected MEFs outside MUC1-rich areas, including adhesive 21 integrins. Cells were treated without or with Mn to activate integrins (scale contacts (right). c, Fraction of immobilized, confined and freely diffusive b3 bar, 3 mm). Right panels: magnified area of interest showing fluorescence integrins outside of adhesive contacts in control MEFs (Ctrl) and MUC1- micrographs of focal adhesions visualized with paxillin–GFP in control MEFs transfected cells (MUC1) before and after Mn21 treatment. Results are the or MUC1 in MUC1–GFP-expressing MEFs, and individual b3 integrin mean 6 s.e.m. Statistical significance is given by *P , 0.05; **P , 0.01; trajectories recorded with sptPALM. Single molecule trajectories are colour- ***P , 0.001. d, Fluorescence micrograph of MUC1–GFP and an illustrative 21 coded to indicate immobile and mobile (confined and freely diffusive) b3 single integrin trajectory in MEFs treated with Mn (scale bar, 1 mm). integrin state and do so, at least in part, independently of cytoskeletal at distant metastatic sites19. Thus, the ability to survive, particularly tension. within unfavourable microenvironments and under minimally adhesive conditions, is a prerequisite for efficient tumour cell metastasis19. Bulky glycoproteins promote growth and survival Given their ability to promote integrin adhesion assembly, we hypoth- Tumour metastasis is a multi-step process that depends on the efficient esized that bulky glycoproteins could facilitate metastasis by promoting dissemination of primary cancer cells and their subsequent colonization focal adhesion signalling to enhance tumour cell growth and survival.

a GFP MUC1 fluorescence Vinculin (ROI) MUC1 height (ROI) b 30 c 527 nm 220 )

–1 25 Mem. 20 Height (nm) 190 YFP 15

10 FRET

160 5 Ctrl Adhesion rate (pN s +MUC1 0 CFP 0 500 1,000 Substrate 130 Compressive force (pN) 440 nm

FRET Vinc. (ROI) Vinc. (ROI) d FRET e f Ctrl +MUC1 60 1.0 200 MUC1(ΔTR) *** 50 0.8 MUC1 FRET efficiency (%) 160 ** )

2 *** 40 0.6 120 **

FRET (ROI) FRET (ROI) 30 0.4 80 area ( μ m

20 0.2 Tensed integrin 40 +MUC1 sensor Normalized counts (no.) +MUC1 (Δ TR) sensor 10 0 0 0020460 FRET efficiency (%) 0 DMSOBlebb. DMSOBlebb. Y27632 Y27632

Figure 4 | Integrins are mechanically loaded and re-enforced by bulky corresponding vinculin–mCherry-labelled focal adhesions (scale bar, 8 mm; glycoproteins. a, GFP-fluorescence and topographic SAIM images of MUC1– ROI scale bar, 1 mm). e, Histogram of observed FRET efficiencies of wild-type GFP (scale bars, 3 mm) and the corresponding focal adhesions visualized MUC1 and MUC1(DTR) strain gauges. f, Quantification of fibronectin- with vinculin–mCherry. b, Adhesion rate versus force of contact between crosslinked a5 integrin in control and MUC1-expressing normal MECs treated cell and substrate (compressive force) measured with single-cell force with solvent alone (DMSO), myosin-II inhibitor (blebbistatin; 50 mM), or Rho spectroscopy for control and MUC1-expressing MECs. Results are the kinase inhibitor (Y-27632; 10 mM) for 1 h followed by detergent-extraction mean 6 s.e.m. of at least 10 cell measurements per point. c, Schematic of FRET- to reveal the fibronectin bound integrin that is under mechanical tension. based MUC1 compressive strain gauge. d, FRET efficiency maps of Results are the mean 6 s.e.m. of three separate experiments. Statistical ectodomain-truncated (1MUC1(DTR) sensor) and wild-type (1MUC1 significance is given by *P , 0.05; **P , 0.01; ***P , 0.001. sensor) strain gauges measured at the ventral cell surface of MECs and the

Consistent with this notion, analysis of human data sets revealed that distribution on the surface of transformed MECs (Extended Data Fig. patients with aggressive breast cancers that presented with circulating 10b). Inhibition of HA synthesis or HA cell-surface retention sig- tumour cells (CTCs) express disproportionately high amounts of bulky nificantly reduced the growth of transformed MECs on compliant glycoproteins and have altered glycosyltransferase expression profiles hydrogels, raising the possibility that bulky cell-surface constituents, (Fig. 5a and Extended Data Fig. 1d, e). Furthermore, analysis of genes in addition to MUC1, could similarly promote tumour aggression (Ex- expressed within CTCs isolated from a cohort of breast cancer patients tended Data Fig. 10b). However, unlike the experiments with tailless with metastatic disease confirmed that several predicted bulky glyco- MUC1 or the glycoprotein mimetics, which lack signalling capability, proteins could be detected in these patient samples (Fig. 5b). we cannot exclude that HA-induced growth and survival phenotypes We nextexamined whether a bulky glycocalyx couldpromote growth are not also, at least in part, induced through HA’s direct biochemical and survival ofnon-malignant MECs. Usingour glycoprotein mimetics, signalling activity20,21. we observed that untreated MECs or MECs incorporated with short We next addressed whether a bulky glycocalyx promotes MEC growth (3 nm) or medium (30 nm) length mimetics were not viable 24–48 h and survival by regulating focal adhesion assembly and crosstalk with after they were plated on highly compliant hydrogel substrates that growth factor signalling pathways5,7. We found that pharmacological mimic the stiffness of soft sites of colonization, like lung or brain (Young’s inhibition of kinases linked to growth factor signalling, including phos- modulus, E 5 140 Pa; Fig. 5c). By contrast, MECs incorporated with long phoinositide 3-kianse (PI(3)K), mitogen-activated kinase, and Src kinase, glycoprotein mimetics (80 nm) remained viable (Fig. 5c). Analysis of each independently inhibited the growth and survival of MUC1-expressing gene expression profiles and immunofluorescence analysis of freshly MECs on highly compliant substrates (Fig. 5e). We also noted that the isolated CTCs in our human metastatic breast cancer cohort revealed MUC1 growth and survival phenotype requires integrin engagement that MUC1 could be detected inmostof the samples examined (Fig. 5b). and integrin signalling through focal adhesion kinase (FAK), which Similar to results with the synthetic mimetics, we observed that ectopic mediates crosstalk between integrin and growth factor signalling path- expression of either full-length or a tailless, signalling-defective MUC1 ways (Fig. 5f and Extended Data Fig. 9b)5,6. Non-malignant MECs ex- innon-malignant MECs permitted their growth and survival even when pressing the MUC1 ectodomain, but not control MECs, assembled plated as single cells on compliant hydrogels (Fig. 5d and Extended Data distinct focal adhesion structures with activated Y397-phosphorylated Fig. 9a). FAK on compliant substrates (Extended Data Fig. 8c). Furthermore, We noted that the CTCs in our cohort also expressed high levels of MECs expressing wild-type or tailless, signalling defective MUC1, and CD44, a receptor that binds and retains bulky hyaluronic acid (HA) plated on the compliant substrates showed enhanced Y118-phosphory- glycan structures on the cell surface (Extended Data Fig. 10a)20. Sim- lated paxillin, ERK and AKT activation in response to epidermal growth ilar to our observations with MUC1 and bulky glycoprotein mimetics, factor stimulation (Fig. 5g and Extended Data Fig. 8d). This response was we observed that HA and integrins exhibit an anti-correlated spatial attenuated by FAK inhibition (Fig. 5g, h and Extended Data Fig. 8d).

a Upregulated in CTCs b Human CTCs c Soft ECM d Soft ECM e Soft ECM, 50 +MUC1(ΔCT) 1.0 30 10 MUC1 MUC1/Nuc. *** 8 CD44 ± *** 0.8 + : 91 1.4% 40 8 EPCAM 6 High 20 0.6 MEGF9 30 6 NOTCH1 4 ✯ 20 4 *** 0.4 SDC1 10 *** ✯ *** One-sided P Muc5B Cell death (%) Cell death (%) 2 0.2 ✯ 10 2

L1CAM Cells per colony (no.) Cells per colony (no.) 0 CHL1 0 0 0 0 MUC4 None 3 nm ΔCT) Src In. Low 30 nm80 nm DMSO Bulky MUC16 Control Control PI(3)K MEKIn. In. All genes Membrane +MUC1(ΔCT) +MUC1( Growth and f Soft ECM, g ERK activation h i Ctrl +MUC1(ΔCT) survival Kinetic trap +MUC1(ΔCT) 8 Adhesion plaque FAK Inhib. CT) 1.25 Δ ΔCT) 1.0 DMSO 6 Control +MUC1( +MUC1(+FAKi 0.75 Integrin 4 pERK 0.5 Mucin Total 2 Proliferation (AU) 0.25 pAKT (fold increase) Spatial control ERK 0 0 0 1 2 3 4 5 Stim. Stim. Stim. Unstim. Unstim. Unstim. Soft Stiff Soft Stiff Day ECM stiffness ECM Reciprocal force Figure 5 | Bulky glycoproteins promote cell survival and are expressed in quantified 48 h after plating on a soft hydrogel. e, Quantification of the number CTCs. a, Violin plots showing that genes encoding bulky transmembrane of vehicle (DMSO), PI(3)K inhibitor, MEK inhibitor, or Src inhibitor-treated proteins are more highly expressed in primary human tumours in patients with control and MUC1(DCT)-expressing MECs per colony 48 h after plating on a circulating tumour cells (CTCs). White dots and thick black lines indicate soft hydrogel. f, Proliferation of solvent (DMSO) or FAK-inhibitor-treated the median and interquartile range of the P-value distribution of transcripts of MUC1(DCT)-expressing MECs quantified at the indicated day after plating on all cellular genes (all genes), all transmembrane proteins (membrane), and soft hydrogels. g, Representative western blots showing phosphorylated and bulky transmembrane proteins (bulky). b, Heat map quantifying gene total ERK in control and MUC1(DCT)-expressing MECs plated on soft expression of bulky glycoproteins in CTCs isolated from 18 breast cancer hydrogels unstimulated or stimulated with EGF. Cells were treated with solvent patients (x axis; left), and representative immunofluorescence micrograph of (control, 1MUC1(DCT)) or FAK inhibitor (1MUC1(DCT) 1 FAKi) before MUC1 detected on human patient CTCs (right; scale bar, 5 mm). Quantification stimulation. h, Bar graphs showing quantification of immunoblots probed for of the percentage of CTCs with detectable MUC1 is shown. c, Cell death in activated AKT in control and MUC1(DCT)-expressing non-malignant MECs control non-malignant MECs and those with incorporated glycomimetics 24 h after plating on soft versus stiff hydrogels. i, Model summarizing quantified 24 h after plating on a soft (140 Pa) fibronectin-conjugated hydrogel biophysical regulation of integrin-dependent growth and survival by bulky substrate. d, Cell death (left graph) and growth (right graph) of control MECs glycoproteins. In all bar graphs, results are the mean 6 s.e.m. of at least 2–3 and those expressing cytoplasmic-tail-deleted MUC1 (1MUC1(DCT)) separate experiments (*P , 0.05; **P , 0.01; ***P , 0.001).

Together, these findings indicate that a bulky glycocalyx can promote 5. Walker, J. L., Fournier, A. K. & Assoian, R. K. Regulation of growth factor signaling and cell cycle progression by cell adhesion and adhesion-dependent changes in tumour aggression by enhancing integrin-dependent growth and sur- cellular tension. Cytokine Growth Factor Rev. 16, 395–405 (2005). vival (Fig. 5i). 6. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nature Rev. Cancer 10, 9–22 (2010). 7. Shibue, T. & Weinberg, R. A. Integrin b1-focal adhesion kinase signaling directs the Discussion proliferation of metastatic cancer cells disseminated in the lungs. Proc. Natl Acad. We present evidence to support a new paradigm for the biological func- Sci. USA 106, 10290–10295 (2009). tion of cell surface glycans and glycoproteins. Independent of, and in 8. Godula, K. et al. Control of the molecular orientation of membrane-anchored biomimetic glycopolymers. J. Am. Chem. Soc. 131, 10263–10268 (2009). addition to, their biochemical properties, we demonstrate how bulky 9. Godula, K., Rabuka, D., Nam, K. T. & Bertozzi, C. R. Synthesis and microcontact constituents of the glycocalyx can physically influence receptor organ- printing of dual end-functionalized mucin-like glycopolymers for microarray ization and activity. Although the current investigation focuses on applications. Angew. Chem. Int. Edn Engl. 48, 4973–4976 (2009). 10. Eng, E. T., Smagghe, B. J., Walz, T. & Springer, T. A. Intact aIIbb3 integrin is extended integrins, a bulky glycocalyx could, in principle, regulate any transmem- after activation as measured by solution X-ray scattering and electron microscopy. brane receptor that interacts with a tethered ligand. Candidate systems J. Biol. Chem. 286, 35218–35226 (2011). include neurological and immunological synapses22,cell–celladhesions23, 11. 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Nature 466, 263–266 (2010). 13,20 19. Nguyen, D. X., Bos, P. D. & Massague´, J. Metastasis: from dissemination to organ- ulatory enzymes, are so frequently elevated in many solid tumours . specific colonization. Nature Rev. Cancer 9, 274–284 (2009). Indeed, the growth and survival advantages afforded by these molecules 20. Toole, B. P. Hyaluronan: from extracellular glue to pericellular cue. Nature Rev. may preferentially select for cancer cells with a prominent glycocalyx Cancer 4, 528–539 (2004). 21. Bono, P., Rubin, K., Higgins, J. M. & Hynes, R. O. Layilin, a novel integral membrane and favour tumour cell dissemination and metastasis. Mechanical per- protein, is a hyaluronan receptor. Mol. Biol. Cell 12, 891–900 (2001). turbations to cell and tissue structure have a causal role in tumour de- 22. Dustin, M. L. Signaling at neuro/immune synapses. J. Clin. Invest. 122, 1149–1155 velopment and progression29,30, and we now implicate the glycocalyx’s (2012). importance in the metastatic mechano-phenotype. Our results suggest 23. Coombs, D., Dembo, M., Wofsy, C. & Goldstein, B. Equilibrium thermodynamics of cell-cell adhesion mediated by multiple ligand-receptor pairs. Biophys. J. 86, that the glycocalyx and its molecular constituents are attractive targets 1408–1423 (2004). for therapeutic interventions aimed at normalizing transmembrane 24. Salaita, K. et al. Restriction of receptor movement alters cellular response: physical receptor signalling. force sensing by EphA2. Science 327, 1380–1385 (2010). 25. Groves, J. T. Bending mechanics and molecular organization in biological membranes. Annu. Rev. Phys. Chem. 58, 697–717 (2007). METHODS SUMMARY 26. Lundmark, R. & Carlsson, S. R. Driving membrane curvature in clathrin-dependent Complete descriptions of the bioinformatics pipeline, computational model and and clathrin-independent endocytosis. Semin. Cell Dev. Biol. 21, 363–370 (2010). 27. Comoletti, D. et al. Synaptic arrangement of the neuroligin/b-neurexin complex expression constructs are presented in Supplementary Notes 1, 2 and 3, respect- revealed by X-ray and neutron scattering. Structure 15, 693–705 (2007). ively. Compliant hydrogels were fabricated from soft polyacrylamide (E 5 140 Pa) 28. Hildebrandt, H., Mu¨hlenhoff, M., Weinhold, B. & Gerardy-Schahn, R. Dissecting functionalized with fibronectin31 and plated with single cells for all hydrogel ex- polysialic acid and NCAM functions in brain development. J. Neurochem. 103 periments. FRET measurements were conducted in living cells on a spinning disk (Suppl. 1), 56–64 (2007). confocal (photobleaching FRET) or confocal (lifetime imaging) microscope32 (see 29. Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing 15 11 33 integrin signaling. Cell 139, 891–906 (2009). also Supplementary Note 4). SptPALM ,SAIM , single cell force spectroscopy , 30. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer integrin crosslinking17, and fibronectin fibrillogenesis34 measurements and assays Cell 8, 241–254 (2005). were conducted as previously described. Glycoprotein mimetics were synthesized 31. Lakins, J. N., Chin, A. R. & Weaver, V. M. Exploring the link between human and characterized as described in Supplementary Note 6 and subsequently incu- embryonic stem cell organization and fate using tension-calibrated extracellular bated with suspended cells (2 mM for 1 h) to incorporate onto the cell surface imme- matrix functionalized polyacrylamide gels. Methods Mol. Biol. 916, 317–350 (2012). diately before experimentation. For gene expression analysis of CTCs, 20 pools of 32. Bruns, N., Pustelny, K., Bergeron, L. M., Whitehead, T. A. & Clark, D. S. Mechanical CTCs were isolated from the blood of 18 metastatic breast cancer patients and quan- nanosensor based on FRET within a thermosome: damage-reporting polymeric tified by qPCR35. Immunofluorescence of CTCs was conducted on samples isolated materials. Angew. Chem. Int. Ed. 48, 5666–5669 (2009). from three patients35. 33. Friedrichs, J., Helenius, J. & Muller, D. J. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nature Online Content Methods, along with any additional Extended Data display items Protocols 5, 1353–1361 (2010). and Source Data, are available in the online version of the paper; references unique 34. Pankov, R. & Momchilova, A. Fluorescent labeling techniques for investigation of to these sections appear only in the online paper. fibronectin fibrillogenesis (labeling fibronectin fibrillogenesis). Methods Mol. Biol. 522, 261–274 (2009). 35. Magbanua, M. J. M. et al. Genomic profiling of isolated circulating tumor cells from Received 3 August 2013; accepted 23 May 2014. metastatic breast cancer patients. Cancer Res. 73, 30–40 (2013). Published online 25 June 2014. Supplementary Information is available in the online version of the paper. 1. Lanctot, P. M., Gage, F. H. & Varki, A. P. The glycans of stem cells. Curr. Opin. Chem. Acknowledgements We thank S. Gendler, J. Goedhart and M. McMahon for cDNAs, as Biol. 11, 373–380 (2007). indicated in the Methods section. We thank A. Walker for bioinformatics support, 2. Haltiwanger, R. S. & Lowe, J. B. Role of glycosylation in development. Annu. Rev. L. Hauranieh for assistance in CTC analysis, H. Aaron for assistance with FLIM, Biochem. 73, 491–537 (2004). J. B. Sibarita and M. Lagarde`re for support in sptPALM analysis, B. Hoffman in design of 3. Varki, A., Kannagi, R. & Toole, B. P. in Essentials Glycobiol. (Varki, A. et al.) http:// the FRET sensor, and T. Wittmann in assistance with pbFRET measurements. Image www.ncbi.nlm.nih.gov/books/NBK1963/ (Cold Spring Harbor Laboratory Press, acquisition was partly performed at the Nikon Imaging Center and Biological Imaging 2009). Development Center at UCSF and the Berkeley Molecular Imaging Center. This work 4. Paszek, M. J., Boettiger, D., Weaver, V. M. & Hammer, D. A. Integrin clustering is was supported by the Kavli Institute and UCSF Program for Biomedical Breakthrough driven by mechanical resistance from the glycocalyx and the substrate. PLOS postdoctoral fellowships to M.J.P.; DoD NDSEG Fellowship to M.G.R.; NIH GM59907 to Comput. Biol. 5, e1000604 (2009). C.R.B.; NIH Pathway to Independence Award K99 EB013446-02 to K.G.; French

Ministry of Research, CNRS, ANR grant Nanomotility, INSERM, Fondation ARC pour la G.G. conducted sptPALM experiments and analysed the results. V.M.W., R.B. and M.J.P. Recherche sur le Cancer, France BioImaging ANR-10-INBS-04-01, and Conseil designed the bioinformatics pipeline and R.B. implemented the pipeline and Re´gional Aquitaine to O.R. and G.G.; NIH AI082292-03A1 to D.A.H.; The Breast Cancer performed the large-scale gene expression analysis. J.K.M., A.C.W. and M.J.P. fabricated Research Foundation to M.J.M., H.S.R. and J.W.P.; NIH 2R01GM059907-13 to C.R.B. and conducted experiments on compliant hydrogels. M.J.M., H.S.R. and J.W.P. isolated and V.M.W.; and BCRP DOD Era of Hope Scholar Expansion grant BC122990, and NIH CTCs and measured CTC gene and protein expression. K.G., J.E.H. and C.R.B. NCI grants U54CA163155-01, U54CA143836-01, 1U01 ES019458-01, and designed, synthesized and characterized the glycoprotein mimetics. M.J.P and CA138818-01A1 to V.M.W. D.A.H. constructed and implemented the computational model. M.J.P. and V.M.W wrote the manuscript with input from all authors. Author Contributions V.M.W. and M.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 and Author Information Reprints and permissions information is available at constructed expression constructs. C.C.D. and M.J.P conducted single cell force www.nature.com/reprints. The authors declare no competing financial interests. spectroscopy measurements. M.J.P. and K.S.T. designed and implemented the Readers are welcome to comment on the online version of the paper. Correspondence interference microscope. M.J.P., L.C. and M.G.R. performed fluorescence, FRET and and requests for materials should be addressed to V.M.W. interferometric imaging and M.J.P. wrote the accompanying analysis software. O.R. and ([email protected]).

METHODS Immunofluorescence and imaging. Cells were fixed and labelled as previously described and imaged at random on a Zeiss LSM 510 microscope system with a Bioinformatics. To estimate protein-level contributions to extracellular membrane 100X Plan Apochromat NA 1.4 objective and 488 nm Argon, 543 nm HeNe, and bulkiness, we used TMHMM to identify extracellular domains within each isoform 633 nm HeNe excitation lines30. sequence (RefSeq v47) and counted the number of putative extracellular glycosyla- Live epithelial cell imaging and FRET. tion sites predicted by NetOGlyc 3.1 and search of N-glycosylation motifs. Gene- Normal growth media was exchanged for wise enrichment of mRNA upregulation among bulkyproteins inclinical data (GEO a similar formulation lacking phenol red and supplemented with 15 mM HEPES buffer, pH 7.4. Cells were imaged on a Ti-E Perfect Focus System (Nikon) equipped accessions GSE12276 and GSE31364) was tested by permuting P values quantifying with a CSU-X1 spinning disk confocal unit; 454 nm, 488 nm, 515 nm and 561 nm evidence for upregulation in the appropriate samples. Variance in mRNA upregu- lasers; an Apo TIRF 60X NA 1.49 objective; electronic shutters; a charged-coupled lation explained by membrane bulkiness was estimated by regressing the negative log- device camera (Clara; Andor) and controlled by NIS-Elements software (Nikon). transformed P values on the square root of the combined N- and O-glycosylation sites and comparing the residuals with the intercept model. Additional details of For measurement of FRET efficiency, the acceptor photobleaching method pbFRET the analysis and models are provided in Supplementary Note 1. was implemented with live cells on the spinning disk confocal. Cyan fluorescent protein (CFP) was first imaged with 454 nm excitation and a 480/20 emission filter, Computational model. A mechanical model of the cell–ECM interface was con- yellow fluorescent protein (YFP) was subsequently bleached using a 100 mW 515 nm structed as described previously4. A summary of the model is described in Supplemen- laser for 10 s, and CFP was imaged again following bleaching of YFP. Microscope tary Note 2 and parameters are detailed in Supplementary Table 1. Z-focus was maintained during image acquisition using the Perfect Focus System. Antibodies and reagents. Antibodies used include: mouse monoclonal antibody Background images were constructed by imaging 10 unique cell-free regions on (mAb) vinculin (MAB674; Millipore), mouse mAb talin (8d4; Sigma), rat mAb b - 1 the coverslip and averaging the intensity at each pixel. The FRET efficiency was integrin (AIIBII), rabbit mAb paxillin (Y113; Abcam), rabbit mAb FAK pY397 calculated on a pixel-by-pixel basis according to: (141-9; Invitrogen), rabbit polyclonal antibody (pAb) a5-integrin (AB1928; Millipore), mouse mAb MUC1 (HMPV; BD Pharminigen), hamster mAb MUC1 (CT2; Thermo I {B FRET efficiency (%) 5 1{ pre pre 100% Scientific), rabbitmAb Src Family pY416 (D49G4;CellSignaling), mouse mAbFAK Ipost{Bpost (77; BD Transduction Laboratories), rabbit pAb paxillin pY118 (2541; Cell

Signaling), rabbit mAb pan-AKT (C67E7; Cell Signaling), rabbit pAb AKT where Ipre is the CFP intensity before bleaching YFP, Bpre is the CFP-channel back- pS473 (9271; Cell Signaling); rabbit mAb ERK1/2 pT202/pT204 (197G2; Cell Sig- ground intensity before bleaching YFP, Ipost is the CFP intensity after bleaching naling); rabbit pAb ERK1/2 (9102; Cell Signaling); rabbit mAb Gapdh (14C10; Cell YFP, and Bpost is the CFP-channel background intensity after bleaching YFP. Ap- Signaling); Alexa 488 and Alexa 568 conjugated goat anti-mouse and anti-rabbit propriate controls were implemented to account for inadvertent CFP photobleach- mAbs (Invitrogen); FITC conjugated anti-hamster mAbs; Cy5-conjugatedgoatanti- ing, incomplete YFP photobleaching, and intermolecular FRET (see Supplementary mouse and rabbit mAbs (Jackson); and HRP conjugated anti-rabbit and anti-mouse Note 4). mAbs. Chemical inhibitors used in these studies include ROCK inhibitor Y-27632 Time-domain fluorescence lifetime imaging microscopy (FLIM) for additional (Cayman Chemical),myosin-II inhibitor (-)-blebbistatin (CaymanChemical),FAK FRET sensor characterization was implemented with an inverted Zeiss LSM510 inhibitor FAK inhibitor 14 (Tocris), MEK inhibitor U0126 (Cell Signaling), PI(3)K Axiovert 200Mmicroscope with a Plan NeoFLUAR 40X/1.3NA DIC oil-immersion inhibitor Wortmannin (Cell Signaling), Src inhibitor Src I1 (Sigma), and DiI (Mo- objective lens, equipped with a TCSPC controller (SPC-830 card; Becker & Hickl, lecular Probes). Berlin, Germany) as described previously32. CFP was excited with 440 nm light Cell culture conditions. All cells were maintained at 37 uC and 5% CO2. MCF10A generated by frequency doubling of 880 nm pulses from a mode-locked Ti:sapphire humanMECs(ATCC)wereculturedinDMEM F12 (Invitrogen) supplemented with laser (Mai-Tai, Spectra-Physics, 120-150 fs pulse width, 80 MHz repetition rate, and 21 5% donor horse serum (Invitrogen), 20 ng ml epidermal growth factor (Peprotech), Frequency Doubler and Pulse Selector, Spectra-Physics, Model 3980). The emission 21 21 21 10 mgml insulin (Sigma), 0.5 mgml hydrocortisone (Sigma), 0.1 mgml cholera lightwas passedthrougha NFT 440beamsplitter, directedtothe fibre-out port of the 21 toxin (Sigma), and 100 units ml penicillin/streptomycin. MCF7 and T47D breast confocal scan-head, filtered with a 480BP40 filter (Chroma Technology, Rockingham, tumour lines (ATCC) were grown in DMEM supplemented with 10% fetal bovine VT) and detected by a PMC-100 photomultiplier (Becker & Hickl). The pinhole was 21 serum (Hyclone) and 100 units ml penicillin/streptomycin. 293T cells (ATCC) settogive anoptical slice of ,4.0 mm.Images of 386 3 386pixelswere averaged over were maintained in DMEM supplemented with 10% donor horse serum, 2 mM ,120 s. Data analysis toproduceanintensity imageand a FLIM imagewas done off- L-glutamine, and penicillin/streptomycin. Mouse embryonic fibroblasts (MEFs) line using the pixel-based fitting software SPCImage (Becker & Hickl), assuming were cultured in DMEM with 10% fetal bovine serum. Cell lines were tested rou- double exponential decay during the first 8.5 ns of the 12.5 ns interval between laser tinely for mycoplasma contamination. For transient gene expression in MECs, con- pulses. Images were scaled to 256 3 256 pixels and no binning was used. Lifetime structs in pcDNA3.1 or Clonetech-style vectors were nucleofected with Kit V distributions were calculated for a masked portion of the FLIM image, generated (Lonza) using program T-024 24 h before experimentation. Transient transfection with a triangle algorithm threshold of the photo count intensity image. in MEFs was conducted 48 h before experimentation using Fugene 6 (Roche) or Scanning angle interference microscopy. Cells were plated overnight on reflective nucleofection. For stable cell lines harbouring tetracycline inducible transgenes, ex- silicon substrates, fixed or roofed to remove the dorsal membrane (for MUC1– 21 pression was induced with 0.2 ng ml doxycycline 24 h before experimentation. GFP imaging) and then fixed, and imaged randomly as previously described, scan- The conditional v-Src oestrogen receptor fusion (v-Src–ER) was activated with 1 mM ning the incident angle of excitation light from 0u to 42u with a one-degree sampling 4-hydroxytamoxifen 48 h before experimentation to achieve transformation. For rate11. Z-positions were localized with custom algorithms previously described and pY118 pERK, paxillin, and pAKT studies, cells were plated on fibronectin-conjugated available on request11. 21 polyacrylamide hydrogels, serum-starved overnight, and stimulated with 20 ng ml Single particle tracking photo-activation localization microscopy (sptPALM). EGF before collecting protein lysates. Data are reported as the fold increase of phos- sptPALM experiments were performed and analysed as previously described15. phorylated protein relative to total protein, following EGF stimulation. Briefly, live MEFs were imaged at 37 uC in a Ludin chamber on a Ti Perfect Focus Preparation of cellular substrates. Glass and silicon substrates were prepared by System equipped with a Plan Apo 100X NA 1.45 objective, and an electron multi- 2 2 glutaraldehyde activation followed by conjugation with 10 mgml 1 (glass) or 20 mgml 1 plying charge-coupled device (Evolve; Photometrics). For photo-activation local- (silicon) fibronectin as described11. Compliant polyacrylamide hydrogel substrates ization, cells expressing mEOS2-tagged constructs were activated using a 405 nm (soft: 2.5% acrylamide, 0.03% Bis-acrylamide; stiff: 10% acrylamide, 0.5% Bis- laser (Omicron) and the photo-activated fluorophores were excited simultaneously acrylamide) were prepared as previously described with one modification: func- with a 561 nm laser (Cobolt Jive). The powers of the activation and excitation lasers tionalization with succinimidyl ester was with 0.01% N6, 0.01% Bis-acrylamide, were adjusted to keep the number of activated molecules constant and well sepa- 0.025% Irgacure 2959, and 0.002% Di(trimethylolpropane) tetraacrylate (Sigma)31. rated. GFP fusions of paxillin or MUC1 were imaged in between each sptPALM Following functionalization with succinimidyl ester, hydrogels were conjugated over- sequence by imaging the GFP signal above the unconverted mEOS2 background. 2 night with 20 mgml 1 fibronectin at 4 uC and rinsed twice with PBS and DMEM The acquisitionwas driven by Metamorph software (Molecular Devices) in stream- before cell plating. ing mode at 50 Hz. For tracking, single-molecules were localized and tracked over Generation of expression constructs. A description of cDNA constructs and their time using a combination of wavelet segmentation and simulated annealing algo- construction is provided in Supplementary Note 3. rithms. Trajectories lasting at least 20 frames were selected for further quantifica- Generation of stable cell lines. Stable transgene expression was achieved through tion, including calculation of immobile, confined and free-diffusing fraction (see retroviral or lentiviral transduction as previously described11,30. Supplementary Note 5)15. Flow cytometry and cell sorting. Cell surface MUC1 was labelled directly with Preparation of glycopolymer-coated cell surfaces. Mucin mimetic glycopolymers FITC-conjugated mAb MUC1 (clone HMPV). Cytometry and sorting were con- with lipid insertion domains were synthesized and characterized as described in ducted on a FACSAria II (BD Biosciences). Supplementary Note 6. For incorporation into the plasma membrane, cells were

©2014 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH suspended in DMEM and incubated with 2 mM glycopolymer for 1 h. Cells were was prepared with donor horse serum that was depleted of fibronectin using gelatin pelleted by centrifugation and re-suspended in growth media to remove unincor- Sepharose 4B (GE Healthcare). MCF10A cells were plated in the depleted media on porated polymer. fibronectin-conjugated glass coverslips and incubated the next day in 10 mgml21 Quantification of adhesion complexes. Images of adhesions in fixed, immuno- labelled fibronectin for one hour. Cells were quickly rinsed in PBS, fixed in 4% labelled cells or cells expressing paxillin–mCherry were randomly acquired, smoothed paraformaldehyde, and imaged at random on a spinning disk confocal. with a median filter, and background subtracted (12 pixel diameter) in ImageJ. Isolation and gene expression profiling of CTCs. Twenty CTC samples were iso- Adhesion sizes and the number of adhesions per cell were subsequently quantified latedfrom the bloodof 18 metastaticbreast cancer patients as previously described35. in ImageJ with the ‘Analyze Particles’ tool. Briefly, whole blood was subjected to EpCAM-based immunomagnetic enrich- Integrin crosslinking assay. Cells were incubated in suspension with inhibitor ment followed by fluorescence-activated cell sorting of CTCs defined as nucleated, (Y-27632 or Blebbistatin) orcontrol solvent for 1 h beforeplating onglass substrates. EpCAM-positive, CD45-negative cells. CTCs were sorted directly onto lysis buffer Integrin was crosslinked to fibronectin with 1 mM 3,39-dithiobis(sulfosuccinimi- (Taqman PreAmp Cells-to-Ct kit, Life Technologies). cDNA of target genes were dylpropionate) (Pierce Chemical) and cells were extracted with SDS buffer as pre- pre-amplified (14 cycles) and measured via qPCR analysis. The mean Ct for ACTB viously described17. Crosslinked a5 integrin was immuno-labelled and imaged at and GAPDH was used for normalization to calculate relative gene expression (DCt). random with a Plan Apo VC 60X objective on a Nikon TE2000 epi-fluorescence Studies involving CTCs were approved by the UCSF Committee on Human Re- microscope equipped with a charged-coupled device camera (HQ2; Photometrics). search. Samples were obtained with IRB approved consent from all patients. Single cell force spectroscopy. Measurements were performed on an Asylum MFP- Immunofluorescence labelling of CTCs. CTC samples were isolated from the 3D-BIO atomic force microscope as previously described33. Briefly, cells were attached blood of three metastatic breast cancer patients as described for gene expression to a streptavidin-coated, tipless cantilever using biotinylated jacalin (MUC1-expressing profiling. Isolated CTCs were mounted and fixed on poly-L-lysine-coated slides cells) or concanavalin A (all other cells) and pressed against the adhesive substrate and labelled with FITC-conjugated MUC1 mAb (Clone HPMV). As a control, with a calibrated force and duration before measuring the force required to detach purified white blood cells from the same patients were prepared similarly, and the cell from the substrate. All measurements were conducted on fibronectin- or their immunofluorescence was compared to CTC samples. BSA-coated glass slides at room temperature. The relative rate of adhesion was cal- Statistics. Statistical significance of experimental data sets was determined by culated as the slope of a linear fit of cellular detachment force against contact time. Student’s t-test after confirming that the data met appropriate assumptions (nor- Assessment of fibronectin–fibrillogenesis. Human recombinant fibronectin was mality, homogenous variance and independent sampling). Statistical analyses of labelled with N-hydroxysuccinimide Alexa568 (Invitrogen) according to manu- microarray gene expression data sets are described in detail in Supplementary Note 1. facturer’s protocol and dialysed extensively in PBS. Conversion of soluble, fluor- All public microarray data were downloaded from the NCBI Gene Expression Om- escently labelled fibronectin from the growth media into insoluble fibrils was nibus website and analysed using custom R scripts (all Perl, PHP and R scripts used imaged according to published protocol34. Briefly, MCF10A complete growth media in this work are available on request).

Extended Data Figure 1 | Large-scale gene expression analysis reveals gene-wise estimates of the compared parameters are similarly ranked (for increased expression of genes encoding bulky glycoproteins and glycan- example, genes with high values of X also tend to have high values of Y). The modifying enzymes in primary tumours of patients with disseminated data indicate that the number of extracellular N-glycosylation sites and disease. a, Bioinformatics pipeline to estimate the extracellular bulkiness of a O-glycosylation sites identified within a gene are only weakly correlated, and protein from its corresponding amino acid sequence. For each isoform neither dominates the total number of sites estimated per gene. c, Violin plots sequence, the transmembrane and extramembrane domains were identified contrasting the distributions of gene-wise one-sided P values (y axis) using a hidden Markov model (TMHMM). A combination of motif searches quantifying evidence for transcriptional upregulation of glycosidases and and neural network prediction then identified likely N- and O-glycosylation glycosyltransferases, and subsets of glycosyltransferases (sialyltransferases and sites within each sequence. Isoform-level bulkiness estimates were generated by N-acetylgalactosaminyltransferases) with the full distribution. White dots summing the number of predicted N- and O-glycosylation sites located within and thick black lines indicate the median and interquartile range of the gene- the extramembrane regions of the isoform. b, Heat map depicting the pairwise wise P-value distribution among category members, and the width of the violin spearman correlation coefficients calculated by comparing all per-gene along the y axis indicates the density of the corresponding values. P values estimates of the total number of extra-membrane amino acids (AAoutside), are derived from comparisons of expression levels in primary tumours of N-glycosylation sites (Nglyc), O-glycosylation sites (Oglyc), and the overall patients with or without distant metastases using a t-test. Indicated P values bulkiness measure (total sites; for example, the sum of extra-membrane N- and were estimated using a one-sided Kolmogorov–Smirnov test. d, Violin plots O- glycosylation sites). Correlation coefficients relating the corresponding quantifying transcriptional upregulation of glycan-modifying enzymes in gene-wise measures are listed in the corresponding cells and depicted on a primary tumours of patients presenting with circulating tumour cells compared colour scale, where white corresponds to perfect correlation (rho 5 1), and the to tumours without detectable circulating tumour cells. e, Table of bulky dendrograms indicate the overall relationship between the parameters, glycoproteins and potential bulk-adding glycosyltransferases whose expression estimated by Euclidean distance. High correlation coefficients indicate that is upregulated in tumours that present with circulating tumour cells.

Extended Data Figure 2 | Computational model of the cell–ECM interface. of which is depicted above. Integrins are tethered to the cell surface and their Schematic of an integrated model that describes how the physical properties of distance-dependent binding to the ECM–substrate is calculated according to the glycocalyx influence integrin–ECM interactions. The cell surface is the Bell model. To calculate integrin-binding rate as a function of lateral modelled as a three-dimensional elastic plate; the ECM as a rigid substrate distance from an adhesion cluster, an adhesion cluster is first constructed by underneath the cell surface; and the glycocalyx as a repulsive potential between assembling a 3 3 3 bond structure. The rates for additional integrin–ECM the plate and substrate. To compute stress–strain behaviour, the model is bonds then are computed at various distances from the cluster. discretized using the three-dimensional lattice spring method, the cross-section

Extended Data Figure 3 | Synthesis and characterization of glycoprotein incorporation of polymer on the surface of mammary epithelial cells (left) and mimetics. a, Scheme for synthesis of lipid-terminated mucin mimetics labelled binding with recombinant Alexa568-labelled galectin-3 with or without with Alexa Fluor 488 (AF488). b, Reagents and yields for the synthesis of competitive inhibitor, b-lactose (right). Although a weak affinity between polymers 3a–c. c, Characteristics of polymers 6a–c based on 1H NMR spectra. galectin-3 and the pendant N-acetylgalactosimes has previously been reported, Glycoprotien mimetics were engineered to have minimal biochemical the results suggest that incorporation of polymer does not significantly change interactivity with cell surface lectins. d, Flow cytometry results quantifying the affinity of the cell surface for lectins.

Extended Data Figure 4 | MUC1 expression constructs. a, Schematic of full-length MUC1 or MUC1(DCT). b, Schematic of MUC1 strain sensor MUC1 expression constructs. Full-length MUC1 consists of a large and control constructs. Cysteine-free mTurqoiuse2 (CFP), Venus (YFP), or a ectodomain with 42 mucin-type tandem repeats, a transmembrane domain, FRET module consisting of the fluorescent proteins separated by an elastic and short cytoplasmic tail. The tandem repeats and cytoplasmic tail are deleted linker (8 repeats of GPGGA) are inserted into the MUC1 ectodomain adjacent in MUC1(DTR) and MUC1(DCT), respectively. For fluorescent protein to the MUC1 tandem repeats. The mucin tandem repeats are deleted in fusions, mEmerald (GFP) and mEOS2 are fused to the C terminus of ectodomain-truncated variants (DTR).

Extended Data Figure 5 | MUC1-mediated adhesion formation. adhesion. Binding of soluble fibronectin to MUC1 was not detected. Scale bar, a, Quantification of the average number of large adhesions, greater than 1 mm2, 10 mm. c, Time lapse images of MUC1–YFP and vinculin–mCherry, showing per area of cell in control epithelial cells (Control) and those ectopically the dynamics of adhesion assembly (Vinc.) and MUC1 patterning (MUC1). expressing ectodomain-truncated MUC1 (1MUC1(DTR)), wild-type MUC1 Scale bar, 1 mm. d, Rate of adhesion measured with single cell force (1MUC1), or cytoplasmic-tail-deleted MUC1 (1MUC1(DCT)). Results are spectroscopy of control (Cont.), a5 integrin-blocked (anti-a5), and MUC1- the mean 6 s.e.m. of three separate experiments. b, Fluorescence micrographs expressing cells (1MUC1) to fibronectin-coated surfaces and control cells to showing immuno-labelled MUC1 and fluorescently labelled fibronectin BSA-coated surfaces (BSA). Results are the mean 6 s.e.m. of at least 15 cell fibrils in control and MUC1-expressing epithelial cells. Soluble, labelled measurements. Statistical significance is given by *P , 0.05; **P , 0.01; fibronectin in the growth media was deposited by cells at sites of cell–matrix ***P , 0.001.

Extended Data Figure 6 | b3 integrin mobility in MUC1-expressing cells. right, panels show GFP-tagged wild-type MUC1 (red) and positions of 21 a, Molecular diffusivity and adhesion enrichment measured with sptPALM in individual b3 integrins (green) in MEFs without Mn treatment (left panel) mouse embryonic fibroblasts (MEFs). Adhesion enrichment is reported as the and individual integrin trajectories recorded with sptPALM within MUC1-rich ratio of the number of molecules detected inside focal adhesions per unit area to regions, outside MUC1-rich regions, and that cross MUC1 boundaries (scale the number of molecules detected outside focal adhesions per unit area. bar, 2 mm). The ratio of integrins crossing out versus crossing in the MUC1 b, Mean diffusion coefficients measured for freely diffusive b3 integrin tracks boundaries per cell is close to one (1.0 6 0.1, n 5 9 cells, 4,145 trajectories) outside of adhesive contacts in control (Cont.) and MUC1-transfected showing that the flux of free diffusing integrins crossing in or out the mucin 21 (1MUC1) MEFs with and without Mn to activate b3. c, Mean diffusion region is the same. g, From left to right, panels show integrin trajectories within coefficients measured for confined b3 integrin tracks outside of adhesive an arbitrary region drawn in a MUC1-rich area (dashed white circles), outside contacts in MEFs with and without Mn21. d, Mean radius of confinement of the circled region, and that cross the circled region (scale bar, 2 mm). The measured for confined b3 integrin tracks outside of adhesive contacts in MEFs ratio of integrins crossing the MUC1-rich boundaries versus the fictive with and without Mn21. e, Fraction of immobilized (Imm.), confined (Conf.), boundaries per cell is close to one (1.2 6 0.2, n 5 9 cells, 9,321 trajectories), and freely diffusive (Free) b3 integrins inside of adhesive contacts in control and showing that the MUC1–adhesive zone boundary does not affect the diffusive MUC1-transfected MEFs with and without Mn21 treatment. f, From left to crossing of integrins. For all bar graphs, results are the mean 6 s.e.m.

Extended Data Figure 7 | MUC1 strain gauge. a, Western blot of indicated photons from CFP and their fluorescence lifetimes in MECs expressing construct expressed in HEK 293T cells and probed with anti-GFP family ectodomain-truncated (MUC1(DTR) sensor) or full-length MUC1 strain antibody, or full-length MUC1 construct expressed in HEK 293T cells and sensors (MUC1 sensor). Shorter lifetimes are indicative of higher energy probed with an antibody against the MUC1 tandem repeats. b, Pseudo- transfer between the CFP donor and YFP acceptor, and thus closer spatial coloured images showing similar FRET efficiencies measured by the proximity of the donor and acceptor (scale bar, 10 mm). f, Representative profile photobleaching FRET method for mammary epithelial cells (MECs) expressing of CFP lifetimes and emitted photons of the full-length MUC1 sensor along low (Low) and high (High) levels of the sensor construct. Scale bar, 5 mm. c, Plot the red line in panel e. Pixels 0 and 40 correspond to the base and tip of the showing the level of CFP bleaching per CFP imaging cycle in MECs. d, Control arrow, respectively. A drop in fluorescence lifetime (Lifetime) is often observed images showing minimal intermolecular FRET in MECs expressing similar before the drop in MUC1 molecular density (Photons) as an adhesive zone levels of both MUC1 CFP and MUC1 YFP. e, Micrographs showing the emitted is approached.

Extended Data Figure 8 | Tension-dependent integrin activation and focal (scale bar, 10 mm). c, Fluorescence micrographs of paxillin–mCherry and adhesion assembly in MUC1-expressing cells. a, Fluorescence micrographs of immuno-labelled activated FAK (pY397) in control and MUC1(DCT) fibronectin-crosslinked a5 integrin in control and MUC1-expressing expressing MECs plated on compliant fibronectin-conjugated hydrogels mammary epithelial cells (MECs) treated with solvent alone (DMSO), myosin- (E 5 140 Pa; scale bar, 3 mm; ROI scale bar, 0.5 mm). d, Western blots showing II inhibitor (blebbistatin; 50 mM), or Rho kinase inhibitor (Y-27632; 10 mM) for phosphorylation of paxillin (pY118) in control and MUC1-expressing MECs 1 h and detergent-extracted following crosslinking. Only fibronectin-bound on compliant substrates (E 5 140 Pa) following overnight serum starvation integrins under mechanical tension are crosslinked and visualized following and stimulation with EGF. MUC1-expressing cells treated with a detergent extraction (scale bar, 15 mm). b, Fluorescence micrographs showing pharmacological inhibitor of focal adhesion kinase (1FAKi) for 1 h before EGF formation of myosin-independent adhesion complexes in MUC1-expressing stimulation did not exhibit robust paxillin phosphorylation. MECs. Cells were pre-treated for 1 h and plated for 2 h in 50 mM blebbistatin

Extended Data Figure 9 | Cell proliferation on soft ECM. a, Fluorescence MUC1(DCT)-expressing epithelial cells on soft hydrogels conjugated with micrographs showing DAPI-stained nuclei of control and MUC1(DCT)- bovine serum albumin (BSA) or fibronectin (Fn). Cells plated similarly on expressing MECs after 24 h of plating on soft, fibronectin-conjugated hydrogels BSA– and Fn–hydrogels, but cell proliferation was significantly enhanced on (E 5 140 Pa; scale bar, 250 mm). The majority of cells plated as single cells, Fn–hydrogels. Results are the mean 6 s.e.m with statistical significance given indicating that multi-cell colonies that formed at later time points were largely by *P , 0.05; **P , 0.01; ***P , 0.001. attributed to cell proliferation. b, Quantification of cell proliferation of

Extended Data Figure 10 | Hyaluronic acid production by tumour cells v-Src-transformed MECs per colony 48 h after plating on soft polyacrylamide promotes cellular growth. a, Quantification of hyaluronic acid (HA) cell gels (fibronectin-conjugated) and treated with vehicle (DMSO), hyaluronic surface levels on control (10A-Cont.), transformed (10A-v-Src, 10A-HRAS) acid synthesis inhibitor 4-methylumbelliferone (14MU; 0.3 mM), or and malignant (MCF7, T47D) mammary epithelial cells (MECs). competitive inhibitor HA oligonucleotides (1Oligo; 12-mer average b, Fluorescence micrographs of HA and immuno-labelled paxillin on the oligonucleotide size; 100 mg ml21). Results are the mean 6 s.e.m with statistical v-Src transformed MECs (scale bars, 3 mm). c, Quantification of the number of significance is given by *P , 0.05; **P , 0.01; ***P , 0.001.