Abstract
Mucus is essential for protection of the airways; however, in chronic airway disease mucus hypersecretion is an important factor in morbidity and mortality. The properties of the mucus gel are dictated in large part by the oligomeric mucins and, over the past decade, we have gained a better understanding of the molecular nature of these complex O-linked glycoproteins. We know now that MUC5AC mucins, as well as different glycoforms of the MUC5B mucin, are the predominant gel-forming glycoproteins in airways mucus. Furthermore, the amount, molecular size, and morphology of these glycoproteins can be altered in disease. From more recent data, it has become clear that oligomeric mucins alone do not constitute mucus, and other mucin and nonmucin components must be important contributors to mucus organization and hence airways defense. Therefore, the challenge over the coming decade will be to investigate how the oligomeric mucins are organized to yield “functional” mucus. Such studies will provide a clearer perception of airways mucosal protection and may highlight specific components as potential targets for therapeutic strategies for the treatment of hypersecretory disease.
The respiratory mucus gel performs a number of essential functions that collectively lead to the protection of the airways. This highly hydrated gel, in conjunction with ciliated epithelial cells, forms the mucociliary “escalator,” which, along with cough, is essential for the maintenance of sterile and unobstructed airways. By contrast, overproduction of mucus with altered rheologic properties is an important factor in the morbidity and mortality of chronic airways disease (e.g., asthma, cystic fibrosis [CF], and chronic obstructive pulmonary disease [COPD]) (1–3). As a result of the change in physical properties of the gel, mucociliary clearance is impaired or abolished with the attendant problems of poor gas exchange and/or inflammation and/or bacterial colonization. Thus, production of mucus with the “correct” properties for airways protection is paramount for health.
A variety of factors have been found to trigger mucus hypersecretion, including proinflammatory cytokines (e.g., interleukin-4, interleukin-13, epidermal growth factor, and tumor necrosis factor-α), ATP, bacterial exoproducts, and host proteases (4–9), but the mechanisms underlying mucus biogenesis and secretion, and its molecular composition and supramolecular organization, are still obscure. The major gel-forming components of the airways mucus gel are the oligomeric mucins, and the hypothesis that drives our research is that these glycoproteins play the vital role in determining the physical properties of this essential barrier in health and disease. However, it should also be pointed out, especially in pathologic conditions, that other molecules such as proteoglycans and DNA may contribute to the physical properties of airways mucus.
Over the last decade, great strides have been made in identifying and characterizing the major oligomeric mucins present in airways sputa. However, there have been no studies to date that relate the ensemble of their physical properties (i.e., length, charge density, macromolecular architecture) to a specific set of rheologic parameters for a mucus gel, never mind its optimization to a specific function, such as ciliary transport. In this article, we have been given the task of reviewing our own contributions to this area. We have also suggested further avenues of investigation that will be necessary to gain a better understanding of the biology and pathobiology of this essential barrier.
Mucins are currently defined as high-Mr glycoproteins that contain at least one and sometimes multiple protein domains that are sites of extensive O-glycan attachment (mucin-like domains). Thus, the composition of these glycoproteins is dominated by carbohydrate, which can total in some cases as much as 80% of the weight of the molecule. There appear to be two major types of mucin, one thought to be monomeric and, primarily, but not exclusively, located at the cell surface, and the other oligomeric. This latter type is secreted and thought to be responsible for the rheologic properties of mucus. The mucin-like domains are enriched in the amino acids serine and threonine, which are the sites for the attachment to the linkage sugar N-acetylgalactosamine. In addition to N-acetylgalactosamine, mucin oligosaccharides may also contain N-acetylglucosamine, galactose, fucose, sialic acids, and sulfate. The oligosaccharides have remarkable structural diversity and all of their specific roles remain to be fully understood. However, a consequence of their attachment to the polypeptide is to cause it to stiffen, resulting in a large expansion of the volume domain of the molecule, which gives mucins their characteristic space-filling, and thus gel-making, properties (10). Furthermore, it has long been known that certain bacteria bind specific oligosaccharide ligands (for a review see [11]). It is likely that the primary function of the oligosaccharide diversity is to enhance the possibility that bacteria bind to mucus, thus facilitating their removal by mucociliary transport. Moreover, 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 play a key role in governing the susceptibility to infection.
The studies we shall summarize have been concerned solely with the gel-forming mucins. From our studies in collaboration with Ingemar Carlstedt in the early 1990s, it was clear that the gel-forming mucins in airways mucus consisted of a heterogeneous mixture of glycoproteins that was physically similar and extremely large (12–16). The mucus gel could be almost completely solubilized by noncovalent bond–breaking agents (e.g., 6M guanidinium chloride), a process usually achieved overnight but in some cases taking days. Thus, entanglement of the long mucin chains was considered to be the primary mechanism for gel formation and, as a consequence, the size, concentration, and chemical nature of the mucins are important factors for determining the properties of the gel. Physical characterization of the mucins isolated from sputum using light scattering and electron microscopy showed them to be polydisperse in both mass (2–40 mD) and size (0.5–10 μm in length). Furthermore, these studies demonstrated that the component mucin monomers or subunits (2–3 mD) are assembled linearly and held together by disulfide bonds (17). Figure 1
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.
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It is now apparent that these oligomeric glycoproteins are members of a larger family of mucins. Of the 13 mucin genes (MUC) currently identified, only 4 (MUC2, MUC5AC, MUC5B, and MUC6) contain the cysteine-rich motifs in their C- and N-terminal domains necessary for oligomerization (18–21). Three of these genes, MUC2, MUC5AC, and MUC5B, are expressed in the airways. A number of important studies on airways mucins have concentrated on messenger RNA expression, in particular focusing on MUC2 and MUC5AC, and highlight specific disease-related factors that upregulate these two mucins (4–9, 22–24). However, the fact that these macromolecules are synthesized and then stored awaiting the appropriate stimulus for secretion implies that messenger RNA studies may be misleading for the amount of mucin in mucus. This is borne out by biochemical data, which have demonstrated that the products of the MUC5AC and MUC5B genes are predominant in sputum (25–29) and that the MUC2 mucin is barely detectable (see subsequent text). Thus, there seems to be a discrepancy between MUC2 messenger RNA levels and the occurrence of the mature MUC2 mucin in sputum. This may be due to the apparent “insolubility” of this glycoprotein, as has been reported for MUC2 isolated from small-intestinal mucus (30), causing it to be retained in the airways. While our data do not rule this out, in the single study we have performed, where the mucus was physically removed from an asthmatic lung postmortem, there was still little evidence for this mucin (3, 31).
Matching the polypeptides of the different mucins present in mucus to their respective genes has been a long process, which was eventually achieved by fractionation of the mucins (after reduction of their disulfide bonds) by anion exchange chromatography and agarose gel electrophoresis, and then by a combination of biochemical analyses (i.e., amino acid composition, peptide sequencing, and peptide fingerprinting) as well as reactivity with mucin-specific antisera (for more details see Figure 2)
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).
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The analysis of multiple sputum samples revealed that the two major mucin gene products, MUC5AC and MUC5B have different chemical properties. The MUC5AC mucin has a more homogeneous charge distribution than MUC5B, which occurs in differently charged forms (26). In most samples analyzed, two different MUC5B forms (glycoforms) are found, and while their charge density is not identical between individuals it is apparent that a “high” and “low” charge form occurs in most cases (Figure 3)
Figure 3. Agarose gel electrophoresis of reduced sputum samples. Sputa, NaCl-induced from healthy airways (lanes 1, 6 and 7) and spontaneous secretions from diseased airways, were solubilized in 6 M GuCl and then dialyzed into 6 M urea. The samples were reduced with 10 mM dithiothreitol, treated with 25 mM iodoacetamide to alkylate-free thiol groups and then subjected to electrophoresis on a 0.7% agarose gel (for more details see Reference [65]). After electrophoresis, gels were transferred to nitrocellulose and probed with antisera specific for (A) MUC5AC and (B) MUC5B mucins (section from the data presented in Reference [39]). These blots were also stained with an antiserum directed against MUC2, but no bands were visualized (data not shown). The results show the greater variation in electrophoretic migration of the reduced MUC5B mucins compared with MUC5AC; furthermore, with the exception of one sample (lane 6), all the others exhibit two different MUC5B charge forms. These data also demonstrate that there is a larger variation in electrophoretic mobility between low-charge populations than there is between highly charged forms.
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Studies performed on the intact MUC5AC and MUC5B mucins suggest that these mucins have different macromolecular characteristics. Sedimentation studies have indicated that both are polydisperse in size, and this may arise from the oligomerization of a variable number of their constituent monomers and/or as a consequence of specific proteolytic processing and/or degradation. Moreover, these studies showed that the MUC5AC mucins exhibited a lower average sedimentation rate than the MUC5B glycoprotein, which was taken to indicate that MUC5AC was smaller than MUC5B (27, 32). However, this is probably not the case, as we have shown that MUC5AC is in fact highly oligomerized and the apparent difference in size may be explained by a difference in physical characteristics of these two mucins (33). The MUC5AC polymer has a low mass per unit length, suggesting small oligosaccharides, and adopts a very stiff extended conformation in solution, whereas the MUC5B mucin appears to adopt a more compact structure. Further evidence that these two mucins have different macromolecular features is indicated by their behavior on agarose gel electrophoresis. Unreduced MUC5AC preparations exhibit a ladder-like banding pattern and oligomeric species containing 16 monomers can be discerned (33). By contrast, unreduced MUC5B mucins barely enter the gel (Thornton and coworkers, unpublished observation).
It is clear that MUC5AC and MUC5B are the predominant gel-forming mucins in the airways and that they have distinct chemical and physical features. What effect these differences between the mucins have on the gel properties has yet to be determined. Moreover, at an even more fundamental level, we do not know whether MUC5AC and MUC5B mucins are blended together to form the mucus gel or if they form the basis of separate gels. The latter has been suggested in gastric mucus, where laminated layers containing different mucins have been reported (34).
Immunolocalization studies on normal airways, using mucin-specific antisera, have shown that MUC5AC mucins are produced predominantly by goblet cells in the surface epithelium. MUC5B mucins on the other hand, originate mainly, but not exclusively, from the mucous cells of the submucosal glands (17, 27, 32), and this distribution is consistent with messenger 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 carbohydrate epitope, the sulfo-Lewisa antigen (SO3-3Galβ1–3GlcNAc) (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 moieties, this will not be possible.
It is interesting to speculate that the spatial separation of mucin production in the normal airways (i.e., MUC5AC on the surface and MUC5B in the glands), coupled to the fact that the two sites are controlled by different secretory mechanisms, may allow for fine-tuning of the mucus composition to modulate mucus properties depending on the challenge in the airways.
On the basis of the immunolocalization and expression data, it seemed logical at that time to believe that the mucin composition of mucus would inform us on the contribution of the various cellular sources to the secretion and, therefore, might throw up possible cellular targets for therapies aimed at modulating mucin, and hence mucus production in hypersecretory diseases. However, in hypersecretory disease, this marked spatial separation of synthesis does not seem to hold. Such diseases are associated 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 these mucins can be coexpressed in the same cell or arise from different cells has not yet been determined. Because we do not know the relative change in expression of the mucins at the different locations, we cannot be certain that their occurrence is not a valid indicator of the major source of their production. Thus, there is an obvious need for further, more in-depth studies on mucin expression in disease.
In a series of studies, we have shown that the gel-forming mucins can be changed in amount, type, and size in airways disease (3, 12, 13, 31). For example, in the viscid mucus obstructing the airways of an individual who died in status asthmaticus, there were changes in all three parameters. There was an approximately seven-fold increase in mucin concentration compared with healthy secretions. Furthermore, we demonstrated that the only mucin type responsible for the aberrant physical properties of this gel was a low-charge glycoform of the MUC5B mucin with unusual structural features and extreme size (3, 31). The clinical outcome in this case demonstrated the dramatic consequences of alterations in the mucin component of mucus and highlighted the need to determine the molecular profile of mucins in sputum.
Therefore, in order to gain a more detailed picture of mucin type (gene product and glycoform) and quantity in normal and pathologic respiratory mucus, we developed a quantitative Western blotting assay to measure the levels of MUC2, MUC5AC, and the different glycoforms of the MUC5B mucins directly from sputum (39). Data from 44 samples (15 NaCl-induced normal, 10 asthma, 10 CF, and 9 COPD) showed, as our previous nonquantitative studies had suggested, that MUC5AC and MUC5B are the major gel-forming mucins in sputum. By contrast, the MUC2 mucin was present in only trace amounts, confirming our earlier observations that MUC2 is only a minor component in sputum. Moreover, this study showed that airways mucus is not a single substance and is comprised of variable amounts of MUC5AC and MUC5B glycoproteins. Somewhat surprisingly, the variation in content was particularly marked in the “normal” samples. A key observation to come from this work was that, compared with secretions from normal subjects and individuals with asthma, there was more MUC5B in CF and COPD sputa and, most notably, there was a significant increase in the amount of the low charge form of the MUC5B mucin in the diseased, possibly infected sputa. This might suggest a link between infection/inflammation and MUC5B production. The functional consequences of mucus with different mucin compositions, and in particular of the different charge forms of the MUC5B mucin, are at present completely obscure. We would suggest the general hypothesis that MUC5AC, being primarily the product of goblet cells, may have the major mechanical function of facilitating ciliary clearance of mucus, whereas MUC5B emanating from the glands may form the basis of a gel, the primary role of which is to help with the clearance of specific pathogens or other irritants. The abnormal mucin composition of the gel in CF and COPD airways may provide an insight into the altered physical nature of these secretions and ultimately suggest potential cellular targets for therapy. These observations again highlight the need for more studies on mucin expression in disease, and in particular on MUC5B expression, which has so far been largely ignored. Furthermore, these findings reemphasize the need to develop reliable probes for the MUC5B mucin glycoforms to establish their cellular origin.
Studies of human airways secretions have been, and will continue to be, essential to highlight changes associated with disease. However, these investigations are fraught with difficulty related to mucus collection, as well as possible changes in the mucins being disguised by nonspecific postsecretion processing. Furthermore, the effects of extraneous environmental influences are difficult to control and, for ethical reasons, it is impossible to perform in vivo studies of mucin/mucus biogenesis and secretion. Thus, to progress our understanding of both normal and diseased airways, there is an urgent need for in vitro systems that mimic in vivo mucus production. For this purpose a number of laboratories have developed airways epithelial cell cultures (40–43). The normal human tracheobronchial air–liquid interface cultures instigated by Nettesheim and colleagues are of particular interest (40). When cultured in the presence of retinoic acid, normal human tracheobronchial cells grow and differentiate into a mucociliary epithelium. They secrete functional mucus onto their apical surface (40) that is clearly capable of ciliary transport. This culture system appears to mimic in vivo airways mucin production in that all three oligomeric airways mucins, MUC5AC, MUC5B, and MUC2 are expressed. Furthermore, their basal levels of expression can be modulated by physiologically relevant regulators (e.g., thyroid hormone and epidermal growth factor) (44–46). We have investigated the apical secretions from such cultures and have shown that MUC5AC and MUC5B are the predominant mucins stored and secreted by normal human tracheobronchial cells (passage 2). Moreover, these mucins have similar macromolecular properties to their counterparts in human airways (44). However, while the MUC5AC mucin exhibits a similar charge profile to MUC5AC in vivo, by contrast, the MUC5B mucin produced is more homogeneous, with evidence of only a single (perhaps high-charge) glycoform. Thus, the mucin phenotype and hence the resultant properties of the secretions may not be a truly accurate reflection of in vivo mucus. Nonetheless, such culture systems provide a powerful and manipulable experimental system and seem set to form the basis of ever widening studies that should revolutionize our understanding of the biology and pathobiology of mucus over the next few years. For example, work from Boucher and collaborators using cultures derived from cells with the CF defect has shown that the dynamics of the attached mucus layer are altered and, in these compared with normal cultures, mucus transport is reduced or even abolished (41).
While size and concentration of the mucins are key factors controlling gel formation, concentrated solutions of mucins alone do not reproduce all the physical properties of the gel (47). This is emphasized in our recent findings where we have shown, using confocal fluorescence recovery after photobleaching, that the network properties of mucus cannot be recapitulated by concentrated mucin solutions even at higher than in vivo mucin concentration (48). Unlike some other methods for looking at mucus properties, confocal fluorescence recovery after photobleaching is nondestructive and provides a powerful approach to investigating the properties of macromolecules in concentrated solution, including complex mixtures, such as in mucus, under equilibrium conditions, and in the absence of concentration gradients and flow and shear forces (48, 49). We have used it to determine lateral tracer diffusion of fluorescent molecular probes (i.e., bovine serum albumin and mucins) and of rigid polystyrene microspheres of different size in mucus at physiological concentrations. It is important to note that the tracer diffusion measurements alone are not a measure of gel formation, which involves polymer cross-links and entanglement and occurs in concentrated solutions of all flexible polymers. However, they provide a quantitative comparison of the “network” caused by gel-formation, or entanglement, that impedes the free diffusion of other macromolecules in a concentration-dependent way. Our initial confocal fluorescence recovery after photobleaching analysis of MUC5B (extracted and purified in the presence of 4–6 M guanidinium chloride) showed that in concentrated solutions its properties resulted from polymer entanglement with no evidence of protein–protein self-association or glycan-mediated interactions (48). A direct comparison of concentrated guanidinium chloride-purified MUC5B mucin solutions with the native saliva from which it was prepared revealed that the MUC5B mucin did not replicate the properties of the parent mucus. At similar MUC5B concentrations to those found in saliva, the guanidinium chloride–purified mucin was approximately 20 times more permeable, suggesting that there was a higher order structure in the native mucus, which may involve other components in the secretion (48). Further analyses have implicated calcium as a key regulator of MUC5B mucin supramolecular organization in salivary mucus (50). This study demonstrated a reversible, calcium-dependent interaction between MUC5B mucins that was sensitive to denaturation by chaotropic solvents (e.g., 6 M guanidinium chloride), treatment with reducing agents and the proteinase trypsin (50). This study also highlighted the need for alternative, nondenaturing solvents to disassemble mucus gels for investigation of their functions.
The fact that gel-forming mucins alone do not constitute mucus may not be totally surprising because it is a complex mixture of ions, mucins, glycoproteins, proteins, and lipids and, like the mucins, the total amount of these other components is increased in hypersecretory disease (51–54). Some of these components contribute to airways protection by targeting pathogens (e.g., secretory IgA, proline-rich proteins, defensins, lysozyme, and transferrin), and others may act to modulate the organization and hence the properties of the gel. For example, in the gastrointestinal tract the mitogenic/reparative trefoil peptides are intimately associated with mucus and have been suggested to bind to oligomeric mucins and increase mucus viscosity (55). Work from our laboratory has demonstrated that a large glycoprotein, gp-340, is found in complexes with MUC5B in airways mucus (56). It is interesting to speculate on the consequences of an interaction of gp-340 with the mucin network. Gp-340 clearly has an important antibacterial role in mucus, either via its direct binding to bacteria (57) or by its association with the bacterial-binding collectin surfactant protein-D (58). Thus, binding of gp-340, and likely other protective factors, to the gel network increases the functionality of the mucus by facilitating clearance of sequestered bacteria from the respiratory tract via the mucociliary escalator or cough.
It is clear from these two examples that nonmucin glycoproteins and proteins play key roles in mucosal protection and mucus organization; but while there have been many studies on the molecular components of mucus, we do not yet know their full complement or which of these are associated with the mucins and what function(s) they perform. These other components may be over- or under-expressed in disease conditions, which would have an effect on the hosts ability to prevent colonization or the rheologic properties of mucus may be compromised with attendant problems. Thus proteomics has a key role to play in identifying the different molecular species impacting on mucus structure and function.
A final but no less important factor that will have a major influence on the properties of airways mucus is the availability of water on the epithelial surface. Currently, the relationship between mucin hydration and mucus properties is not well understood. We know that oligomeric mucins are preassembled into large oligomers within the cell, where they are stored within granules in a largely dehydrated form. However, after secretion the requirements of a specific mucin to hydrate are unknown but are likely affected by a variety of factors, including their size, charge density, and organization. Furthermore, their hydration will be intimately coupled with the ionic composition and water availability of the environment into which they are secreted. This will in turn be affected by the other biomolecules already present in that environment. Aberrant mucin hydration may be of particular importance in the airways of patients with CF. In CF, the competition for water on the airways epithelium appears to be more severe, and the water imbalance in the epithelial fluid (41, 59, 60) may well have a strong negative impact on mucin unpackaging and hydration. This may explain the abnormal properties of the mucus, which are a feature of this disease.
While this article has focused on the oligomeric mucins, these are not the only mucins expressed in the airways. Other members of the mucin family (MUC1, MUC4, MUC7, MUC11, and MUC13) are expressed and these mucins, with the exception of MUC7, are thought primarily, though not exclusively, to occur in transmembrane forms, which can arise in mucus due to shedding from the cell surface (61–63). However, in the case of MUC1 and MUC4, there are alternatively spliced forms that are secreted (61–63). There is little biochemical characterization of these mucins, and their amounts in mucus are unknown. Furthermore, their role in mucus has yet to be defined and there is clearly an urgent need to understand their function in mucus.
As far as the oligomeric mucins are concerned, an extensive array of mucin-specific probes are available, the methodologies necessary for their separation and characterization have been developed, and the air–liquid cultures offer a more physiologically relevant in vitro system for their study. Thus, we are well placed to perform new studies of the cell and structural biology of the mucins and mucus, which is essential for a more coherent picture of how this essential barrier functions to protect the airways. Finally, there are currently few effective therapies to alleviate mucus hypersecretion, and these studies may highlight specific components as potential targets for future therapeutic strategies for the treatment of hypersecretory conditions.
The authors acknowledge the substantial contributions to the research reviewed here by Ingemar Carlstedt (University of Lund, Sweden), Paul Richardson (St. Georges Hospital, London, UK), Paul Nettesheim, Tom Gray, and Peter Koo (NIEHS, U.S.A.), and Sara Kirkham, Marj Howard, Nagma Khan, David Knight, Bertrand Raynal, and Tim Hardingham (University of Manchester, UK).
| 1. | Phillips GJ, James SL, Lethem MI. Rheological properties and hydration of airway mucus. In Rogers DF and Lethem MI, editors. Airway mucus: basic mechanisms and clinical perspectives. Basel: Birkhauser Verlag; 1997. pp. 117–147. |
| 2. | Puchelle E, Zahm JM, de Bentzmann S, Gaillard D. In Rogers DF and Lethem MI, editors. Airway mucus: basic mechanisms and clinical perspectives. Basel: Birkhauser Verlag; 1997. pp. 301–326. |
| 3. | Sheehan JK, Richardson PS, Fung DCK, Howard M, Thornton DJ. Analysis of respiratory mucus glycoproteins in asthma: a detailed study from a patient who died in status asthmaticus. Am J Respir Cell Mol Biol 1995;13:748–756. |
| 4. | Levine SJ, Larivee P, Logun C, Angus CW, Ognibene FP, Shelhamer JH. Tumor necrosis factor-α induces mucin hypersecretion and MUC2 gene expression in airway epithelial cells. Am J Respir Cell Mol Biol 1995;12:196–204. |
| 5. | Li D, Gallup M, Fan N, Szymkowski DE, Basbaum CB. Cloning of the amino-terminal 5′-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts. J Biol Chem 1998;273:6812–6820. |
| 6. | Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol 1999;276:L835–L843. |
| 7. | Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999;103:779–788. |
| 8. | Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol 1999;276:L835–L843. |
| 9. | Nadel J. 2001. Role of epidermal growth factor receptor cascade in airway hypersecretion and proposal for novel therapy. In Salathe M, editor. Cilia and mucus. New York: Marcel Dekker; 2001. pp. 315–338. |
| 10. | Jentoft N. Why are proteins O-glycosylated. Trends Biochem Sci 1990;15:291–294. |
| 11. | Scharfman A, Lamblin G, Roussel P. Interactions between human respiratory mucins and pathogens. Biochem Soc Trans 1995;23:836–839. |
| 12. | Thornton DJ, Davies JR, Kraayenbrink M, Richardson PS, Sheehan JK, Carlstedt I. Mucus glycoproteins from normal human tracheobronchial secretions. Biochem J 1990;265:179–186. |
| 13. | Thornton DJ, Sheehan JK, Lindgren H, Carlstedt I. Mucus glycoproteins from cystic fibrotic sputum: macromolecular properties and structural architecture. Biochem J 1991;276:667–675. |
| 14. | Thornton DJ, Sheehan JK, Carlstedt I. Heterogeneity of mucus glycoproteins from cystic fibrotic sputum. Biochem J 1991;276:677–682. |
| 15. | Thornton DJ, Devine PL, Hanski C, Howard M, Sheehan JK. Identification of two major populations of mucins in respiratory secretions. Am J Respir Crit Care Med 1994;150:823–832. |
| 16. | Sheehan JK, Thornton DJ, Somerville M, Carlstedt I. The structure and heterogeneity of respiratory mucus glycoproteins. Am Rev Respir Dis 1991;144:S4–S9. |
| 17. | Thornton DJ, Davies JR, Carlstedt I, Sheehan JK. Structure and biochemistry of human respiratory mucins. In Rogers DF and Lethem MI, editors. Airway mucus: basic mechanisms and clinical perspectives. Basel: Birkhauser Verlag; 1997. pp. 19–39. |
| 18. | Gum JR, Hicks JW, Toribara MW, Siddiki B, Kim YS. Molecular cloning of the human intestinal mucin (MUC2) cDNA: identification of the amino terminus and the overall sequence similarity to prepro-von Willebrand factor. J Biol Chem 1994;269:2440–2446. |
| 19. | Van de Bovenkamp JHB, Hau CM, Strous GJAM, Buller HA, Dekker J, Einerhand AWC. Molecular cloning of human gastric mucin MUC5AC reveals reserved cysteine-rich D-domains and a putative leucine zipper motif. Biochem Biophys Res Commun 1998;245:853–859. |
| 20. | Desseyn J-L, Buisine MP, Porchet N, Aubert JP, Laine A. Genomic organization of the human mucin gene MUC5B cDNA and genomic sequences upstream of the large central exon. J Biol Chem 1998;273:30157–30164. |
| 21. | Toribara NW, Ho SB, Gum E, Gum JR, Lau P, Kim YS. The carboxyl-terminal sequence of the human secretory mucin, MUC6: analysis of the primary amino acid sequence. J Biol Chem 1997;272:16398–16403. |
| 22. | Takeyama K, Dabbagh K, Lee HM, Agusti C, Lausier JA, Ueki IF, Grattan KM, Nadel JA. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 1999;96:3081–3086. |
| 23. | Takeyama K, Jung B, Shim JJ, Burgel PR, Pick TD, Ueki IF, Protin U, Kroschel P, Nadel JA. Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol. 2001;280:L165–L172. |
| 24. | Shim JJ, Dabbagh K, Ueki IF, Pick TD, Burgel PR, Takeyama K, Tam DCW, Nadel JA. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol Lung Cell Mol Physiol 2001;280:L134–L140. |
| 25. | Thornton DJ, Carlstedt I, Howard M, Devine PL, Price MR, Sheehan JK. Respiratory mucins: identification of core proteins and glycoforms. Biochem J 1996;316:967–975. |
| 26. | Thornton DJ, Howard M, Khan N, Sheehan JK. Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. J Biol Chem 1997;272:9561–9566. |
| 27. | Wickström C, Davies JR, Eriksen GV, Veerman ECI, Carlstedt I. MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem J 1998;334:685–693. |
| 28. | Hovenberg HW, Davies JR, Herrmann A, Linden CJ, Carlstedt I. MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions. Glycoconj J 1996;13:839–847. |
| 29. | Davies JR, Svitacheva N, Lannefors L, Kornfalt R, Carlstedt I. Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis secretions. Biochem J 1999;344:321–330. |
| 30. | Herrmann A, Davies JR, Lindell G, Martensson S, Packer NH, Swallow DM, Carlstedt I. Studies on the “insoluble” glycoprotein complex from human colon: identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage. J Biol Chem 1999;274:15828–15836. |
| 31. | Sheehan JK, Howard M, Richardson PS, Longwill T, Thornton DJ. Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug. Biochem J 1999;338:507–513. |
| 32. | Hovenberg HW, Davies JR, Carlstedt I. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from goblet cells. Biochem J 1996;318:319–324. |
| 33. | Sheehan JK, Brazeau C, Kutay S, Pigeon H, Kirkham S, Howard M, Thornton DJ. Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions. Biochem J 2000;347:37–44. |
| 34. | Ota H, Katasuyama T. Alternating laminated array of two types of mucin in the human gastric surface mucous layer. Histochem J 1992;24:86–92. |
| 35. | Copin MC, Devisme L, Buisine MP, Marquette CH, Wurtz A, Aubert JP, Gosselin B, Porchet N. From normal respiratory mucosa to epidermoid carcinoma: expression of human mucin genes. Int J Cancer 2000;86:162–168. |
| 36. | Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer NH, Sheehan JK. Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology 1999;9:293–302. |
| 37. | Davies JR, Carlstedt I. Respiratory tract mucins. In Salathe M, editor. Cilia and mucus. New York: Marcel Dekker; 2001. pp. 167–178. |
| 38. | Groneberg DA, Eynott PR, Oates T, Lim S, Wu R, Carlstedt I, Nicholson AG, Chung KF. Expression of MUC5AC and MUC5B mucins in normal and cystic fibrosis lung. Respir Med 2002;96:81–86. |
| 39. | Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J 2002;361:537–546. |
| 40. | Gray T, Guzman K, Davis CW, Abdullah LH, Nettesheim P. Mucocilliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 1996;14:104–112. |
| 41. | Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. Evidence for periciliary layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998;95:1005–1015. |
| 42. | Berger JT, Voynow JA, Peters KW, Rose MC. Respiratory carcinoma cell lines, MUC genes and glycoconjugates. Am J Respir Cell Mol Biol 1999;20:500–510. |
| 43. | Rose MC, Piazza FM, Chen YA, Alimam MZ, Bautista MV, Letwin N, Rajput B. Model systems for investigating mucin gene expression in airway diseases. J Aerosol Med 2000;13:245–261. |
| 44. | Thornton DJ, Gray T, Nettesheim P, Howard M, Koo JS, Sheehan JK. Characterisation of the MUC5AC and MUC5B mucins synthesised by cultured normal human tracheobronchial epithelial cells. Am J Physiol 2000;278:L1118–L1128. |
| 45. | Gray T, Nettesheim P, Basbaum C, Koo JS. Regulation of mucin gene expression in human tracheobronchial epithelial cells by thyroid hormone. Biochem J 2001;353:727–734. |
| 46. | Gray T, Koo PS, Nettesheim P. Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium. Toxicology 2001;160:35–46. |
| 47. | Sellers A, Allen A. Gastrointestinal mucus rheology. In Chantler E and Ratcliffe NA, editors. Mucus and related topics. Cambridge: The Company of Biologists Limited; 1989. pp. 65–71. |
| 48. | Raynal BD, Hardingham TE, Thornton DJ, Sheehan JK. Concentrated solutions of salivary MUC5B mucin do not replicate the gel-forming properties of saliva. Biochem J 2002;362:289–296. |
| 49. | Gribbon P, Hardingham TE. Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching. Biophys J 1998;75:1032–1039. |
| 50. | Raynal B, Hardingham TE, Sheehan JK, Thornton DJ. Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus. J Biol Chem 2003;278:28703–28710. |
| 51. | Lopez-Vidriero MT, Das I, Reid LM. Airway secretion: source, biochemical and rheological properties. In Brain JD, Proctor DF, Reid LM, editors. Respiratory defence mechanisms. New York: Marshall-Dekker; 1977. pp. 289–356. |
| 52. | Lopez-Vidriero MT, Reid LM. Chemical markers of mucous and serum glycoproteins and their relation to viscosity in mucoid and purulent sputum from various hypersecretory diseases. Am Rev Respir Dis 1978;117:465–476. |
| 53. | Boat TF, Cheng PW, Leigh MW. Biochemistry of mucus. In Takishima T and Shimura S, editors. Airway secretion. New York: Marcel Dekker; 1994. pp. 217–282 |
| 54. | Widdicombe JG. Airway surface liquid: concepts and measurements. In Rogers DF and Lethem MI, editors. Airway mucus: basic mechanisms and clinical perspectives. Basel: Birkhauser Verlag; 1997. pp. 1–18 |
| 55. | Thim L, Madsen F, Poulsen SS. Effect of trefoil factors on the viscoelastic properties of mucus gels. Eur J Clin Invest 2002;32:519–527. |
| 56. | Thornton DJ, Davies JR, Kirkham S, Gautrey A, Khan N, Richardson PS, Sheehan JK. Identification of a nonmucin glycoprotein (gp-340) from a purified respiratory mucin preparation: evidence for an association involving the MUC5B mucin. Glycobiology 2001;11:969–977. |
| 57. | Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, Frangsmyr L, Holmskov U, Leffler H, Nilsson C, et al. Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 2000;275:39860–39866. |
| 58. | Holmskov U, Lawson P, Teisner B, Tornoe I, Willis AC, Morgan C, Kock C, Reid BM. Isolation and characterisation of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule. J Biol Chem 1997;272:13743–13749. |
| 59. | Joo NS, Irokawa T, Wu JV, Robbins RC, Whyte RI, Wine JJ. Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands. J Biol Chem 2002;277:50710–50715. |
| 60. | Ballard ST, Trout L, Mehta A, Inglis SK. Liquid secretion inhibitors reduce mucociliary transport in glandular airways. Am J Physiol Lung Cell Mol Physiol 2002;283:L329–L335. |
| 61. | Moniaux N, Escande F, Batra SK, Porchet N, Laine A, Aubert JP. Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins. Eur J Biochem 2000;267:4536–4544. |
| 62. | Williams SJ, Wreschner DH, Tran M, Eyre HJ, Sutherland GR, McGuckin MA. MUC13, a Novel human cell surface mucin expressed by epithelial and hemopoietic cells. J Biol Chem 2001;276:18327–18336. |
| 63. | Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollingsworth MA, Engelhardt JF. MUC5B and MUC7 are differentially expressed in mucous and serous cells of submucosal glands in human bronchial airways. Am J Respir Cell Mol Biol 1998;19:30–37. |
| 64. | Sheehan JK, Boot-Handford RP, Chantler E, Carlstedt I, Thornton DJ. Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. Biochem J 1991;274:293–296. |
| 65. | Thornton DJ, Khan N, Sheehan JK. Identification and separation of mucins and their glycoforms. In Corfield AP, editor. Mucin methods and protocols; methods in molecular biology series. Totowa, NJ: Humana; 2000. pp. 77–86. |