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Research Center for Genetic Medicine, Children's National Medical Center and Departments of Pediatrics and of Biochemistry and Molecular Biology, George Washington University, Washington, District of Columbia; and Department of Pediatrics, Duke University Medical Center, Durham, North Carolina
ABSTRACT I. INTRODUCTION A. Overview B. Mucin Glycoproteins 1. Mucin classification 2. MUC protein backbones: TRs 3. Mucin glycosylation C. Molecular Tools for MUC Identification D. Mucin Expression in Epithelial Tissues/Cells and Airway Cells E. Mucins in Airway Secretions/Lung Mucus F. Comparative Biology of Mucin Gene Expression in the Respiratory Tract II. MUCIN OVERPRODUCTION IN CHRONIC AIRWAY DISEASES A. Overview B. Mucin Overproduction in Asthma 1. Overview 2. MUC mRNA and protein in asthmatic cells 3. MUC mucins in asthmatic secretions and mucus plugs 4. Genetic association of MUC genes and asthma 5. Goblet cell metaplasia and mucin gene expression in murine models of asthma C. Mucin Overproduction in COPD 1. Definition of COPD/chronic bronchitis: the inflammatory milieu 2. Mucin changes in vivo 3. Mucin regulation by inflammatory mediators in vitro and in vivo in animal models of chronic bronchitis D. Mucin Overproduction in CF 1. The ''mucus abnormality'' 2. Mucin mRNA/protein expression in CF cells 3. MUC mucins in CF airway secretions 4. Alterations in terminal glycosylation of O-glycans 5. Models of airway obstruction in CF III. REGULATION OF MUCIN GENES A. Overview B. Transcriptional Regulation by Infectious Mediators of Mucin Genes 1. MUC2 gene regulation 2. MUC5AC gene expression C. Transcriptional Regulation by Inflammatory/Immune Response Mediators D. Posttranscriptional Regulation of Mucin Genes IV. SUMMARY AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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50% carbohydrate, wt/wt). MUC protein backbones are characterized by numerous tandem repeats that contain proline and are high in serine and/or threonine residues, the sites of O-glycosylation. Secretory and membrane-tethered mucins contribute to mucociliary defense, an innate immune defense system that protects the airways against pathogens and environmental toxins. Inflammatory/immune response mediators and the overproduction of mucus characterize chronic airway diseases: asthma, chronic obstructive pulmonary diseases (COPD), or cystic fibrosis (CF). Specific inflammatory/immune response mediators can activate mucin gene regulation and airway remodeling, including goblet cell hyperplasia (GCH). These processes sustain airway mucin overproduction and contribute to airway obstruction by mucus and therefore to the high morbidity and mortality associated with these diseases. Importantly, mucin overproduction and GCH, although linked, are not synonymous and may follow from different signaling and gene regulatory pathways. In section I, structure, expression, and localization of the 18 human MUC genes and MUC gene products having tandem repeat domains and the specificity and application of MUC-specific antibodies that identify mucin gene products in airway tissues, cells, and secretions are overviewed. Mucin overproduction in chronic airway diseases and secretory cell metaplasia in animal model systems are reviewed in section II and addressed in disease-specific subsections on asthma, COPD, and CF. Information on regulation of mucin genes by inflammatory/immune response mediators is summarized in section III. In section IV, deficiencies in understanding the functional roles of mucins at the molecular level are identified as areas for further investigations that will impact on airway health and disease. The underlying premise is that understanding the pathways and processes that lead to mucus overproduction in specific airway diseases will allow circumvention or amelioration of these processes. |
I. INTRODUCTION |
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The apical epithelial surfaces of mammalian respiratory, gastrointestinal, and reproductive tracts are coated by mucus, a mixture of water, ions, glycoproteins, proteins, and lipids. Mucosal components are secreted apically by goblet cells in polarized epithelium and by secretory cells in the submucosal glands (SMG). Mucus provides a protective barrier against pathogens and toxins and contributes to the innate defensive system in mucosal immunology (54).
Mucin glycoproteins (mucins) are the major macromolecular constituents of epithelial mucus and have long been implicated in health and disease. Mucins historically are large, highly glycosylated, viscoelastic macromolecules that are difficult to isolate and purify (218). The application of recombinant DNA technology to the mucin field resulted in the cloning of MUC genes that encode mucin protein backbones.1 This enlarged the definition of mucins to include membrane-tethered, as well as secreted, mucins. The development of molecular tools to identify mucins by their MUC protein backbone enabled studies on the expression and localization of MUC gene products in tissues, cells, and secretions. Currently, investigations on specific mucins and their roles in diseases, mucosal defense, and epithelial repair/remodeling are underway in many laboratories. Mucins implicated in cancer (110), lung diseases (153, 286), gastrointestinal diseases (53, 70), as well as mucins expressed in the eye (90) and ear (164), have recently been reviewed.
This review focuses on mucins in airway health and disease, which are expressed in the conducting airways (trachea, bronchi, bronchioles) of the lower respiratory tract. Airway mucins are major components of the soluble layer and/or viscoelastic gel that comprise lung mucus in healthy airways and contribute to the mucociliary defense system that protects the lungs against pathogens and environmental toxins (219). Mucin production entails several biological processes (Fig. 1), as the biosynthesis of even the simplest mature glycosylated mucin requires transcription of a MUC gene to encode a MUC mRNA, which is then translated into a MUC protein backbone that is posttranslationally modified by one or several glycosyltransferases (GT). Secretory mucins are stored in secretory granules and released at the apical surface in response to mucin secretagogues, while membrane-tethered mucins are integrated into the cell membrane.
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In section I, current information on the structure and properties of mucins, especially those expressed in the lower respiratory tract, as well as molecular tools used to identify MUC gene products in tissues, cells, and secretions, is presented. Information on mucin overproduction in chronic airway diseases is overviewed in section II and then addressed in disease-specific subsections on asthma, chronic bronchitis, and CF. Upregulation of MUC genes and goblet cell hyperplasia (GCH) are fundamental processes that contribute to and sustain mucin overproduction in the airways of patients with chronic lung diseases. Information on GCH and secretory cell metaplasia in animal models of airway diseases is integrated into each disease-specific subsection in section II, together with pertinent information on mucin gene regulation. However, the rapid increase in information on this latter topic warranted a separate section on "regulation of mucin genes" in section III. These studies have typically been carried out in airway epithelial cancer cell lines and/or primary normal human bronchial epithelial (NHBE) cells. When grown on an extracellular matrix and maintained at an air-liquid interface (ALI), NHBE cells differentiate to morphologically mimic conducting airway epithelium (reviewed in Refs. 2, 305), thus providing a useful model system for airway studies. A brief summary and overview of future directions for research in lung mucin biology are provided in section IV.
Mucins are complex glycoproteins synthesized in epithelial cells and typically characterized by large molecular weight (2–20 x 105 Da), high carbohydrate content (50–90% by weight) reflecting a large number of O-glycans, and an extensive number of tandem repeats (TR) in the protein backbone (Fig. 2). The TR domains are the characteristic feature that distinguishes mucins from other glycoproteins, especially from membrane-bound glycoproteins/receptors that have extracellular regions high in serine, threonine, and proline and may be members of the immunoglobulin superfamily. Mucins are classified by their MUC protein backbone, which is encoded by a MUC gene. MUC genes are localized to chromosomes 1, 3, 4, 7, 11, 12, and 19 (Table 1). MUC gene products vary in size. MUC transcripts range from 1.1 to >15 kb, while MUC proteins, which account for 10–50% (wt/wt) of mucin mass, have 377 to 11,000 amino acids in their protein backbones.
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Additionally, the definition of what determines a mucin gene is not always consistent. Perhaps serine/threonine/proline-rich TR domains (see sect. IB2) should define a mucin, while a significant amount of serines, threonines, and a large number of O-glycosides would define a mucinlike glycoprotein. Three of the mucin genes in GenBank encode numerous serine and threonine residues, but do not encode TR domains in their protein backbones (Tables 2 and 3). These are MUC14, a membrane-bound O-sialoglycoprotein (131), MUC15, a membrane-tethered glycoprotein (197), and MUC18/CD146, a neural cell adhesion member of the immunoglobulin superfamily (151). If these three macromolecules are considered as serine/threonine-rich glycoproteins rather than mucins, then the present list of human mucins would have 18 members all of which had TR, e.g., MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC16, MUC17, MUC19, and MUC20. Clarification of what constitutes a MUC gene product is evolving and should eventually be standardized. This process will be facilitated by the establishment of a mucin website (www.mucin.org), initiated by Dr. Yin Chen while in Dr. Reen Wu’s group at the University of California, Davis.
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Eighteen of the >20 mucin genes that are deposited in GenBank encode TRs, the exceptions being MUC14, MUC15, and MUC18 (see above and Tables 2 and 3). The tandem repeating nucleotide sequences are typically encoded by a single central exon in a MUC gene. Many MUC genes are polymorphic, with alleles having a variable number of TRs (VNTR) (81), as indicated in Table 3. Additional variations occur with minor changes in the length of the repeat unit (length polymorphisms) and repeating sequence (sequence polymorphisms). Thus different individuals can express different forms of the same MUC gene product. In addition, MUC genes may undergo alternative splicing (183). Changes in VNTR are reported in cancer (reviewed in Ref. 110), ulcerative colitis (143), and respiratory diseases (124).
TRs are unique in sequence and size (Table 3) and thus are the defining characteristic of each MUC protein. Among different mucins, TRs vary in length (5–375 amino acids) and in number (5–395 repeats). Some MUC proteins have two domains with different TR sequences, e.g., MUC2, MUC3A, MUC3B, and MUC5AC. The two major airway mucins, MUC5AC and MUC5B, have TR domains that are repeated four or five times in their protein backbones, respectively, and are separated by cysteine-rich domains (Fig. 3). Each TR contains proline and is rich in serine and/or threonine residues, the sites of O-glycosylation. Thus TRs largely determine a mucin's degree of glycosylation, as longer or more highly repeated TR sequences can allow for hundreds of O-glycans per molecule (Figs. 2 and 3). MUC5B, for example, contains a repeat of 29 amino acids with a total of 16 serines and threonines. Seventy-two repetitions of the 29 amino acid sequence contribute 1,152 possible O-glycosylation sites to the MUC5B backbone. The number of TRs in a MUC protein also determines mucin size as they elongate the protein backbone and thus increase mucin molecular mass. They also likely contribute to the structural and functional diversity of mucins in ways that are not yet appreciated at the molecular or physiological level. For example, the TR domains of MUC2 are uninterrupted by cysteine-rich domains, in contrast to MUC5AC and MUC5B (Fig. 3). This perhaps reflects the need for MUC5AC/B to couple to the mucociliary escalator in the airways, whereas MUC2 may need to be more rigid to better protect the intestinal epithelium against proteolytic attack. Indeed, MUC2 is considered an insoluble mucin biochemically (10).
TRs are the primary domains to which O-glycans are attached, and O-glycans can alter mucin conformation. This has been shown for ovine submaxillary mucin, which undergoes a change from an extended filament to a globular form following enzymatic removal of the disaccharide O-glycans (228). Airway mucins typically present as flexible filamentous structures (227, 247), which on aggregation form a large interwoven network (227) quite different from the massive ropelike structures characteristic of sheep submaxillary mucin aggregates (50). Thus TR and their attached O-glycans influence the size, shape, and mass of mucins, thereby contributing to the biophysical and biological properties of mucus itself. For example, larger, bulkier, heavier mucins might result in more viscous mucus. Future studies should reveal the molecular details wherein MUC-specific TR contribute to the biochemical and physical properties of mucins.
TR domains are typically not conserved between human MUC and rodent Muc genes, whereas the non-TR motifs at the amino- and carboxy-terminal domains of many mucins are typically highly conserved between species, as first shown for MUC1/Muc1 (259).
Like mucins, many macromolecules, including cell adhesion markers and selectin ligands, have O-glycans attached to serine or threonine residues in their protein backbone (60, 219). However, mucins are 50–90% carbohydrate by mass and have numerous (dozens to several hundred) O-glycosylation sites per molecule due to the high number of TR in each MUC protein backbone (Table 3). Studies to determine and/or predict which serines or threonines in TRs or in mucins are O-glycosylated are challenging (88).
O-glycosylation is a major part of mucin biosynthesis and requires an N-acetylgalactosaminyl peptidyltransferase and one or more GTs depending on the final O-glycan structure. O-glycosylation is initiated in the Golgi when an N-acetylgalactosaminyl peptidyltransferase transfers N-acetylgalactosamine (GalNAc) to a serine or threonine when a nascent MUC polypeptide chain traverses the Golgi, as depicted in Figure 1. Each O-glycan is then elongated by the stepwise addition of hexoses [galactose (Gal), N-acetylglucosamine (GlcNAc), fucose] or sialic acid by specific GT (33, 235). More than 30 GT have now been identified, of which at least a dozen participate in the synthesis of mucin O-glycans (34).
Four major core structures (Fig. 4) have been identified in O-glycans isolated from mucins (114). The transfer of Gal to C-3 of GalNAc yields a structure that will be core type 1 or 2. The next transferase can either add to Gal (elongation of core 1) or to GalNAc to form a branch (core 2). Similarly, GlcNAc can be added to GalNAc to form core 3, which can be branched to form core 4. Core structures can then be elongated by Gal and GlcNAc transferases to form Gal
1,3/4GlcNAc units, which can be terminated by blood group determinants, sialic acid, sulfate, or fucose. Sialic acid moieties and sulfates on Gal or GlcNAc moieties impart negative charges to mucins, which account for their low pI values, whereas fucose imparts hydrophobicity. Terminal sugars, because of their hydrophobicity or charge, are thought to contribute to or determine the physical and/or biological properties of mucins. Thus alterations in terminal glycosylation of mucins, which may occur in disease states (see sect. IID4), have the potential to alter the physical properties of mucins and the rheological properties of mucus.
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A similar situation prevails for airway mucins. Over the last 30 years, several dozen O-glycans have been cleaved from mucins isolated from sputum from patients with airway disease and their structures determined by mass spectrometry or NMR (147). The four major core types in specific O-glycan structures (Fig. 4) have been identified in mucins from patients with airway diseases (278), whereas structural information from mucins isolated from healthy airways has not been reported. The identities of the MUC mucins to which specific O-glycans are attached are unknown, as O-glycans were cleaved from mucin-rich fractions. Thus it is not yet established whether the complexity of O-glycan structures reflects altered levels of specific mucins in airway secretions and/or alterations in glycosylation of specific MUC proteins in disease or inflammation (see sect. IID4). To add to the complexity, more than one core type O-glycan may be attached to a single MUC backbone, especially in mucins that have more than one TR type domain. A comprehensive analysis of O-glycan structures of specific MUC protein backbones will prove useful in determining whether specific mucins (defined both by MUC backbone and O-glycan core structures) are altered in airway diseases consequent to inflammation and/or in a recessive genetic disease, like CF (148).
While O-glycosylation is predominant in mucins, several MUC proteins contain one or more sites with the Asn-X-Ser/Thr sequence requisite for N-glycosylation. N-glycosylation typically occurs cotranslationally in the ER; however, chemical identification of N-glycosides isolated from mucins secreted in vivo or in vitro has not been reported. Recently, a newly characterized linkage of C-mannose to tryptophan was identified in MUC5AC and MUC5B mucins synthesized in vitro (202). Low levels of mannose have occasionally been reported in secreted mucins, but they have generally been considered a contaminant. However, if mannose reflects a C-mannose linkage in airway mucins synthesized in vivo, these short glycans may contribute to the biological or physical properties of airway mucins.
C. Molecular Tools for MUC Identification
Mucin gene products are identified by nucleotide probes to specific MUC mRNA sequences or by antibodies that recognize specific MUC protein determinants. The sequence of the TR domains of each MUC gene product is unique (Table 3), which facilitated the use of MUC-specific oligonucleotide probes to evaluate MUC mRNA localization and expression in lower respiratory tract epithelial cells (8, 36, 212).
Antibodies raised against cognate sequences of MUC TR domains have been used in immunocytochemical studies to localize MUC protein expression, although antigen retrieval is generally required. However, these antibodies often do not detect mucins on Western blot analyses or ELISA, presumably because extensive O-glycosylation masks the TR epitopes and/or blocks accessibility of antibodies. In contrast, polyclonal antibodies raised against cognate sequences in the non-TR domains of secreted mucins identify mature, i.e., glycosylated, mucins in epithelial secretions by Western blot analyses (225, 271). MUC-specific polyclonal antibodies raised against cognate sequences in the cysteine-rich domains of MUC5AC (18, 225, 271), MUC2 (116), and MUC5B (273) recognize mature secreted airway mucins in epithelial secretions. Monoclonal antibodies raised against sequences in the non-TR domain of MUC5B and MUC7 and assayed in salivary secretions have recently been reported (230). Information on MUC-specific polyclonal or monoclonal antibodies used to investigate human MUC or rodent Muc mucins in respiratory tract cells or secretions is provided in Table 4. Detailed information on monoclonal antibodies used to investigate mucin expression in epithelial tissues and in cancer studies is available (304).
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Mucin genes are expressed in most epithelial tissues (85, 287). MUC1 is a pan-epithelial membrane-tethered mucin expressed at the cell surface in numerous epithelial tissues, as well as in hematopoietic tissues (85). Expression of all other mucin genes is somewhat, but not exclusively, restricted in healthy tissues (reviewed in Ref. 222) and can be altered in diseases, especially in cancer (reviewed in Ref. 110). Secreted mucins are typically markers for goblet cells in the surface epithelium and glandular cells in the respiratory, salivary, gastrointestinal, and reproductive tracts. MUC genes are differentially expressed in goblet cells in diverse epithelial tissues. MUC5AC is the predominant mucin normally expressed in goblet cells in the lung, eyes, and stomach, while MUC2 is expressed in intestinal goblet cells. MUC5B and MUC19 mucins are typically restricted to glandular cells.
In the lower respiratory tract, expression of at least 12 human mucin genes (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11, MUC13, MUC15, MUC19, and MUC20) have been observed at the mRNA level in tissues from healthy individuals. This is reviewed or described in the following references: MUC1–MUC8 (212, 222), MUC11 (153), MUC13 (302), MUC15 (197), MUC19 (46), and MUC20 (108). In situ hybridization studies show that ciliated and basal cells in the surface epithelium of the lower (8, 212) and upper (9) respiratory tract, as well as goblet cells and SMG secretory cells, can express MUC genes.
In airway tissues from healthy individuals, goblet cells typically express MUC5AC mRNA or protein, while glandular mucosal cells express MUC5B and MUC8 (reviewed in Ref. 222) and MUC19 mRNA (46). MUC5B mRNA, which is typically expressed only in mucosal cells of SMG and not in goblet cells in tissues from normal adults, is also expressed in glandular neck cells and some goblet cells during midtrimester fetal development (212). Current thinking is that expression of MUC5B gene products in goblet cells in human adult lower respiratory tract epithelium is atypical and may be a marker of airway diseases (44). However, MUC5B mucin has also been reported in goblet cells in normal lung tissues from patients who died without pulmonary involvement (95, 96). These discrepancies may reflect regional variations within the airways and will require more attention to the locus from which tissues are obtained, as well as systematic morphometric analyses.
Interestingly, MUC7 mucin, which is well-expressed in mucosal and serosal cells in salivary glands (205), is typically localized to a subset of serous cells in SMG, but only in 15–20% of airway tissue from normal individuals (245). Studies on MUC gene expression in tissues from patients with airway diseases are limited in number and overviewed in section II.
Mucin expression in the in vitro NHBE and NHNE model cell systems has also been evaluated in cells derived from healthy airway tissues (19). Upregulation of MUC3, MUC4, MUC5AC, MUC5B, and MUC6 mRNA occurs during differentiation of NHBE cells. MUC1, MUC2, MUC7, and MUC8 mRNAs are also expressed but do not appear to be strongly regulated as a function of differentiation. Expression of MUC5B mRNA is localized to goblet cells in these differentiated NHBE cell cultures that lack SMG (44). MUC5B expression in vitro thus differs from expression in vivo in adult airway tissue, but is similar to expression patterns observed during development where MUC5B is expressed in goblet, as well as glandular, cells (212).
E. Mucins in Airway Secretions/Lung Mucus
Isolation and characterization of lung mucins have traditionally utilized the copious amounts of sputum from patients with airway diseases. However, comparative studies of mucins in healthy and diseased airways require procurement of airway secretions or mucus from individuals without airway disease. Healthy individuals produce much smaller amounts of airway secretions and do not expectorate mucus, but mucus samples from normal airways can be obtained following a standardized protocol of hypertonic saline exposure (270). Mucus samples can also be obtained by endotracheal suctioning of patients without airway diseases who are undergoing surgical procedures (231).
Identifying specific mucins in airway samples is now feasible because of the availability of MUC-specific antibodies. The large, cysteine-rich, gel-forming mucins MUC5AC (116, 194, 271), MUC5B (273), and MUC2 (116) mucins have been identified immunochemically in induced mucus or bronchial washings from normal airways, while MUC5AC (179, 194, 223, 271), MUC5B (57, 273, 300), MUC8 (241, 242), and MUC2 (57) mucins have been identified in sputum samples or bronchial washings from patients with chronic airway diseases.
Comparative analyses of mucin levels are more challenging than simply identifying specific mucins because of the inherent difficulties in solubilizing mucus or sputum samples (218) and in determining the best method (weight, volume, or protein) of normalizing mucin levels. The limited number of studies carried out are summarized below and presented in more detail in the disease-specific subsections in section II. Briefly, MUC5AC and MUC5B appear to be present at lower levels in mucus from nondiseased airways than in sputum from patients with asthma, chronic bronchitis, or CF (133). In contrast, in a recent study in which mucins are normalized on a weight basis, MUC5AC and MUC5B mucin levels are decreased in CF sputum compared with normal mucus (107). MUC2 mucin is detected only at very low levels in airway samples from control and diseased airways (116, 133), which is somewhat surprising as the MUC2 gene is upregulated in vitro by inflammatory mediators present in the airway secretions of patients with chronic lung disease (see sect. IIIB1). As indicated above, this may reflect the less soluble nature of MUC2 mucin.
Other mucins may be present in airway secretions, as 12 mucins have been identified at the mRNA level in the lower respiratory tract (see sect. ID). We have recently shown that MUC7 mucin is expressed in the airway secretions of most asthmatic pediatric patients but is absent in nonasthmatic pediatric patients (296). Neither levels of MUC19, a recently identified gel-forming mucin localized to mucosal cells in SMG (46), nor of MUC8, a mucin typically expressed in mucosal cells of SMG (242), have been evaluated in normal or diseased airway tissues or secretions. The question of whether extracellular glycopeptide fragments of membrane-tethered mucins are cleaved by proteases and thus present in the mucosal layer of inflamed or infected airways has also not yet been evaluated.
Levels of MUC5B and MUC5AC mucin have also been determined in secretions from differentiated NHBE cells. MUC5B mucins are present at >10-fold higher levels than MUC5AC mucins (111), in agreement with the higher level of MUC5B mRNA expression in vitro (19).
F. Comparative Biology of Mucin Gene Expression in the Respiratory Tract
Respiratory tract expression of mucin genes in non-human species has been investigated mainly in the context of perturbations during disease, especially murine models. This is overviewed briefly below, and addressed in more detail in subsections in section II on asthma, COPD, and CF.
Normal, e.g., naive, murine lungs express membrane-tethered mucins. Muc1 protein (201) and Muc4 mRNA (63) have been identified. The glandular secretory mucins Muc5b (48) and Muc19 (46) have been localized at the mRNA level to the few SMG located in the proximal trachea of some murine strains (67). In contrast to human airways, only rare superficial epithelial cells express Muc5ac mRNA in naive airway epithelium (55). However, following allergen sensitization and exposure, Muc5ac mRNA is well-expressed (4) and Muc5ac mRNA/protein is localized to allergen-induced goblet cells in the surface airway epithelium (239). Additionally, mucin gene products typically localized to murine salivary glands, e.g., Muc5b or Muc10, are expressed in murine airway goblet cells induced following allergen sensitization by ovalbumin (OVA) (48) or interleukin (IL)-13 exposure (220), respectively.
Similar to human airways, rat lungs have been reported to express Muc1 protein (201), Muc2 mRNA (193), and Muc4 protein (178) in their conducting airway epithelium. Muc5ac protein/mRNA is expressed in rat goblet cells (103, 118). Expression of other Muc genes has not been evaluated in rat, as far as we are aware.
To date, there are little data on mucin expression in other species due to the limited number of Muc clones yet identified. The Muc1 gene is expressed in hamster lung (198), rhesus lung (GenBank accession no. AF176947), and across many other mammalian species (201). The Muc2 gene is expressed in guinea pig (160) and monkey (6) lungs. The Muc5ac gene is expressed in guinea pig (160) and horse (87) lungs. As more mammalian orthologs of human respiratory tract mucin genes are identified and characterized, non-murine animal models of chronic inflammatory airway disease will be useful for evaluating mucin gene regulation and secretory cell remodeling.
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II. MUCIN OVERPRODUCTION IN CHRONIC AIRWAY DISEASES |
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Mucin glycoproteins function as part of the normal mucociliary clearance and contribute to the innate immune system in the respiratory tract. Acute challenges to the respiratory tract, including environmental toxins, allergens, or infectious pathogens, activate lung inflammatory/immune response mediators. Some mediators initiate mucin hypersecretion by activating a secretory cascade that results in the rapid release (within minutes) of mucins from secretory granules of goblet cells in the surface epithelium and/or secretory cells in the SMG (Fig. 5). These hypersecreted mucins contribute to the innate mucosal defense system by protecting the airway epithelium in ways that are not yet well understood at the molecular level. They may entrap particles in the viscoelastic mucus gel, acting perhaps in conjunction with tethered mucins at the apical surface of epithelial cells (110), or bind receptors on inflammatory cells using motifs in specific MUC domains or in O-glycans. For example, the blood group Leb oligosaccharides associated with MUC5AC gastric mucin are the primary receptor for Helicobacter pylori in the human stomach (277). Functional studies on bacterial interactions with specific O-glycan epitopes in airway mucins have not yet been carried out.
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Airway mucins are overproduced by patients with chronic airway diseases like asthma, chronic bronchitis/COPD, and CF that expectorate copious amounts of mucus. In these patients, increased mucin production in the absence of exacerbation likely reflects GCH in the airway epithelium, which increases the baseline level of mucin production (Fig. 5). Inflammatory mediators and by-products identified in the airway secretions or sputum of patients with chronic airway diseases are implicated in GCH, which is a characteristic feature and common outcome in chronic airway diseases (Table 5). However, for each airway disease, specific and unique factors appear to be responsible for initiating pathways and processes that ultimately lead to airway obstruction. That GCH reflects airway remodeling is now an accepted paradigm (75); however, the mechanism(s) that lead to GCH in specific airway diseases are not well understood. Briefly, asthma is a multifactorial disease characterized by airway inflammation, hyperresponsiveness, and GCH/mucus obstruction that is likely initiated by an imbalance of TH cytokines (39, 75). CF airway pathophysiology, which results from mutations in the CFTR gene, is characterized by airway inflammation, bacterial infection, neutrophilia, and GCH/mucus obstruction, but the underlying mechanisms that result in CF airway disease are not yet elucidated (30, 206). Inflammation associated with cigarette smoke or pollutants likely mediate the development of GCH in chronic bronchitis. While the etiology and pathogenesis leading to GCH are specific to each airway disease, mechanisms that lead to GCH are likely to converge at a common pathway.
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B. Mucin Overproduction in Asthma
Asthma, a major cause of chronic illness worldwide, has increased in prevalence by >80% in all age and ethnic groups over the past two decades (109, 196). Asthma is characterized in the airways by inflammation (TH2-driven eosinophilia), airway hyperresponsiveness, obstruction by mucus, and remodeling due to fibroblast and smooth muscle cell proliferation and GCH (38, 75). A hallmark feature of asthmatic patients who die after an acute exacerbation is the almost complete occlusion of airways by excessive mucus (3). Mucus plugging does not occur in all patients dying of asthma; however, incomplete plugs often encrust the airways of chronic asthmatics who have died from causes other than asthma (253). This indicates that mucus plug formation is a chronic process in asthmatic patients with varying disease severity, which progresses to airway occlusion and therefore may be a major contributor to disease mortality.
Excessive mucus in asthmatic patients reflects overproduction of mucins due to GCH and SMG hypertrophy, which are major pathological features of asthma. However, GCH is now thought to be a more consistent pathological feature compared with SMG hypertrophy (216). The latter feature is not common to all patients, even those with sputum production (120), whereas GCH is the most characteristic clinical finding even among newly diagnosed asthmatics (144) and in most (194), but not all (172), mild and moderate asthmatics. Airway GCH is especially marked in patients who die from status asthmaticus, with a 30-fold increase in the percentage of goblet cells compared with patients dying from nonasthmatic respiratory diseases (3). Thus airway remodeling by GCH alters airway mucin composition, contributes to the obstruction of the conducting airways with mucus, and culminates in obstruction of the airways with mucus plugs, thereby greatly contributing to the severity of asthma (21, 109, 123, 215, 216, 224, 286).
2. MUC mRNA and protein in asthmatic cells
Expression and localization of some secretory mucins have been evaluated in airway tissues from asthmatic patients. Localization of MUC5AC mRNA or protein expression is not altered in airway cells of asthmatic patients, since MUC5AC gene products are restricted to goblet cells in normal (8, 212) and asthmatic (94) airway epithelium. However, a marked increase in MUC5AC expression has been observed in asthmatic epithelial samples obtained by fiberoptic bronchoscopy and bronchial biopsy (112), and a trend toward overexpression of MUC5AC mRNA has been observed in goblet cells of bronchial brushings of asthmatic patients (194).
On the other hand, altered localization of MUC5B gene products has been noted in airway tissues from asthmatic patients where MUC5B is expressed in goblet cells and glandular neck cells, as well as in mucosal SMG cells (94). Because dual localization of MUC5B in surface epithelium and mucosal glandular cells occurs during midtrimester fetal development (212), mediators in asthmatic airways may recapitulate events that occur during development. Neither MUC2 nor MUC19 mRNA/protein expression has yet been evaluated in asthmatic airway tissues, although MUC2 mucin has been evaluated in airway secretions, as discussed in the following subsection.
3. MUC mucins in asthmatic secretions and mucus plugs
Overproduction of airway mucins in asthmatic patients is a well-established paradigm clinically, and "mucus abnormalities" are considered a feature of asthma. However, as recently pointed out in a seminal review (216), it is not yet established whether these abnormalities result from overproduction of specific mucins, an intrinsic biochemical abnormality in asthmatic mucins, or interactions between mucins and other mucosal components.
Studies to date have focused on the secreted cysteine-rich mucins: MUC5AC, MUC5B, and MUC2. MUC5AC and MUC5B mucins have been identified in asthmatic airway secretions. MUC5AC mucin, initially isolated as tracheobronchial mucin from the lung mucus of a patient with bronchial asthma (179, 223), was subsequently shown to be present in airway secretions from both healthy individuals and asthmatic patients (116, 194, 271). MUC5B mucin has also been identified immunochemically and biochemically in asthmatic sputum, as well as in normal airway mucus (273, 300). Overall, MUC5AC and MUC5B mucin levels appear higher in secretions from asthmatic airways compared with nondiseased airways (133), likely reflecting GCH as well as SMG hypertrophy/hyperplasia in some asthmatic patients (216).
MUC2 mucin is present at very low levels in normal or asthmatic airway secretions (57, 116, 133), although it is well-detected in gastrointestinal tract secretions using the same antibody (271). Because MUC2 mRNA expression is increased ex vivo in human bronchial explants from nondiseased airway tissues (157) and in vitro in airway epithelial cell lines (13) by inflammatory mediators present in asthmatic airway secretions, it is surprising that MUC2 mucin is only a minor component of asthmatic airway secretions. This may reflect difficulties in clearing "insoluble" MUC2 mucin from the airways. MUC2 is considered to be an almost insoluble mucin, which protects the intestinal epithelium against damage (10). This likely reflects the very large TR domain of MUC2, which is not interspersed by cysteine-rich domains, in contrast to MUC5AC and MUC5B mucins (Fig. 3), which predominate in the airways. Interestingly, MUC2 mucin is detected in a higher percentage of secretions from asthmatic patients (3/10) than from induced sputum of healthy individuals (1/11) (133), suggesting that analysis of MUC2 mucin warrants further analysis in a larger cohort of patients.
Recent biochemical data indicate that MUC7 mucin is present in the airway secretions of asthmatic, but not nonasthmatic, pediatric patients (296). Other secretory mucins (MUC6, MUC8, and MUC19) may be present in asthmatic secretions but have not yet been evaluated.
The molecular identity of mucins in asthmatic mucus obtained from inspissated airways following exacerbations has not been thoroughly investigated. A study carried out to biochemically investigate mucins from a patient who died in status asthmaticus (248) later identified MUC5B mucin in the mucus plug (246). MUC5AC and MUC5B mucins have been identified immunohistochemically in the lumen of patients with fatal asthma (94). A systematic evaluation of mucins that comprise the mucus plugs occluding the airways of patients who have died in status asthmaticus would be useful. The resultant data would provide fundamental information on mucin profiles following asthmatic exacerbations and thus direct future studies on regulation of MUC gene(s) activated by inflammatory/immune response genes during asthmatic exacerbations.
4. Genetic association of MUC genes and asthma
Genetic studies on mucin genes have been carried out to evaluate the potential involvement of mucins in airway obstruction in asthma and other airway diseases. Polymorphisms in the number and lengths of TR in MUC1, MUC2, MUC4, MUC5AC, and MUC5B genes have been identified. Additionally, polymorphisms have been evaluated in atopic patients with or without asthma. No correlations of VNTR polymorphism in MUC1, MUC4, MUC5AC, or MUC5B genes were observed in patients with asthma, although a longer allele in the MUC2 gene is associated with a cohort of atopic, nonasthmatic patients (284). In contrast, an association of asthma with the MUC7 gene is reported, with a significantly lower frequency of the MUC7*5 allele in a small cohort of Northern European asthmatics (132).
5. Goblet cell metaplasia and mucin gene expression in murine models of asthma
Mice have proven useful models over the last decade for investigating the pathogenesis of allergic asthma. In vivo studies using OVA sensitization and challenge or direct cytokine exposure have linked Th2 cytokine-mediated inflammation and GCM. IL-13 is a central mediator of GCM in murine models of allergic asthma (97, 140, 303), and GCM induced by other Th2 cytokines is stimulated through a common, IL-13-mediated pathway (299). GCM is a characteristic phenotype used to monitor the pathophysiology of asthma, since goblet cells are very rarely seen in the airway epithelium of naive mice, while airway goblet cells predominate in mice exposed to inflammatory or immune-response mediators. Briefly, mice exposed to single or multiple doses of allergen or IL-13 develop GCM within 1–3 days after one or more allergenic challenges (4, 239, 303), although a few goblet cells are evident by 6 h after a single exposure to IL-13 (239) or OVA challenge (74).
Without continual exposure to allergens, GCM is transient in murine airways, although the temporal processes are impacted by allergen or mediator, dose, and murine strain. The epithelium reverts nearly to baseline levels 11 days after multiple challenges with OVA (22) and GCM, which is maximal at 3 days after a single OVA exposure, is minimal 8 days following OVA exposure (239). IL-13-induced GCM also reverts to baseline following multiple IL-13 exposures (97, 140, 303). Taken together, these results suggest that sustained GCM requires ongoing exposure of the airways to IL-13, either directly or indirectly following an allergenic exposure, and that a fairly rapid reversal of the processes leading to GCM occurs in the absence of IL-13 or genes activated by IL-13. Thus GCH manifested in patients with asthma or obstructive airway diseases may also be reversible if allergens or inflammatory mediators are removed or inhibited. If this were not so, the prevalence of GCH would be nearly 100% at any time in allergenic environments, whereas in the absence of environmental insults, GCH can revert to a normal morphology in conducting airways (238).
Ligand binding of IL-13 to the IL-4R
/IL-13 receptor activates Stat6, which is a key regulator of IL-13-mediated GCM (140, 141). While the downstream genes and pathways that lead to GCM in murine models of allergic asthma remain to be identified, Foxa2, a winged helix transcription factor, is implicated in this pathway as mice that lack Foxa2 manifest GCM (294). Mechanisms that contribute to the reversal of GCM in rodents are also being investigated. Members of the Bcl-2 family, which inhibit apoptosis, play a role in restoring a metaplastic airway epithelium to a normal phenotype following allergenic challenges (250). A better understanding of the mechanisms that directly initiate or reverse GCM in murine airways may lead to pharmacological intervention that would ultimately decrease or circumvent GCH, and thus airway obstruction by mucus in human airways.
Cytokine-induced GCM correlates with increased expression of Muc5ac, an airway goblet cell marker. However, this correlation is not a demonstration that the cytokine of interest regulates mucin gene expression. For example, IL-4 (269) and IL-9 (171) are reported to increase Muc5ac gene expression and mucus production in vivo, but this likely reflects the IL-13-induced increase in goblet cells (299), which endogenously express Muc5ac mRNA/protein, rather than cytokine regulation of mucin gene expression. IL-4 does not upregulate MUC5AC/Muc5ac gene expression; the IL-9 story is less clear (Table 6, see sect. IIIC). In addition to inducing GCM in murine airways, IL-13 may, however, directly or indirectly upregulate Muc5ac gene expression, as IL-13 increases the promoter activity of Muc5ac following transfection of a Muc5ac-promoter-luciferase plasmid into murine transformed Clara cells (74). However, IL-13 does not upregulate MUC5AC mRNA expression in three human lung cancer cell lines (226) or in differentiated NHBE cells (45), suggesting differences in the sequences or regulation of Muc5ac and MUC5AC promoters.
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Two models of chronic asthma in which GCM is sustained and not transient have recently been established and may prove useful in investigating pathways that maintain GCH. In an OVA-induced model, airway remodeling manifested by GCM persists after resolution of acute allergen-induced airway inflammation (152). In a virally induced model, GCM develops following infection of murine airways with Sendai virus (SV) and subsequent viral clearance (293). Sendai virus, a murine RNA virus of the family Paramyxoviridae, is a natural pathogen of mice and causes reversible bronchiolitis and pneumonia in susceptible strains of mice (35, 199, 261), which parallels clinical features of childhood and adult asthma in the lower respiratory tract (149, 298). GCM is maintained for more than a year in C57BL/6 mice following SV infection, suggesting that genes and pathway(s) that lead to a chronic asthma phenotype are activated and maintained by the murine host in response to infection by a murine virus. Related work in rat strains has shown that Brown Norway rats are more likely than F344 rats to develop an asthma phenotype following SV inoculation (258).
C. Mucin Overproduction in COPD
1. Definition of COPD/chronic bronchitis: the inflammatory milieu
COPD is defined clinically as a complex of diseases characterized by airflow obstruction due to chronic bronchitis or emphysema. It is the fourth leading cause of patient deaths in adults in the United States. Approximately 14 million people in the United States have COPD; the majority of these patients have chronic bronchitis (237). The diagnostic criteria for chronic bronchitis are the presence of cough and sputum on most days for at least 3 mo per year in two consecutive years without another explanation (5). Epidemiologically, the major risk factor associated with the development of chronic bronchitis is exposure to environmental inhalant toxins, especially cigarette smoke. Exacerbations of airway disease may be provoked by viral or bacterial infections or by exposure to air pollutants.
The central airways of patients with chronic bronchitis are chronically inflamed with increased numbers of macrophages and T lymphocytes (237). Corresponding to the cellular inflammatory profile, there are increased levels of IL-6, IL-1, IL-8, tumor necrosis factor- (TNF-), and monocyte chemotactic protein-1 in induced sputum or bronchoalveolar lavage from patients with stable COPD (52). During acute exacerbations, neutrophils and neutrophil chemokine and receptor expression increase in the airways of COPD patients (211). Neutrophils and macrophages release proteases that contribute to the inflammatory milieu and overwhelm antiprotease defenses. In addition, reactive oxygen species (ROS) generated by cigarette smoke or by inflammatory cells may produce airway and parenchymal injury (237). Together, these mediators injure the airway, activating programs for remodeling (including GCH) and/or MUC gene regulation.
A major pathological feature of chronic bronchitis is an increase in secretory cells due to hypertrophy of SMG and to GCH, which can extend to the peripheral airways. Secretory cell hyperplasia is a prerequisite for sustained mucus hypersecretion/mucin overproduction. These phenotypes are a significant component of airway obstruction in chronic bronchitis and directly correlate with reduced pulmonary function (283).
Altered cellular localization and increased expression of mucins have been reported in patients with COPD. In airway tissues from patients with obstructive airway diseases, MUC5B mRNA is expressed in goblet, as well as glandular, cells, while MUC5B is not expressed in goblet cells of control patients. MUC5B mRNA is also expressed in the goblet cells of differentiated NHBE cells derived from these patient tissues, but expression levels are similar in cells from control patients and patients with obstructive airway diseases (44). In another study, no differences in alcian blue or periodic acid Schiff (PAS) staining are observed in peripheral lung sections from 9 smokers with COPD, 11 age-matched controls including smokers, and 6 lifelong nonsmokers with normal lung function, although intraluminal PAS staining is significantly more frequent among COPD subjects. MUC5AC protein expression is significantly higher in the bronchiolar epithelium of patients with COPD, and MUC5B expression is increased in the bronchiolar lumen MUC5B of COPD patients relative to controls (41).
Airway mucins from chronic bronchitis patients overall are similar in size and structure to mucus from healthy individuals, but appear to be less acidic (56). However, glycosylation patterns vary during acute exacerbations, as chronic bronchitis mucins are highly sialylated with increased sialylated and sulfated Lex structures (147). Despite biochemical characterization of the mucus gel, information about specific mucin glycoproteins in chronic bronchitis airway secretions is limited to a few studies. MUC5AC and MUC5B mucins are highly expressed constituents both in normal and chronic bronchitis mucus (133). The ratio of MUC5B:MUC5AC is increased, and MUC5B differs in charge in chronic bronchitis sputum compared with normal airway mucus (133). These observations suggest that both goblet and SMG cell secretions contribute to the mucus hypersecretion observed during exacerbations of chronic bronchitis.
3. Mucin regulation by inflammatory mediators in vitro and in vivo in animal models of chronic bronchitis
Because cigarette smoke is the single common factor related to development of chronic bronchitis, several in vivo models have been established to investigate the mechanism of smoke-induced airway inflammation and secretory cell metaplasia. Either inhaled tobacco smoke or cigarette smoke extract induce secretory cell metaplasia in rat main stem bronchi (146) or in BALB/c mice airways (181). In mice, exposure to smoke increases neutrophilic inflammation, mucin expression, and airway reactivity to methacholine. An aldehyde component of cigarette smoke, acrolein, induces expression of Muc5ac gene in vivo in rat (27) and mouse (28) lungs, concomitant with secretory cell metaplasia. In mice, these effects are dependent on macrophage, rather than neutrophil, inflammation (28). One mediator that regulates the secretory metaplastic response to cigarette smoke in guinea pig airways is platelet activating factor (137).
Air pollutants also induce secretory cell metaplasia in animal models in vivo. Sulfur dioxide exposure induces superficial secretory cell hyperplasia in rats (145) and mucus gland hypertrophy in dogs (236). Ozone induces mucus cell metaplasia in rat nasal airway epithelium (106). Neutrophil elastase (NE) proteolytic activity stimulates bronchial secretory cell metaplasia in hamsters (32) and in mice (288). It is noteworthy that inflammation associated with cigarette smoke or pollutants appears to mediate the development of secretory cell metaplasia. This concept is supported by evidence that glucocorticoid treatment mitigates the effect of cigarette smoke (214) or ozone (113) on secretory cell metaplasia.
Bacteria, including Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), and Haemophilus influenzae (H. influenzae), are common pathogens that trigger exacerbations of bronchitis. Bacterial products regulate mucin gene expression in epithelial cell lines in vitro, as discussed in section III. In vivo, endotoxin or lipopolysaccharide (LPS), a component of Gram negative bacterial cell wall membranes, increase goblet cell number in rat nasal epithelia (251) and in murine respiratory epithelia (306). Endotoxin also enhances ozone-induced secretory cell metaplasia in rat nasal epithelia (292), suggesting that infections and pollutants synergize to exacerbate secretory cell metaplasia. Paramyxoviruses also induce secretory cell metaplasia in vivo as a separate trait independent of intracellular adhesion molecule (ICAM)-1 expression and acute inflammation in a chronic model of murine asthma (293). These studies demonstrate the powerful effect of infectious agents on the induction of secretory cell metaplasia, thereby resulting in increased mucin production since goblet cells endogenously express mucin genes and secrete mucin glycoproteins.
The complement of inflammatory mediators expressed in the airways of patients with stable COPD and asthma are different, as TH1 cytokines predominate in COPD and TH2 cytokines predominate in asthma (Table 5). Interestingly, viral infections, cigarette smoke, and pollutants cause exacerbations in both patient populations, which are marked by neutrophil inflammation. Mucus secretion is an important component of the innate immune system. Therefore, teleologically, it is possible that both TH1 and TH2 cytokines regulate mucin gene expression and secretory cell metaplasia in vivo and that individual cytokines have different functions in epithelial and immune cells. These possibilities are being investigated by numerous laboratories, and current data on mucin gene expression are presented in Table 6. It is likely that some mediators synergistically interact with pollutants, infectious agents, and neutrophilic mediators to significantly enhance remodeling that culminates in mucin overproduction and hypersecretion in airway disease states.
CF was initially called "mucoviscidosis" because of copious amounts of "mucoproteins" in the respiratory and gastrointestinal tracts of CF patients (76). Evidence for a fundamental mucus abnormality in CF is supported by ultrasound studies, wherein 90% of CF fetuses during the 17- to 19-wk period of gestation manifest inspissated meconium in the distal ileum, which is generally reabsorbed by birth (68). Gastrointestinal mucus obstruction is not life threatening except to the small number of CF infants born with meconium ileus.
In contrast, mucus obstruction in CF airways occurs postnatally (309), even though pulmonary complications resulting from airway mucus obstruction and predisposition to infection are the major cause of morbidity and mortality in CF patients (297). There are no marked morphological abnormalities in the airways of CF fetuses or neonates and the numbers and distributions of goblet cells in the epithelium, as well as the numbers and sizes of SMG, are within normal ranges at birth (263). Dilated acinar and duct lumens in SMG are observed in CF airways early in life; the earliest consistent pathological lesion and evidence of mucus obstruction presents in the bronchioles (262).
CF is a recessive genetic disease caused by mutations in the CFTR gene, which encodes the cystic fibrosis transmembrane regulator glycoprotein, CFTR (213). CFTR is expressed in mucus-rich epithelial tissues (respiratory, gastrointestinal, and reproductive tracts), non-mucus rich tissues (sweat glands and kidney), as well as nonepithelial tissues (heart and brain) (274). However, obstruction is typically observed only in mucus-rich tissues. The question of how CFTR initiates the primary pathogenetic event in CF airways is still unresolved and remains a question of paramount interest. There are several current hypotheses on the pathogenesis of CF lung disease. 1) Expression of mutant CFTR in CF respiratory cells produces defective chloride secretion and elevated sodium absorption (29), resulting in altered salt concentrations in airway secretions. However, the question of whether salt concentrations in CF airways are hypotonic or hypertonic is still unresolved (98, 268). 2) The concept that alterations in mucus volume impact on mucus hydration, and thus on the rheology of CF airway mucus to increase susceptibility to infection in CF airways, continues to gain credence (30). A marked decrease in the airway surface liquid volume in CF bronchial explants in vitro reflects the a
) and cotranslationally inserted into the endoplasmic reticulum (ER). O-glycosylation of the MUC protein backbone is initiated posttranslationally in the cis-Golgi following transfer of GalNAc to serine or threonine amino acids by an N-acetylgalactosaminyl peptidyltransferase. The addition of GalNAc likely alters the conformation of globular apomucins to a more linear molecule (
) residues in the TR domains. O-glycans exhibit size heterogeneity, which may reflect incomplete biosynthesis during elongation and termination of O-glycans or differences in substrate preferences of various GTs at specific serine or threonine sites.
