Mucins are a heterogeneous group of polydisperse and high molecular weight O-glycosylated proteins, ranging from 300,000 to >1 × 10⁷ Da [1]. Mucin genes encode polypeptide monomers that are synthesized as rod-shape apomucin cores and are post-translationally modified by exceptionally abundant and terminally sialylated glycan moieties. Mucins are secreted or inserted to the apical surface membrane of epithelial cells via a membrane-spanning hydrophobic domain [2, 3]. Secreted mucins constitute the most abundant glycoprotein component of mucus, a gel-like substance that covers all wet-surfaced epithelia, including those of the respiratory, gastrointestinal, and genitourinary tracts [4–7]. Eighteen mucin (MUC) genes have been cloned, 12 of which are expressed in the respiratory tract [3]. Airway secreted mucins (MUC2, 5AC, 5B, 7, 8, 11, 13, 19, and 20) are produced by goblet cells on the surface epithelium and mucous cells from sub-mucosal glands [4, 5, 7]. Membrane-associated mucins in the airways include MUC1, MUC4, and MUC16. Mucin gene and protein structures, goblet cell mucin secretion and hypersecretion in airway diseases as well as the regulation of goblet cell hyperplasia have been well-documented in the literature [7–9]. However, the complex interactions between mucins and other protein components of airway mucus remain to be elucidated.

In vitro cultures of human and animal airway epithelial cells have been used to characterize mucin production under various experimental conditions. Human cell cultures from tracheal glands were reported to express a mixed serous and mucous phenotype [10, 11]. Normal and cystic fibrosis cells from human tracheal glands isolated by the explant/outgrowth procedure secreted high molecular weight mucin-like glycoproteins and expressed the MUC2 gene [12]. The development and characterization of primary cell cultures from tracheal surface epithelium [13] that were highly enriched for secretory cells allowed the first identification of authentic airway mucins that were produced in vitro [14]. Confluent cultures of hamster tracheal epithelial cells cultured on a type I collagen gel synthesized and secreted high molecular weight glycoconjugates which eluted in the void volume upon Sepharose CL-4B column chromatography [14, 15]. Biochemical analysis of these mucous glycoproteins showed both size and charge microheterogeneities that were remarkably similar to those of mucins produced in vivo.

The in vitro model of mucin production by airway epithelial cell cultures first established in hamsters was subsequently extended to guinea pig and human airway epithelium [16, 17]. Proteomic studies have revealed that the protein composition of in vitro human airway epithelial cell secretions is particularly complex. Candiano et al. [18] used a two-dimensional SDS-PAGE/proteomics approach to identify the proteins secreted by in vitro cultures of polarized monolayers of human airway epithelial cells at resting and following stimulation with IL-4, IL-1β, TNF-α, or IFN-γ. Approximately 175 polypeptides were identified, among which were immune-related proteins, structural proteins, proteases, and protease inhibitors. Comparisons between treated and untreated conditions showed that the expression of several proteins was significantly modified by the different cytokines. Kesimer et al. [19] identified 134 proteins from apical secretions of primary airway epithelial cells, with 84 proteins (62.7%) being common with the proteins identified in in vivo human tracheobronchial sputum. Separation of the proteins by density gradient ultracentrifugation in the presence of 4 M guanidine hydrochloride identified 29 proteins that were present in the high-density, mucin-rich fraction. The nonmucin proteins in this fraction were also present in the lower-density pool, suggesting to the authors that they associated and copurified with the mucins. However, given that the proteins were isolated under denaturing conditions, the current study was conducted to identify mucin-associated proteins in in vitro human airway epithelial cell secretions isolated under more physiological conditions.

Among the 55 proteins that were identified in the high molecular weight fraction of hAECS from in vitro cultures of primary NHBE cells collected under physiological conditions, 43 (78.2%) were unique to this study and 12 (21.8%) were common to proteins identified in the mucin-rich apical secretions isolated under denaturing conditions by Kesimer et al. [19]. The shared proteins of the two studies are: MUC1, MUC4, MUC5B, MUC16, CD59, complement C3, clusterin, glutathione S-transferase, lactotransferrin, long palate, PLUNC (palate, lung and nasal epithelial clone) protein, LPLUNC1 (long PLUNC), and the polymeric immunoglobulin receptor. All of the proteins unique to our study comprise important inflammatory, anti-inflammatory, anti-microbial and/or anti-oxidant components that are elicited by the respiratory tract in response to exposure to infectious or injurious insults [21]. On the other hand, among the proteins that were identified by Kesimer et al. [19] but not in this study are alpha-defensin 1 and secretory leukocyte peptidase inhibitor (SLPI), both of which are thought to have broad-spectrum antibiotic activities [22].

While it is tempting to speculate that the proteins we identified may form a physiologically relevant association with mucins, it is important to emphasize that they may or may not be actively secreted by the cells, or may simply constitute membrane-bound proteins that passively adhere to the mucins, although other membrane components, such as lipids, were not identified. Further, given that the apical washings were dialyzed against distilled water, we cannot exclude the possibility that some of the identified polypeptides are intracellular proteins from sloughed cells and thus are not necessarily components of airway mucus. We also cannot rule out that our results may not reflect the proteome of in vivo airway lining fluid given the probable contributions of other cell types and blood-borne proteins that were not present in the NHBE cell cultures. However, the contribution of the cell culture media appears negligible since serum albumin, the most abundant protein in culture media, was not identified in the current analysis.

The presence of MUC1, MUC4, MUC5B, and MUC16 in in vitro hAECS confirms prior studies of mucin glycoprotein secretion by human and animal airway epithelial cell cultures [13–19]. Mucins constitute the main protein components of mucus and provide a multifaceted function to the mucosal surface from which they are produced, ranging from lubrication to cell signaling to forming protective physical barriers against chemical or biological damage [4–6]. But whether or not these functions are provided by mucins alone or in association with other proteins remains an unanswered question, which we and others [18, 19] have attempted to address. Specifically, several reports have promoted the concept that the association between mucins and other proteins plays a vital role in defense of the respiratory and gastrointestinal mucosa against microbial infections [23–25], including an early model put forth for mucin-protein interactions in lung mucus by Rose et al. [26].

The inability to identify MUC5AC mucin by proteomic analysis may have been due to insensitivity of the detection instrumentation, incomplete trypsin digestion, a relatively low abundance of this protein, and/or failure of TCA to precipitate MUC5AC. Other proteomic studies of hAECS collected from in vitro cell cultures have demonstrated that MUC5B is the predominant mucin with much lesser amounts of MUC5AC [19]. By microarray hybridization analysis, expression of MUC1 and MUC5B mucins, but not MUC5AC, were up-regulated during ALI culture of human bronchial epithelial cells [27]. Therefore, these or other technical artifacts are the most likely explanation for the current result given that MUC5AC was clearly present in the hAECS by Western blot analysis and the fact that our prior study identified human MUC5AC in the spent culture medium of A549 lung epithelial cells and rat Muc5ac in apical washings of primary tracheal epithelial cell cultures at an ALI [28].

The family of proteins identified in this study were categorized according to molecular and biological functions by Pather analysis. According to the original definitions of Gene Ontology by Ashburner et al. [29], molecular function is defined as the elemental activity of a gene product at the molecular level, such as binding or catalysis, while a biological function refers to the collected operations or sets of molecular events with a defined beginning and end, and pertinent to the functioning of integrated living units (cells, tissues, organs, and organisms). A biological function is accomplished via one or more ordered assemblies of molecular functions. In this study, the molecular functions associated with the greatest number of proteins that were identified in the high molecular weight fraction of the hAECS samples were regulatory (11 proteins), calcium binding (9 proteins), transfer/carrier (6 proteins), and defense/immunity (6 proteins). The biological functions associated with the greatest number of proteins were defense/immunity (18 proteins), protein metabolism and modification (11 proteins), and signal transduction (9 proteins). As noted above, some of these functions have been previously ascribed to airway mucins.

In addition, this collection of proteins could be further classified into inflammatory, anti-inflammatory, anti-oxidative, and anti-microbial categories based upon search of the published literature and molecular/biological function analyses. Some of the proteins fall into more than one category because they perform more than one function. For example, protease inhibitors may act both as inflammatory and anti-inflammatory components of mucus, depending upon the context of their expression [21]. Protease inhibitors that were identified in the present study were alpha-1-antichymotrypsin, alpha-2-macroglobulin, alpha-2-antiplasmin, inter-alpha-trypsin inhibitor heavy chain H2, pigment epithelium-derived factor, cystatin-B, and tissue inhibitor of metalloproteinase 1. Many of these proteins have been shown to regulate protease activities in various diseases that afflict airway epithelia [30–35].

The anti-oxidative proteins identified herein were ceruloplasmin, clusterin, glutathione S-transferase (GST), pigment epithelium-derived factor, peroxiredoxin 1 (Prdx1), lactate dehydrogenase, lactotransferrin, ubiquitin, and ubiquitin-conjugating enzyme E2 D2. Amongst these, GST and Prdx1 are probably the best characterized in the respiratory tract. GST provides redox balance in response to production of reactive oxygen species and has been reported to be involved in host defense in the lung [36]. The Prdx family of anti-oxidant enzymes reduces peroxides, lipid hydroperoxides and peroxynitrites, and the role of Prdx1 in airway inflammation has been reviewed [37]. Within the group of anti-microbial proteins, CD59 is involved in inflammatory signal transduction in T cells in response to pathogenic insults [38], while components of the complement system act as antibiotic components during host defense against bacteria that commonly infect the respiratory tract, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Streptococcus pneumoniae [39–41]. The palate, lung and nasal epithelial clone (PLUNC) family can be subdivided into short (SPLUNC) and long (LPLUNC) proteins [42]. In this classification, the original protein called PLUNC is now SPLUNC1, which is expressed in sub-mucosal glands of normal individuals and expression is increased in cystic fibrosis lungs, especially in the surface epithelia of the conducting airways. PLUNC was reported to protect the airway epithelial sodium channel (ENaC) from proteolytic cleavage, to prevent respiratory infections, and to inhibit lung allergic responses [43, 44]. Finally, the S100 series of Ca²⁺ binding proteins are highly expressed in neutrophils and monocytes [45, 46] and have been reported as anti-microbial agents against P. aeruginosa [47] as well as novel TLR ligands important in modulating inflammation [45, 48].

We identified 55 proteins that were present in the high molecular weight fraction of in vitro human airway epithelial cell apical secretions isolated under physiological conditions. Among the mucin gene products that were identified were MUC1, MUC4, MUC5B, and MUC16. By proteomic analysis, these proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial. Given that the majority of these proteins in their monomeric form possess molecular weights far less than that selected for analysis, it is theoretically possible that they may associate with the highly sialylated and negatively charged mucins through ionic and hydrophobic interactions to form a highly ordered structural organization that may be important for maintaining the proper function of airway mucus.