We have compared the protein composition of pooled AS from air-liquid interface cultures of normal human bronchial epithelial cells with IS from normal healthy donors. Such primary cell cultures are commonly employed as a model system for a host of biological studies, from CF pathogenesis to viral infection (3
), and our particular focus is the innate protective properties of mucus, specifically, the contribution of mucins to the structure of this complex environment. As a consequence, our approach was developed to take into account these major host defense components, which we show (by refractometry) contribute 20–30% of the mass of the total biomolecules in IS and AS from cell culture. This is an important finding, since proteomic analyses of human IS (25
), as well as other mucous secretions such as cervicovaginal (33
), nasal (8
), saliva (42
), and airway surface liquid (7
), have either not identified mucins or have identified few peptides. For instance, a proteomics study by Candiano et al. (7
) on the airway surface liquid of the bronchial epithelial cell culture reports no mucins. The study by Nicholas et al. (25
) on sputum reports the presence of MUC5B (13 peptides), MUC5AC (6 peptides), and MUC1 (1 peptide). In contrast, we have identified 98 peptides from MUC5AC and MUC5B from IS (see supplemental Fig. 1 for comparison); 5 of these peptides may have arisen from either MUC5B or MUC5AC, since these mucins share some identical sequence. The reason for the discrepancy is likely to be in the approaches taken to separate the components in the mucous secretions before MS-based identification. Previous studies have typically employed one- and two-dimensional PAGE methods, in which large glycoproteins, such as mucins, would be largely excluded and, therefore, not represented in subsequent MS analyses. However, the approach taken here separates the molecules in solution on the basis of their buoyant density; therefore, they are not excluded from the MS analysis. Thus future proteomic studies on mucus, from whatever tissue, should ensure that the methodology does not exclude these important glycoproteins.
Mucins represent a large proportion of the mass of biomolecules in the cell culture and in vivo secretions, but using the refractometry-based analysis alone, we cannot discriminate between the gel-forming mucins and the large epithelial mucins MUC1, MUC4, and MUC16. However, the MS data of the mucin-rich pool show, on the basis of the number of peptides identified from each mucin, that the gel-forming mucins MUC5AC and MUC5B are more abundant than the epithelial mucins. Furthermore, the cell culture secretion contains more MUC5B than MUC5AC, whereas IS contains similar levels of these two mucins. It is noteworthy that MUC2, which has been shown to be present in trace amounts in sputum (18
), was not identified in AS or IS, indicating that this mucin is not a major component of either secretion.
No specific functional association has been identified for MUC5AC and MUC5B in airway mucus, but the MUC5B-rich mucus produced in culture is at least able to be transported by cilia (22
). Although no distinct function has been attributed to each mucin type, they are produced in distinct locations within the normal airways: MUC5B is mainly a product of the submucosal glands, and MUC5AC is produced by the surface epithelium (15
). Thus our data suggest that the culture may actually have aspects of a ductal/glandular phenotype, rather than simply representing the epithelial surface, where the normal goblet cell mucin is shown to be MUC5AC. In support of this notion, other proteins such as DMBT1 (40
) and a number of other protective antibacterial proteins, such as lysozyme (11
) and PLUNC and LPLUNC1 (5
), found in this study are also products of glandular cells. The presence of known serous cell-derived proteins, such as lysozyme, α1
-antichymotrypsin, neutrophil gelatinase-associated lipocalin, and heat shock cognate 71 (16
), suggests that the cultures possess a mixed mucous and a serous secretory phenotype.
The contribution of epithelial mucins (MUC1, MUC4, and MUC16) to both secretions is minor, but, interestingly, their contribution (based on the number of peptides identified and coverage of the nonglycosylated regions) to the cell culture secretions is greater, as confirmed by Western blotting after agarose electrophoresis (unpublished data). Why this should be the case is not clear; it might result from the different methods used to collect the two samples. The cell culture sample was collected from the apical surface of the cells after 24–48 h, whereas in vivo mucus elicited by the hypertonic saline treatment was collected within minutes of stimulation. Thus the in vivo secretions may contain a higher level of freshly secreted material; furthermore, the mucus is not confined to a culture dish but can be moved from the local area by mucociliary clearance. Thus the culture sample may contain a higher level of cell surface molecules as a result of epithelial cell turnover or increased proteolysis-induced shedding as a consequence of its inability to be moved from the local area.
Overall, the composition of the in vivo and in vitro secretions is similar. However, there are many differences in the constituent proteins, likely because of the diversity of cell types contributing to the in vivo mucus. Whereas the cell culture mucus is dominated by molecules released from ciliated epithelial cells and mucin-secreting cells (13
), IS contains secretions from these cells and extra contributions from cells in the submucosal glands and peripheral lung (e.g., surfactant-related proteins derived from alveolar type II cells). Furthermore, it will also contain proteins from plasma exudate and saliva (see supplemental Table 2). Once the contribution of salivary, immune cell, and blood-related proteins has been taken into account, the similarity of the protein composition of the two secretions becomes increasingly evident. Given that the culture secretions have been shown to form flowing mucus (22
), we infer that the cohort of proteins identified is sufficient to make a gel with properties that enable transport by cilia. However, we do not know if it provides an effective biological barrier and how, or even if, these properties differ from those of mucus produced in vivo. One might speculate that the “extra” components in the sputum confer different biological properties to mucus; for example, the role of plasma-derived proteins as complementary factors to epithelium-derived proteins in the physiology of normal mucus is likely to be important. Furthermore, the extra components are likely to make a greater contribution to the secretion in disease, e.g., albumin and fibrinogen in asthmatic sputum (31
). Thus, although we acknowledge some limitations of this in vitro model system, the secretions from these cultures, with their absence of proteins from plasma and migratory cells, provide a good starting model to address questions relating to the innate defense of the airway epithelium. In particular, the cultures will provide a unique insight into the role of airway cell-derived proteins, many of which have no proposed function within the context of mucus in airway defense.
Removing the mucins as a separate category, we divided the remainder of the proteins into nine categories according to their proposed general functions by the UniProtKB/Swiss-Prot database (). The proportion assigned to each category is based on the total number of peptides of all the proteins identified and contributing to that category; although this method is not completely rigorous, it does permit comparison of the two samples. Clearly, proteins in the innate immune category form the major group. In support of this notion, transcript profiling data of air-liquid interface cultures of CF and non-CF airway epithelial cells show that transcripts for host defense proteins, some of which are major proteins identified in our analysis (polymeric immunoglobulin receptor, SPLUNC, and LPLUNC1) are highly abundant (32
). From a comparison of all proteins considered to be important for innate immunity, we found that 33 such proteins were common to IS and AS, 41 are present in AS, and 39 are present in IS (putative serum and saliva-derived host defense proteins are not included in these numbers). The preponderance of such proteins in the cell culture secretion suggests that it will be a useful tool for study of innate immunity.
Proteins identified in the culture secretion and the IS were further analyzed to determine whether they were secretory proteins; for this analysis, proteins were checked for signal peptide cleavage sites with the SignalP 3.0 server (). In AS secretion 73 (54%) proteins were found to have signal peptide cleavage sites compared with 70 (52%) in IS. The remainder of the proteins not considered secreted proteins by this analysis comprise cytoplasmic, membrane, lysosomal, Golgi, and cytoskeletal-related components according to the UniProtKB/Swiss-Prot database annotations. A substantial proportion of the cell-derived proteins in the nonsecreted category are cytoskeletal in origin. Of these, proteins such as actin and ezrin are mainly associated with the glycocalyx, especially around the microvilli on ciliated cells. This raises the following questions: 1) How do these proteins arise in the secretion? 2) What is the impact of these proteins on the protective properties of the secretion? Since no major cellular organelle proteins, such as cytochrome c from mitochondria, glucose-6-phosphatase from endoplasmic reticulum, or ribosomal proteins, were detected, it seems possible that these proteins, of epithelial origin, may arise as part of the secretion process from the cells, rather than a generalized disruption process. Whether these proteins are a further part of the protective function of mucus is not clear but warrants further investigation.
Fig. 5. Analysis and comparison of cellular origin of proteins in AS and IS. Presence of secretory signals was checked by SignalP 3.0 and SecretomeP 2.0 server. Nonsecreted proteins were classified according to UniProtKB/Swiss-Prot database annotations. Potential (more ...) Summary and conclusions.
We have described a compositional comparison of proteins from normal primary human respiratory cell culture washings and IS. These data suggest that the culture is a valid model for study of innate immune protection functions of mucins and mucus (derived wholly from airway cells) on a ciliated epithelium. Except for the mucins, the sputum compositional data presented here are in good agreement with data from other compositional studies (23
) and provide a set of reference data for comparison of secretory proteins from pathological states of the lung, such as chronic obstructive pulmonary disease, asthma, CF, and lung cancer, in culture and sputum. This study was motivated by two questions: 1
) Is the protein and mucin composition of the cell culture secretion similar to that of in vivo mucus? If we exclude the proteins that could not originate in the airway epithelia, there is a high level of identity (90%) of proteins; thus we take the answer to be yes. 2
) Is it a valid model to investigate aspects of innate defense of the airways? The percentage of shared proteins associated with innate immunity (87%) also indicates a positive answer.