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Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

Kesimer M, Kirkham S, Pickles RJ, Henderson AG, Alexis NE, Demaria G, Knight D, Thornton DJ, Sheehan JK - Am. J. Physiol. Lung Cell Mol. Physiol. (2008)

Bottom Line: Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS).When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins.This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry and Biophysics, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. kesimer@med.unc.edu

ABSTRACT
Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

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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 salivary- and blood-sourced proteins were excluded.
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f5: 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 salivary- and blood-sourced proteins were excluded.

Mentions: 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 (Fig. 5). 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.


Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

Kesimer M, Kirkham S, Pickles RJ, Henderson AG, Alexis NE, Demaria G, Knight D, Thornton DJ, Sheehan JK - Am. J. Physiol. Lung Cell Mol. Physiol. (2008)

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 salivary- and blood-sourced proteins were excluded.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636953&req=5

f5: 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 salivary- and blood-sourced proteins were excluded.
Mentions: 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 (Fig. 5). 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.

Bottom Line: Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS).When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins.This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry and Biophysics, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. kesimer@med.unc.edu

ABSTRACT
Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

Show MeSH
Related in: MedlinePlus