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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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Mucins (A) and protein groups (B) in AS and IS. Values in A are averages over 4 independent experiments; error bars represent SD. All peptides from proteins from extraneous sources, e.g., saliva, serum, and lung, were excluded from B (see supplemental Table 2 for excluded proteins).
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f4: Mucins (A) and protein groups (B) in AS and IS. Values in A are averages over 4 independent experiments; error bars represent SD. All peptides from proteins from extraneous sources, e.g., saliva, serum, and lung, were excluded from B (see supplemental Table 2 for excluded proteins).

Mentions: MS/MS analysis of tryptic peptides derived from the mucin-rich fraction identified a total of 29 proteins in AS and 28 in IS (Table 2); as expected, this fraction was dominated by mucins. The same five mucins, namely, MUC1, MUC4, MUC16, MUC5B, and MUC5AC, were identified in AS and IS (Fig. 4A), together with another glycoprotein, DMBT1/gp340, which has been shown to copurify with mucins under these conditions (40) and is also considered to be a mucin (24). Four of these mucins (MUC4, MUC5AC, MUC5B, and MUC16) were unique in this pool; MUC1 and DMBT1 were also present in the protein-rich pool. The nonmucin proteins identified in this fraction were also present in the lower-density, protein-rich pool. To compare the mucin peptide coverage, we have only considered the nonglycosylated regions of the protein. In the AS sample, the gel-forming mucin MUC5B was, from this perspective, the dominant mucin (61 peptides, 45% coverage); lesser amounts of MUC5AC (32 peptides, 21% coverage) and the epithelial mucins MUC1 (5 peptides, 19% coverage), MUC4 (8 peptides, 10% coverage), and MUC16 (21 peptides 11% coverage) were found. In the IS sample, MUC5B was again the major mucin detected (51 peptides, 38% coverage), but MUC5AC appeared to be present at a comparable level (47 peptides, 30% coverage). The epithelial mucins MUC1 (2 peptides, 7% coverage), MUC4 (1 peptide, 1% coverage), and MUC16 (6 peptides, 4% coverage), although identified in IS, contributed fewer peptides and less coverage (of the nonglycosylated regions) than in the AS sample, suggesting that they were present at a lower level in IS. A measure of the enrichment of the mucins against all other proteins may be taken as the ratio of total protein peptide to mucin peptide in the two pools: 72 in pool 1 and 0.8 in pool 2 (AS).


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)

Mucins (A) and protein groups (B) in AS and IS. Values in A are averages over 4 independent experiments; error bars represent SD. All peptides from proteins from extraneous sources, e.g., saliva, serum, and lung, were excluded from B (see supplemental Table 2 for excluded proteins).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Mucins (A) and protein groups (B) in AS and IS. Values in A are averages over 4 independent experiments; error bars represent SD. All peptides from proteins from extraneous sources, e.g., saliva, serum, and lung, were excluded from B (see supplemental Table 2 for excluded proteins).
Mentions: MS/MS analysis of tryptic peptides derived from the mucin-rich fraction identified a total of 29 proteins in AS and 28 in IS (Table 2); as expected, this fraction was dominated by mucins. The same five mucins, namely, MUC1, MUC4, MUC16, MUC5B, and MUC5AC, were identified in AS and IS (Fig. 4A), together with another glycoprotein, DMBT1/gp340, which has been shown to copurify with mucins under these conditions (40) and is also considered to be a mucin (24). Four of these mucins (MUC4, MUC5AC, MUC5B, and MUC16) were unique in this pool; MUC1 and DMBT1 were also present in the protein-rich pool. The nonmucin proteins identified in this fraction were also present in the lower-density, protein-rich pool. To compare the mucin peptide coverage, we have only considered the nonglycosylated regions of the protein. In the AS sample, the gel-forming mucin MUC5B was, from this perspective, the dominant mucin (61 peptides, 45% coverage); lesser amounts of MUC5AC (32 peptides, 21% coverage) and the epithelial mucins MUC1 (5 peptides, 19% coverage), MUC4 (8 peptides, 10% coverage), and MUC16 (21 peptides 11% coverage) were found. In the IS sample, MUC5B was again the major mucin detected (51 peptides, 38% coverage), but MUC5AC appeared to be present at a comparable level (47 peptides, 30% coverage). The epithelial mucins MUC1 (2 peptides, 7% coverage), MUC4 (1 peptide, 1% coverage), and MUC16 (6 peptides, 4% coverage), although identified in IS, contributed fewer peptides and less coverage (of the nonglycosylated regions) than in the AS sample, suggesting that they were present at a lower level in IS. A measure of the enrichment of the mucins against all other proteins may be taken as the ratio of total protein peptide to mucin peptide in the two pools: 72 in pool 1 and 0.8 in pool 2 (AS).

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