Analysis of the proteome of human airway epithelial secretions

Abstract

Background: Airway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles and chemicals entering the lungs. Airway mucus contains various macromolecules, the most abundant being mucin glycoproteins, which contribute to its defensive function. Airway epithelial cells cultured in vitro secrete mucins and nonmucin proteins from their apical surface that mimics mucus production in vivo. The current study was undertaken to identify the polypeptide constituents of human airway epithelial cell secretions to gain a better understanding of the protein composition of respiratory mucus.

Results: Fifty-five proteins were identified in the high molecular weight fraction of apical secretions collected from in vitro cultures of well-differentiated primary human airway epithelial cells and isolated under physiological conditions. Among these were MUC1, MUC4, MUC5B, and MUC16 mucins. By proteomic analysis, the nonmucin proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial.

Conclusions: Because the majority of the nonmucin proteins possess molecular weights less than that selected for analysis, it is theoretically possible that they may associate with the high molecular weight and negatively charged mucins to form a highly ordered structural organization that is likely to be important for maintaining the proper defensive function of airway mucus.

Figure 1

Sepharose CL-4B gel filtration chromatography of hAECS. The percent optical densities at 280 nm (solid line) and 555 nm (dashed line) are indicated on the x-axis while individual column fractions are indicated on the y-axis. The void volume (V₀) and total volume (V_t) are indicated.

Figure 2

One-dimensional SDS-polyacrylamide gel electrophoresis of V₀ fraction from gel filtration chromatography. Proteins were separated on a 12% gel under reducing conditions and visualized by silver staining. Lane 1, hAECS sample. Lane 2, prestained molecular weight marker proteins. Molecular weights in kDa are indicated on the right.

Figure 3

MUC5AC Western blot analysis. Equal protein aliquots (100 μg) of lysates of NHBE cells, COS-7 cells (negative control), or hAECS from NHBE cells were resolved by agarose gel electrophoresis, transferred to PVDF membrane, and reacted with anti-MUC5AC antibody (45M1). The positions of MUC5AC and the 250 kDa marker protein are shown on the right.

Figure 4

Pie chart of molecular functions of proteins in high molecular weight hAECS identified by nano-LC-IT MS. Proteins are listed by category, number of proteins in the category and specific proteins identified.

Figure 5

Pie chart of biological functions of proteins in high molecular weight hAECS identified by nano-LC-IT MS. Proteins are listed by category, number of proteins in the category and specific proteins identified.

Figure 6

Categorization of proteins in high molecular weight hAECS identified by nano-LC-IT MS.