Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Abstract

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.

Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

“…3A). This is in agreement with our recent study of the MUC5AC and MUC5B mucins synthesized by normal human tracheobronchial cells in culture (13) that suggests that mucins associated with the cells (mainly dimers) were smaller than those secreted into the medium (large oligomers). From this study we initially concluded that the mucins were oligomerized during or after secretion (13); however, an alternative interpretation of the data could be that the mucins were depolymerized during extraction because of their exposure to intracellular reductants.…”

“…Whereas intracellular proteolytic cleavage has been observed for the MUC5B mucin, its structural consequence has yet to be established (13,14). It is likely that in disease alterations in the biosynthetic process may yield mucins with altered macromolecular structure.…”

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

“…NHBE cell cultures, where the predominant gel-forming mucin is MUC5B (Thornton et al, 2000), were thus used for studies of the structure and properties human airway MUC16. As expected from the predicted sequence, which lacks the D-domains required for oligomerization (O'Brien et al, 2001;Yin, Dnistrian & Lloyd, 2002), MUC16 from the cells was monomeric.…”

Regulation of Airway Mucin Gene Expression

“…Among these, MUC5AC and MUC5B are felt to be the predominant mucins in airway mucus because they are the only ones consistently found at the protein level in mucus and in sputum (10)(11)(12). Thus, most of the data on mucins in the respiratory tracts have focused on these two mucins.…”