Mucin biosynthesis and secretion in tracheal epithelial cells in primary culture

Density-gradient centrifugation of bovine tracheal epithelial cell extracts revealed a ‘high-density’ (1.48g/ml) sialic-acid-rich population as well as a ‘low-density’ (1.42g/ml) one that reacted more strongly with a periodate–Schiff (PAS) assay. The sialic-acid-rich mucins were oligomeric molecules containing disulphide- bond-linked subunits and large glycosylated domains, whereas the PAS-reactive component seemed to be smaller and ‘monomeric’. Only the ‘high-density’ population was secreted from cells cultured for 5 days on plastic or a collagen type 1, Matrigel or Vitrogen substrate. Release was less from cells grown on plastic than from those on a substrate and the amount was unaffected by increasing the thickness of the collagen layer. For cells grown on collagen, the amount of the sialic-acid-rich mucin increased over 10 days, whereas the PAS-reactive component was largely absent after 24h, which was consistent with an initial release of stored PAS-reactive molecules and synthesis of the sialic-acid-rich mucins de novo. Both [3H]proline and [35S]sulphate were poorly incorporated into mucins detected with the chemical assays but molecules with a higher buoyant density than that of either of the previously identified species were labelled with [35S]sulphate. The [35S]sulphate-labelled material yielded large trypsin-resistant fragments and contained O-linked glycans but was not affected by digestion with chondroitin ABC lyase or heparan sulphate lyase, suggesting that it is a mucin rather than a proteoglycan. [35S]Sulphate is thus a poor marker for the major oligomeric mucins produced by bovine tracheal epithelial cells but the radiolabel is incorporated into a heavily labelled mucin-like component.