Mucin biosynthesis and secretion in tracheal epithelial cells in primary culture

Density-gradient centrifugation of bovine tracheal epithelial cell extracts revealed a 'high-density' (1.48 g/ml) sialic-acid-rich population as well as a 'low-density' (1.42 g/ml) one that reacted more strongly with a period ate-Schiff (PAS) assay. The sialic-acid-rich mucins were oligomeric molecul es containing disulphide-bond-linked subunits and large glycosylated domain s, whereas the PAS-reactive component seemed to be smaller and 'monomrric'. Only the 'high-density' population was secreted from cells cultured for 5 days on plastic or a collagen type 1, Matrigel or Vitrogen substrate. Relea se 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 inc reased over 10 days, whereas the PAS-reactive component was largely absent after 24 h, which was consistent with an initial release of stored PAS-reac tive molecules and synthesis of the sialic-acid-rich mucins de novo. Both [ H-3]proline and [S-35]sulphate were poorly incorporated into mucins detecte d with the chemical assays but molecules with a higher buoyant density than that of either of the previously identified species were labelled with [S- 35]sulphate. The [S-35]sulphate-labelled material yielded large trypsin-res istant fragments and contained O-linked glycans but was not affected by dig estion with chondroitin ABC lyase or heparan sulphate lyase,suggesting that it is a mucin rather than a proteoglycan. [S-35]Sulphate is thus a poor ma rker for the major oligomeric mucins produced by bovine tracheal epithelial cells but the radiolabel is incorporated into a heavily labelled mucin-lik e component.