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 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 24 h, which was consistent with an initial release of stored PAS-reactive molecules and synthesis of the sialic-acid-rich mucins de novo. Both [(3)H]proline and [(35)S]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 [(35)S]sulphate. The [(35)S]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. [(35)S]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.
Svitacheva
N
N
Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P.O. Box 94, S-22100 Lund, Sweden.
Davies
J R
JR
D000478:Alkylation; D000818:Animals; D002417:Cattle; D002457:Cell Extracts; D002478:Cells, Cultured; D002500:Centrifugation, Isopycnic; D002501:Centrifugation, Zonal; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009077:Mucins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D002457:Cell Extracts; D009077:Mucins; D019163:Reducing Agents; D014357:Trypsin; D019158:N-Acetylneuraminic Acid