title: Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin.
abstract: MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.
authors.affiliation: School of Biological Sciences, University of Manchester, Michael Smith Building, Manchester M13 9PT, United Kingdom. dave.thornton@man.ac.uk
authors.forename: John K
authors.initials: JK
authors.lastname: Sheehan
authors.forename: Sara
authors.initials: S
authors.lastname: Kirkham
authors.forename: Marj
authors.initials: M
authors.lastname: Howard
authors.forename: Philip
authors.initials: P
authors.lastname: Woodman
authors.forename: Saduman
authors.initials: S
authors.lastname: Kutay
authors.forename: Caroline
authors.initials: C
authors.lastname: Brazeau
authors.forename: Jeanette
authors.initials: J
authors.lastname: Buckley
authors.forename: David J
authors.initials: DJ
authors.lastname: Thornton
mesh_terms: D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008854:Microscopy, Electron; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D013997:Time Factors
chemical_list: D002712:Chlorides; D004220:Disulfides; D006023:Glycoproteins; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D002586:Cesium; C028019:cesium chloride