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.
Highlights
In vertebrates, epithelia are protected on their luminal side by mucus gels synthesized and secreted by specialized cells in the surface and/or underlying submucosa
We have previously demonstrated that oligomeric MUC5AC mucins are secreted from an HT-29 cell line in the form of long linear threads containing in excess of 20 subunits, which can be depolymerized by reduction to yield-reduced subunits of molecular mass 2.2 MDa and a length of 570 nm [18]
Most of the evidence in the literature supports the view that oligomeric mucins are linear polymers assembled in a similar fashion to the von Willebrand factor (VWF) glycoprotein
Summary
Epithelia are protected on their luminal side by mucus gels synthesized and secreted by specialized cells in the surface and/or underlying submucosa. In some experiments the various forms of the MUC5AC mucin were immunoprecipitated with MAN-5ACI, and the precipitate was fractionated by CsCl density gradient centrifugation prior to agarose gel electrophoresis.
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