Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [3H]threonine or [3H]glucosamine and chased in complete media without radiolabel for various lengths of time. From cell and media extracts obtained at each time point, mucin proteins were immunoprecipitated with specific anti-mucin antibodies and analysed by SDS/PAGE and fluorography. At short labelling times with [3H]threonine, without chase, a monomeric thiol-reduction-resistant mucin precursor of apparent molecular mass > 670 kDa was identified. The precursor, in contrast to oligomeric species, was not labelled by [3H]glucosamine but exhibited binding to Vicia villosa isolectin B4, suggesting the presence of some core GalNAc residues. Treatment with tunicamycin to inhibit N-glycosylation had no effect on the apparent mass of the precursor. Identity of the mucin antigen with MUC2 mucin was established by immunoprecipitation with antibodies specific for a MUC2 tandem repeat and C-terminal regions. With increasing chase time the precursor was replaced by thiol-reduction-sensitive mucin oligomers that reached peak intracellular radiolabelling with [3H]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [3H]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [3H]glucosamine incorporation, sialylation and baseline (non-regulated) mucin secretion without affecting initial [3H]threonine incorporation or oligomerization. Oligomerization and Golgi transport are therefore essential early steps in MUC2 mucin secretion. Oligomerization may follow some core O-glycosylation with GalNAc, but precedes elongation of oligosaccharide chains.
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Research Article| August 15 1994
Synthesis and secretion of mucin by the human colonic tumour cell line LS180
D J McCool;
J F Forstner;
Biochem J (1994) 302 (1): 111–118.
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D J McCool, J F Forstner, G G Forstner; Synthesis and secretion of mucin by the human colonic tumour cell line LS180. Biochem J 15 August 1994; 302 (1): 111–118. doi: https://doi.org/10.1042/bj3020111
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