SYNTHESIS AND SECRETION OF MUCIN BY THE HUMAN COLONIC TUMOR-CELL LINE LS180

Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [H-3]threonine or [H-3]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 labeling times with [H-3]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 [H-3]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 [H-3]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [H-3]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [H-3]glucosamine incorporation,in secretion without affecting initial [H-3]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.