Synthesis and secretion of mucin by the human colonic tumour cell line LS180.
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.
McCool
D J
DJ
Hospital for Sick Children, Toronto, Ontario, Canada.
Forstner
J F
JF
Forstner
G G
GG
D003110:Colonic Neoplasms; D016521:Electrophoresis, Gel, Pulsed-Field; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D008985:Monensin / Q000494:pharmacology; D055262:Mucin-2; D009077:Mucins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D011233:Precipitin Tests; D011498:Protein Precursors / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D011498:Protein Precursors; D008985:Monensin; D009439:Neuraminidase