O-GLYCOSYLATED MUC2 MONOMER AND DIMER FROM LS 174T CELLS ARE WATER-SOLUBLE, WHEREAS LARGER MUC2 SPECIES FORMED EARLY DURING BIOSYNTHESIS ARE INSOLUBLE AND CONTAIN NONREDUCIBLE INTERMOLECULAR BONDS

The MUC2 mucin is the major gel-forming mucin in the small and large i ntestine. Due to its sequence similarities with the von Willebrand fac tor, it has been suggested to dimerize in the endoplasmic reticulum an d polymerize in the trans-Golgi network. Using an O-glycosylation-sens itive MUGS antiserum, a dimerization has been shown to occur in the en doplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., O lofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 278, 18857-18 863). Using an antiserum immunoprecipitating O-glycosylated MUGS mucin , monomers and dimers mere shown to occur in soluble form in the lysat e of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, m ost of the labeled MUGS mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitatio n after reduction of disulfide bonds. Analysis by agarose gel electrop horesis revealed two bands, of which the smaller migrated as the O-gly cosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chl oride could also be obtained from LS 174T cells. Such mucins have earl ier been found in the small intestine (Carlstedt, I., Herrmann, A., Ka rlsson, H., Sheehan, J., Fransson, L.-A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by pu rification by isopycnic density gradient ultracentrifugation and analy sis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands m igrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These resu lts suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.