DIMERIZATION OF THE HUMAN MUC2 MUCIN IN THE ENDOPLASMIC-RETICULUM IS FOLLOWED BY A N-GLYCOSYLATION-DEPENDENT TRANSFER OF THE MONOMERS AND DIMERS TO THE GOLGI-APPARATUS

Pulse-chase experiments in the colon cell line LS 174T combined with s ubcellular fractionation by sucrose density gradient centrifugation sh owed that the initial dimerization of the MUC2 apomucin started direct ly after translocation of the apomucin into the rough endoplasmic reti culum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated fro m the fractions that comigrated with the galactosyltransferase activit y during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin tr eatment slowed down the rate of dimerization and introduced further ol igomerization of the MUC2 apomucin in the endoplasmic reticulum, Resul ts of two-dimensional gel electrophoresis demonstrated that these olig omers (putative tri- and tetramers) were stabilized by disulfide bonds . The non-N-glycosylated species of the MUC2 mucin were retained in th e endoplasmic reticulum because no O-glycosylated species were precipi tated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MU C2 mucin.