Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus.
Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum 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 from the fractions that comigrated with the galactosyltransferase activity 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 treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
Asker
N
N
Department of Medical Biochemistry, University of Göteborg, Medicinaregatan 9A, S-413 90 Gothenburg, Sweden.
Axelsson
M A
MA
Olofsson
S O
SO
Hansson
G C
GC
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001693:Biological Transport, Active; D014408:Biomarkers, Tumor; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005752:Gastric Mucins / Q000096:biosynthesis; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D055262:Mucin-2; D009077:Mucins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D014461:Ultracentrifugation
D000900:Anti-Bacterial Agents; D014408:Biomarkers, Tumor; D005752:Gastric Mucins; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D009363:Neoplasm Proteins; C053366:apomucin; D014415:Tunicamycin