THE HUMAN MUC2 MUCIN APOPROTEIN APPEARS TO DIMERIZE BEFORE O-GLYCOSYLATION AND SHARES EPITOPES WITH THE INSOLUBLE MUCIN OF RAT SMALL-INTESTINE

Rabbit antiserum against a synthetic peptide corresponding to a tandem ly repeated amino acid sequence in the human intestinal mucin apoprote in MUC2 was used in immunoprecipitation to study the biosynthesis of M UC2 in the colon-carcinoma cell line LS 174T. Under non-reducing condi tions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electro phoresis after reduction, the larger band migrated to the same positio n as the smaller band and was interpreted as a putative disulphide-bon d-stabilized dimer. Pulse-chase experiments showed only the monomer af ter 5 min and the appearance of the putative dimer after 30 min. The M UC2 apoprotein was also precipitated by antisera against the HF-deglyc osylated peptides of the two highly glycosylated domains of the 'insol uble' mucin complex of rat small intestine [Carlstedt, Herrmann, Karls son, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, 18771-18 781]. Endoprotease Lys-C cleavage of the immuno-purified apoprotein ga ve a large fragment of about 250 kDa that was detected by both the ant iserum against the MUC2 tandem repeat and one of the glycopeptide anti sera. This supports the view that the 'insoluble' mucin of rat small i ntestine is encoded by the Muc2 gene, as recently indicated by a parti al cDNA sequence [Hansson, Baeckstrom, Carlstedt and Klinga-Levan (199 4) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation sta rts after dimerization. The results indicate that the biosynthetic pat hway of the MUC2 mucin may be similar to that of the von Willebrand fa ctor with which MUC2 shares sequence similarities at its C- and N-term ini.