MOLECULAR CHARACTERIZATION OF THE LARGE HEAVILY GLYCOSYLATED DOMAIN GLYCOPEPTIDE FROM THE RAT SMALL-INTESTINAL MUC2 MUCIN

The largest high-glycosylated domain, glycopeptide A, of the 'insolubl e' mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedt ef al., 1993, J Biol Chem 268: 18771-81). A rabbit antiserum raised against deglycosylated glycopeptide A was u sed to clone part of a mucin showing homology to the human MUC2 mucin (Hansson et al., 1994, Biochem Biophys Res Commun 198: 181-90). This s erum specifically stained goblet cells (paranuclear) in the mouse smal l intestine. The size of the coding sequence of glycopeptide A was est imated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal part s of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the prime rs were subtracted. This is slightly larger than the size estimated ea rlier by biochemical studies. The mRNA encoding the rat Muc2 was sligh tly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the 'insoluble' mucin of the rat small intestine is enco ded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A w ere either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 of N-acetylglucosamine linked to C-6 of the N-acetylgalactosaminitol as revealed by tandem m ass spectrometry of the perdeuteroacetylated oligosaccharides. Eightee n oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowle dge of the core peptide of the rat intestinal goblet cell mucin and pr ovide a relatively complete picture of the glycosylation of a defined mucin domain.