Evidence that a peptide corresponding to the rat Muc2 C-terminus undergoes disulphide-mediated dimerization.

We have investigated the possibility that the intestinal mucin rat Muc2 forms dimers during biosynthesis via intermolecular disulphide bridging of its C-terminal domains. Since the cysteine alignment of RMuc2 (and other secretory mucins) is similar to that of human von Willebrand factor, a similar C-tail to C-tail dimerization may occur in mucins. The C-terminal domain of RMuc2 (534 amino acids) was expressed in COS-1 cells, and the products monitored by SDS/PAGE and western blotting with three antibodies to different regions of the C-terminal domain. In cells, the expressed domain was glycosylated and formed disulphide-dependent dimers centred at approximately 150 kDa. The domain dimer, but not its precursor monomer, was secreted into the culture medium. The dimers in the media however, appeared to be 12-15-kDa heavier (i.e. had a slower mobility) than in cell lysates. Initial N-glycosylation, dimerization and secretion were inhibited by addition of tunicamycin to incubations, whereas benzyl-alpha-GalNAc did not interfere with these processes. However benzyl-alpha-GalNAc resulted in a decrease in the apparent size of secreted dimers, such that they now had the same mobility on gels as dimers normally seen in cell lysates (i.e. 150 kDa). A similar change in dimer size was observed after incubating untreated media samples with N-acetylneuraminidase. This suggests that benzyl-alpha-GalNAc caused inhibition of sialylation of cell dimers just before they were secreted. In summary, the C-terminal domain of RMuc2 can form disulphide-dependent dimers, and N-glycosylation is required for dimerization and subsequent secretion. A late sialylation event appears to precede the secretion of mucin domain dimers.