EVIDENCE THAT A PEPTIDE CORRESPONDING TO THE RAT MUC2 C-TERMINUS UNDERGOES DISULFIDE-MEDIATED DIMERIZATION

We have investigated the possibility that the intestinal mucin rat Muc 2 forms dimers during biosynthesis via intermolecular disulphide bridg ing 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 w ith three antibodies to different regions of the C-terminal domain. In cells, the expressed domain was glycosylated and formed disulphide-de pendent 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. ha d a slower mobility) than in cell lysates. Initial N-glycosylation, di merization 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 a pparent size of secreted dimers, such that they now had the same mobil ity on gels as dimers normally seen in cell lysates (i.e. 150 kDa). A similar change in dimer size was observed after incubating untreated m edia samples with N-acetylneuraminidase. This suggests that benzyl-alp ha-GalNAc caused inhibition of sialylation of cell dimers just before they were secreted. In summary, the C-terminal domain of RMuc2 can for m disulphide-dependent dimers, and N-glycosylation is required for dim erization and subsequent secretion. A late sialylation event appears t o precede the secretion of mucin domain dimers.