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

Abstract

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 12Ϫ15-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-A-GalNAc did not interfere with these processes. However benzyl-A-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-A-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.Keywords : mucin ; Muc2; disulfide ; dimer; cystine knot.The internal epithelial surfaces of most organisms are cov-coprotein von Willebrand factor [6,8]. Other secretory mucins ered by a gel that is rich in secreted mucus glycoproteins (mu-show a similar cysteine distribution throughout their C-termini, cins). The gel lubricates the mucosal surface, forms a barrier to including frog integumentary mucin B.1 [9], bovine submaxilvarious chemicals, proteases, toxins and infectious agents, and is lary mucin [10], porcine submaxillary mucin [11] and human secreted in response to a wide variety of stimuli [1Ϫ4]. Several lung mucin MUC5AC [12,13]. The similarity includes a region domains of mucin molecules contribute to their function. The termed the 'cystine knot' motif, which is also present and plays protein core (or apomucin) usually consists centrally of a vari-an active role in the dimerization of transforming growth factorable number of imperfect tandem repeats which contribute to β2 [14], nerve-growth factor [15], Norrie disease protein [16, molecular rigidity because of extensive O-glycosylation of their 17], human chorionic gonadotropin [18] and glial cell line-deserine and threonine residues. The flanking end regions of most rived neurotrophic factor [19]. The involvement of the cystine secretory mucins are enriched in cystein...

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

“…Studies on the pig submaxillary mucin (PSM) and the rat Muc2 mucin suggest that the C-terminal parts of these proteins are responsible for dimer formation [10][11][12]. These findings support the hypothesis that dimerization of the human MUC2 takes place via its C-terminus.…”

“…This proposes that this cysteine might be involved also in the dimerization of mucins. Studies on rat Muc2 [11,12] point out that this cysteine residue is essential for dimer formation, whereas studies on vWF [15], PSM [16] and Norrin [17] indicate that additional cysteine residues may be of importance. The mucin genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11p15.5 [18], all of which contain the cystine-knot motif and show large similarities in the positions of other cysteine residues also outside of the cystine-knot motif, indicating that these mucin genes are part of a family having a common ancestral gene.…”

A sticky end for gastrointestinal helminths; the role of the mucus barrier

“…Both the N‐ and C‐terminal domains are enriched in cysteine residues that facilitate both inter‐ and intramolecular disulphide bond formation; the intermolecular disulphide linkages are responsible for mucin polymerization 28, 29, 30…”

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

“…In fact, we have recently found that CRT is already bound to MUC2 in LS180 cells after a 5 min pulse of [$&S]Promix and that the complex is present at 1 h and 1.5 h chase times. Also, it would be of interest to study the involvement of CRT and CLN during the disulphide-mediated dimerization of rat Muc2 C-terminus peptides in transfected cells as described in [10].…”

“…shown recently [10] that a peptide corresponding to the rat Muc2 C-terminus undergoes disulphide-mediated dimerization. Potential N-glycosylation sites are also located in the N-and Cterminal regions, and some are known to be occupied, since monomeric precursors of both MUC2 and MUC5AC contain Nglycans [1,2].…”