The N Terminus of the MUC2 Mucin Forms Trimers That Are Held Together within a Trypsin-resistant Core Fragment

Abstract

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of ϳ240 kDa. Upon reduction, peptides of ϳ85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.Mucins are highly glycosylated proteins protecting the mucosal surfaces of the body. All mucins are characterized by their heavy O-glycosylation, which is clustered in mucin domains rich in the amino acids Ser, Thr, and Pro. Mucins can be structured in different ways, either as membrane-bound with a transmembrane domain or secreted as monomers or as polymers (1, 2). The latter mucins are also described as gel-forming, as these give the mucus its viscoelastic properties. Four human gel-forming mucins have been found, MUC2, MUC5AC, MUC5B, and MUC6, which are clustered on chromosome 11p15 (3). However, the best studied mucin is the porcine submaxillary mucin (PSM), 1 which has become a model also for the human mucins of this type (3-6).The MUC2 mucin is the main gel-forming mucin of the small and large intestines and is produced by the intestinal goblet cells (2, 7-10). It is a major structural component of the mucous barrier covering the epithelium, protecting the epithelial cells from microorganisms as well as digestive enzymes. In fact, one of the remarkable features of this mucin is that it can withstand the pancreatic digestive enzymes. That the highly glycosylated mucin domains are totally resistant to these enzymes is easy to understand. However, the globular, less glycosylated ends must also be relatively proteolytically resistant, as the polymers must remain intact to maintain the mucous gel. The MUC2 mucin is difficult to solubilize, and it is insoluble in 4 M guanidinium chloride (8, 10), a property that might be related to the appearance of nonreducible ...

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

“…The observations made here suggest that this could also be the case for the MUC2 mucin. This interpretation is supported by the observation that no secretion of the recombinant MUC2 N-terminus [30] was observed when expressed in the LS 174T cells (M.E.V. Johansson and G.C.…”

“…This appears white on the micrographs, suggesting a denser and more condensed protein. A similar phenomenon was observed recently for the oligomerization domains of the MUC2 N-terminus [30]. The central part is attached to the gold-labelled ends via a single flexible thread, which may be a single peptide chain.…”

“…The pictures also reveal that the protein is dense and compact in the domains holding the dimer together. In a recent study [30], we described also that the MUC2 N-terminal trimer is also held together within a compact and dense domain. This interpretation was supported further by the observation that this part of the N-terminus was resistant to trypsin digestion.…”

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

“…These covalent linkages give rise to very large and highly glycosylated polymers,37, 38 which are packaged in dehydrated form inside secretory granules (Figure 3, step 6). This storage mechanism allows for the release of fully synthesized MUC2 polymers which undergo rapid hydration and expansion on the intestinal epithelial surface to maintain mucus barrier integrity during homeostasis or barrier breach 39.…”

The protein disulfide isomerase AGR2 is essential for production of intestinal mucus

“…The major intestinal mucin is MUC2, which contains Ͼ5000 aa and is processed in the ER and the Golgi apparatus. Processing leads to extensive O-glycosylation of the central mucin repeats and formation of intrachain and interchain disulfide bonds involving Ͼ200 cysteine residues concentrated in the cysteine-rich amino-terminal and carboxylterminal domains (9)(10)(11). After mucins are glycosylated, folded, and multimerized, they enter secretory granules where they can be stored before release into the lumen.…”