Studies on the “Insoluble” Glycoprotein Complex from Human Colon

Abstract

The "insoluble" glycoprotein complex was isolated from human colonic tissue and mucin subunits were prepared following reduction. Antibodies raised against peptide sequences within MUC2 revealed that virtually all of this mucin occurs in the insoluble glycoprotein complex. In addition, reduction released a 120-kDa Cterminal MUC2 fragment, showing that proteolytic cleavage in this domain may occur and leave the fragment attached to the complex via disulfide bonds. The variable number tandem repeat region and the irregular repeat domain were isolated after trypsin digestion and shown to have molecular weights of 930,000 and 180,000, respectively, suggesting a molecular weight for the entire MUC2 monomer of approximately 1.5 million. Gel chromatography and agarose gel electrophoresis revealed several populations of MUC2 subunits, and analytical ultracentrifugation showed that these have molecular weights on the order of 2 million, 4 million, and 5 million, corresponding to monomers, dimers, and trimers, respectively. Agarose gel electrophoresis of subunits from individuals expressing both a "long" and a "short" MUC2 allele revealed a larger number of populations, consistent with the presence of short and long monomers and oligomers arising from permutations of the two types of monomers. In addition to disulfide bonds, MUC2 monomers are apparently joined by a "novel," reduction-insensitive bond.The mucosal surface of the gastrointestinal tract is protected by a visco-elastic mucus gel formed by high molecular mass (0.5-25 ϫ 10 6 ) glycoproteins referred to as mucins. The protein backbones of mucins are heavily substituted with O-linked oligosaccharides attached to serine and/or threonine residues, and these amino acids are, together with proline, typically enriched within so-called mucin domains. Several mucin domains may occur in a single mucin subunit and these regions are flanked by less heavily glycosylated segments of the protein core. Large secreted mucins from the respiratory tract, stomach, and endocervix have been shown to be linear oligomers formed by subunits joined via disulfide bonds (1, 2). However, less is known about the macromolecular architecture of intestinal mucins.The mucin genes MUC2, MUC3, MUC4, MUC5B, and MUC6 are expressed in normal human colon (3-7). MUC2 is believed to be the dominating mucus-forming species in this tissue and is, so far, the only "large" mucin of which the cDNA has been fully sequenced (8). The apoprotein contains two mucin domains, which differ in length and are separated by a cysteinecontaining region (Fig. 1A). The longer domain, which is referred to as the variable number tandem repeat (VNTR) 1 region is composed largely of tandemly repeated 23 amino acid peptide units, which vary in number between alleles and are rich in threonine and proline (9, 10). The shorter mucin domain comprises a 347-amino acid-long irregular repeat rich in threonine, serine, and proline. The regions flanking the mucin domains each show a high degree of similarity to the N-and C-terminal cyst...

Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

“…However, data from studies utilizing N-terminal constructs of MUC2 and PSM suggest the potential for non-linear, branched mucin assemblies (8 -10). For MUC2 mucin this may not be surprising because it has been proposed to form an "insoluble" mucin network that is stabilized by disulfide bonds plus additional reduction-insensitive linkage(s) (11,12). A non-linear architecture has also been demonstrated for a population of the MUC5B mucins isolated from the sputum of an individual in status asthmaticus that is shown to possess branched network morphology (15).…”

“…However we cannot discount that another biosynthetic pathway is followed in airway cells in vivo or that the ladders resulted from a change in the reducing status of the secretory cells within the airway. It is interesting to note that proteolytic processing has been demonstrated for mucins (12)(13)(14)27), and we cannot discount that specific proteolytic cleavage is not a factor in generating the MUC5AC mucin polydispersity.…”

“…Moreover, recent data in the literature indicate that oligomeric mucins are not solely linear disulfide-linked assemblies (8 -10). For example, oligomeric MUC2 is assembled from disulfide bonds as well as another reduction-insensitive linkage and may form branched structures (11,12).The polydispersity of oligomeric mucins may be because of proteolytic degradation within the mucus, but it may also arise as a direct consequence of the biosynthetic process, thus allowing cellular control over the physical properties of the mucus. Whereas intracellular proteolytic cleavage has been observed for the MUC5B mucin, its structural consequence has yet to be established (13,14).…”

“…Moreover, recent data in the literature indicate that oligomeric mucins are not solely linear disulfide-linked assemblies (8 -10). For example, oligomeric MUC2 is assembled from disulfide bonds as well as another reduction-insensitive linkage and may form branched structures (11,12).…”

N-terminal Cleavage of the Salivary MUC5B Mucin

“…However, the assembly of the various mucins may follow dissimilar pathways. For example, MUC2 occurs as an insoluble complex that resists extraction with guanidinium chloride unless disulfide bonds are cleaved (2,14,15), and in addition, human salivary MUC5B has been shown to form such insoluble assemblies (16). The formation of insoluble MUC2 and MUC5B is likely to involve "cross-links" in addition to those that join mucin monomers end-to-end into linear structures.…”