Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.
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The MUC4 gene, which encodes a human epithelial mucin, is expressed in various epithelial tissues, just as well in adult as in poorly differentiated cells in the embryo and fetus. Its N-terminus and central sequences have previously been reported as comprising a 27-residue peptide signal, followed by a large domain varying in length from 3285 to 7285 amino acid residues. The present study establishes the whole coding sequence of MUC4 in which the C-terminus is 1156 amino acid residues long and shares a high degree of similarity with the rat sialomucin complex (SMC). SMC is a heterodimeric glycoprotein complex composed of mucin (ascites sialoglycoprotein 1, ASGP-1) and transmembrane (ASGP-2) subunits. The same organization is found in MUC4, where the presence of a GlyAspProHis proteolytic site may cleave the large precursor into two subunits, MUC4alpha and MUC4beta. Like ASGP-2, which binds the receptor tyrosine kinase p185(neu), MUC4beta possesses two epidermal growth factor-like domains, a transmembrane sequence and a potential phosphorylated site. MUC4, the human homologue of rat SMC, may be a heterodimeric bifunctional cell-surface glycoprotein of 2.12 micrometers. These results confer a new biological role for MUC4 as a ligand for ErbB2 in cell signalling. (+info)
Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells.
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To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression. (+info)
Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug.
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We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748-756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact 'island' structures composed of linear threads often emanating from globular 'nodes' rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3x10(6)) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr=4x10(6) to 30x10(6) that in the electron microscope appeared as polydisperse threads (500-3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3x10(6) and length 200-600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology. (+info)
Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
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The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin. (+info)
The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection.
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Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one alpha1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and alpha1,3 fucosyltransferases in the human bronchial mucosa. (+info)
Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis.
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BACKGROUND: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. AIMS: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. METHODS: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. RESULTS: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. CONCLUSIONS: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced. (+info)
Mucin expression and function in the female reproductive tract.
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Reproductive tract epithelia are characterized by the presence of a thick, apical glycocalyx. This glycoprotein coat is drastically reduced in the uterus of many species during the time of embryo implantation. Recent studies indicate that mucin glycoproteins constitute a large proportion of the apical glycocalyx. One of these mucins, Muc-1, has particularly important functions at the luminal surface of the uterus and other female reproductive tract tissues. Muc-1 appears to play a dominant role in maintaining a functionally non-receptive uterine surface with regard to blastocyst attachment. Conversion to a receptive uterine state is brought about by the concerted actions of ovarian steroid hormones that in several species also strongly modulate Muc-1 protein and mRNA expression. Muc-1 also appears to serve a general function in protecting reproductive tract mucosa since Muc-1 null mice are particularly prone to bacterial infection. Collectively, these studies indicate that mucins, including Muc-1, play important barrier roles in reproductive processes and protection from bacterial pathogenesis in the female reproductive tract. (+info)
pH-dependent conformational change of gastric mucin leads to sol-gel transition.
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We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated. (+info)