| 1. |
- Asker, Noomi, 1968, et al.
(författare)
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Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin.
- 1998
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Ingår i: The Biochemical journal. - 0264-6021. ; 335:2, s. 381-7
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse-chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.
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| 2. |
- Asker, Noomi, 1968, et al.
(författare)
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The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the 'insoluble' mucin of rat small intestine.
- 1995
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Ingår i: The Biochemical journal. - 0264-6021. ; 308:3, s. 873-80
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Tidskriftsartikel (refereegranskat)abstract
- Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the 'insoluble' mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the 'insoluble' mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.
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| 3. |
- Karlsson, Niclas G., 1966, et al.
(författare)
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Glycosylation differences between pig gastric mucin populations: a comparative study of the neutral oligosaccharides using mass spectrometry.
- 1997
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Ingår i: The Biochemical journal. - 0264-6021. ; 326 ( Pt 3), s. 911-7
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Tidskriftsartikel (refereegranskat)abstract
- Five mucin populations were isolated from the cardiac region,corpus and antrum of pig gastric mucosa. The released neutral oligosaccharides were permethylated and analysed using high-temperature gas chromatography-mass spectrometry (GC-MS) as well as matrix-assisted laser-desorption mass spectrometry (MALDI-MS). Thirty different oligosaccharides with up to six monosaccharide residues were characterized using both techniques, but the presence of an additional 49 structures was suggested on the basis of their molecular mass by MALDI-MS. Oligosaccharides based on core-1 (Galbeta1-3GalNAcalpha1-) and core-2 [Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] structures were widely distributed, whereas core-3 structures (GlcNAcbeta1-3GalNAcalpha1-) were present only in mucins from the cardiac region and corpus, and core-4 structures [GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] were present exclusively in mucins from the cardiac region. Furthermore the oligosaccharides from one of the mucins from the corpus were significantly longer than those from the other populations. The results illustrate vast structural diversity, but the relative abundances show only a few dominating structures, suggesting that many oligosaccharides may be quite rare in pig gastric mucins. Well-defined mucin populations with distinctly different glycosylation can thus be identified in pig stomach, suggesting that glycosylation of the large secreted mucins from this tissue is not a random event.
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| 4. |
- Karlsson, Niclas G., 1966, et al.
(författare)
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Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis.
- 2000
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Ingår i: The Biochemical journal. - 0264-6021. ; 350 Pt 3, s. 805-14
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Tidskriftsartikel (refereegranskat)abstract
- The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague-Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the 'insoluble' mucin complex revealed a relative decrease (4-7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague-Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcalpha2-3(GalNAcbeta1-4)Galbeta1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sd(a)- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcbeta1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.
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| 5. |
- Lidell, Martin, 1970, et al.
(författare)
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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.
- 2003
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Ingår i: The Biochemical journal. - 0264-6021. ; 372:Pt 2, s. 335-45
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Tidskriftsartikel (refereegranskat)abstract
- The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.
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| 6. |
- Nordman, H, et al.
(författare)
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Mucus glycoproteins from pig gastric mucosa: identification ofdifferent mucin populations from the surface epithelium.
- 1997
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Ingår i: The Biochemical journal. - 0264-6021. ; 326 ( Pt 3), s. 903-10
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Tidskriftsartikel (refereegranskat)abstract
- Pig gastric mucins were isolated from the surface epithelium of the cardiac region, corpus and antrum using density-gradient centrifugation after extraction in 6 M guanidinium chloride. In CsCl/0.5 M guanidinium chloride, mucins solubilized from the cardiac region appeared as a broad unimodal band at 1.52 g/ml whereas those from the corpus and antrum occurred as high- and low-density populations at 1.50 and 1.45 g/ml respectively. High-iron diamine reacted more strongly with the cardiac mucins and the high-density populations from corpus and antrum than with the two low-density ones. In keeping with this, approx. 60% of the oligosaccharides from the former mucins and 20% from the latter contained sulphate. All surface epithelial cells of the cardiac region stained with high-iron diamine, whereas in the corpus only the epithelium in the bottom of the pits reacted, suggesting that the high-density population from this region originates from these cells. Mucins from all regions were composed of subunits, each containing highly glycosylated domains. The mucins from the cardiac region were larger than those from the corpus and antrum, and reduced subunits as well as high-molecular-mass glycopeptides from the cardiac mucins were larger than the corresponding fragments from the other regions. Ion-exchange HPLC showed that reduced subunits from the cardiac mucins and the high-density populations from the corpus and antrum were more 'acidic' than reduced subunits from the two low-density ones. All mucins contained a 'neutral'fraction, in particular those from the antrum. Pig gastric mucus thus contains a number of distinctly different mucin populations varying in buoyant density, size, 'acidity', glycosylation, sulphation and tissue origin.
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| 7. |
- Schneider, Hannah, et al.
(författare)
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The human transmembrane mucin MUC17 responds to TNF alpha by increased presentation at the plasma membrane
- 2019
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 476, s. 2281-2295
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Tidskriftsartikel (refereegranskat)abstract
- Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNF alpha resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.
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| 8. |
- Thomsson, Kristina A, 1969, et al.
(författare)
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Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced Fucalpha1-2 glycosyltransferase.
- 2002
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Ingår i: The Biochemical journal. - 0264-6021. ; 367:Pt 3, s. 609-16
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Tidskriftsartikel (refereegranskat)abstract
- In gene-targeted mouse models for cystic fibrosis (CF), the disease is mainly manifested by mucus obstruction in the intestine. To explore the mucus composition, mucins insoluble and soluble in 6 M guanidinium chloride were purified by three rounds of isopycnic ultracentrifugation from the small and large intestines of CF mice (Cftr(m1UNC)/Cftr(m1UNC)) and compared with wild-type mice. The amino acid composition was typical of that for mucins and showed increased amounts of the insoluble (2.5-fold increase) and soluble (7-fold increase) mucins in the small intestine of the CF mice compared with wild-type mice. Mucins from the large intestine of both wild-type and CF mice showed a high but constant level of fucosylation. In contrast, the insoluble and soluble mucins of the small intestine in CF mice revealed a large increase in fucose, whereas those of wild-type mice contained only small amounts of fucose. This increased fucosylation was analysed by releasing the O-linked oligosaccharides followed by GC-MS. NMR spectroscopy revealed that the increased fucosylation was due to an increased expression of blood group H epitopes (Fucalpha1-2Gal-). Northern-blot analysis, using a probe for the murine Fucalpha1-2 fucosyltransferase (Fut2), showed an up-regulation of this mRNA in the small intestine of the CF mice, suggesting that this enzyme is responsible for the observed increase in blood group H-type glycosylation. The reason for this up-regulation could be a direct or indirect effect of a non-functional CF transmembrane conductance regulator (CFTR) caused by the absence of CFTR channel.
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