| 1. |
- Asker, Noomi, 1968, et al.
(författare)
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Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus.
- 1998
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 273:30, s. 18857-63
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Tidskriftsartikel (refereegranskat)abstract
- Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Axelsson, Magnus A. B., et al.
(författare)
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Deglycosylation by gaseous hydrogen fluoride of mucus glycoproteins immobilized on nylon membranes and in microtiter wells.
- 1998
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Ingår i: Glycoconjugate journal. - 0282-0080. ; 15:8, s. 749-55
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Tidskriftsartikel (refereegranskat)abstract
- Strongly reacting antibodies specific for defined mucin gene products are often directed against the mucin protein backbone of the heavily glycosylated serine/threonine rich regions. A prerequisite for the use of such antibodies is often the complete removal of the oligosaccharides from the protein. This paper describes an efficient one-step deglycosylation method using gaseous hydrogen fluoride on nylon blotting membranes and microtiter wells.
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| 5. |
- Axelsson, Magnus A. B., et al.
(författare)
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Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.
- 2001
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:8, s. 633-44
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Tidskriftsartikel (refereegranskat)abstract
- Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.
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| 6. |
- Axelsson, Magnus A. B., et al.
(författare)
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O-glycosylated MUC2 monomer and dimer from LS 174T cells are water-soluble, whereas larger MUC2 species formed early during biosynthesis are insoluble and contain nonreducible intermolecular bonds.
- 1998
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 273:30, s. 18864-70
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Tidskriftsartikel (refereegranskat)abstract
- The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.
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| 7. |
- Baeckström, Dan, 1956, et al.
(författare)
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Purification and characterization of sialyl-Le(a)-carrying mucins of human bile; evidence for the presence of MUC1 and MUC3 apoproteins.
- 1994
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 269:20, s. 14430-7
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Tidskriftsartikel (refereegranskat)abstract
- Purification of sialyl-Le(a)-carrying mucins from primary human bile by trichloroacetic acid precipitation, delipidation, and gel filtration in guanidinium chloride gave three separable fractions, one of which was further purified by affinity chromatography. These fractions, named SBG1 (for soluble bile glycoprotein), SBG2, and SBG3 had molecular masses of > 1100, 800-950, and 100-250 kDa, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their mucin characteristics were indicated by a high carbohydrate content, ranging from 74 to 95%. The carbohydrate compositions indicated the presence of very long fucosylated polylactosamine chains. Amino acid analyses showed high abundance of serine and threonine in all three fractions (19-36%), confirming their mucin-like nature. Immunochemical analyses of deglycosylated samples detected the MUC1 mucin apoprotein in SBG2 and the MUC3 protein in SBG1. To our knowledge, this is the first report of a MUC3 mucin being purified. This mucin showed no significant reduction in size upon trypsin treatment or disulfide bond reduction and alkylation. Gel filtration of three samples of secondary bile showed that the size distribution of sialyl-Le(a)-carrying glycoproteins was similar to that found in primary bile, and immunochemical analysis showed that the MUC1 protein was present in all three samples. In one sample an additional fraction was isolated, which was insoluble in 6 M guanidinium chloride, but was solubilized upon reduction and alkylation. mRNAs from gallbladder epithelia were analyzed in Northern blot hybridizations showing that the MUC1 and MUC3 but not the MUC2 mucin apoprotein genes were expressed.
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| 8. |
- Baeckström, Dan, 1956, et al.
(författare)
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The transcripts of the apomucin genes MUC2, MUC4, and MUC5AC are large and appear as distinct bands.
- 1996
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Ingår i: Glycoconjugate journal. - 0282-0080. ; 13:5, s. 833-7
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Tidskriftsartikel (refereegranskat)abstract
- RNA from four colorectal carcinoma cell lines was prepared and analysed in Northern blots using probes for the MUC2, MUC4, and MUC5AC mucin apoprotein genes. The sizes of the transcripts were very large, in the order of at least 12-16 kb. The presence of distinct bands is in contrast to earlier reports, where these transcripts showed extensive polydispersity. RNA from rat small intestine was also prepared and probed with cDNA for the rat Muc2 mucin gene. This analysis also showed a large and discrete hybridizing band, indicating that apomucin mRNA of well-defined size can be obtained also from a tissue with high endogenous RNase activity.
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| 9. |
- Bouhours, Danièle, et al.
(författare)
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Structure and genetic polymorphism of blood group A-active glycosphingolipids of the rat large intestine.
- 1995
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Ingår i: Biochimica et biophysica acta. - 0006-3002. ; 1255:2, s. 131-40
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Tidskriftsartikel (refereegranskat)abstract
- Study of blood group A- and B-active glycosphingolipid content of the epithelium of the large intestine of 16 strains of inbred rats led to the discovery of two related strains, SHR and WKY, devoid of A-active glycolipids, whereas all strains expressed B-active glycolipids. This finding evidenced a new A/non-A genetic polymorphism in the rat. Blood group A-active glycolipids were isolated from the large intestine of F344 rats and purified by affinity chromatography on immobilized Helix pomatia lectin. Three glycolipid fractions were separated by preparative thin-layer chromatography and characterized by electron-impact mass spectrometry of their permethylated and permethylated-LiAlH4-reduced derivatives. They were identified as a tetraglycosylceramide (A-4), a hexaglycosylceramide (A-6), and a difucosylated heptaglycosylceramide (A-7) with small amounts of monofucosylated octaglycosylceramide (A-8). Methylation analysis and fragmentation indicated that A6 and A-8 had a lacto- and A-7 a neolactotetraosylceramide core, respectively, identical to the core structures of B-6 and B-7 previously characterized in the large intestine of WF rats (Angström et al. (1987) Biochim. Biophys. Acta 926, 79-86). Upon methylation analysis, B-6 and B-7 purified from SHR (A-deficient) and F344 (A-expressing) were found identical to those of WF rats. This result indicated that precursor substrates for the synthesis of A-active glycolipids were available in SHR rats and thus the genetic deficiency of A-active glycolipid expression probably originated in a defect of the termination of the blood group A determinant by the alpha-3-N-acetylgalactosaminyltransferase.
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| 10. |
- Bäckström, Malin, 1967, et al.
(författare)
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Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.
- 2003
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Ingår i: The Biochemical journal. - 1470-8728. ; 376:Pt 3, s. 677-86
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Tidskriftsartikel (refereegranskat)abstract
- We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.
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