| 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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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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| 6. |
- Baeckström, Dan, 1956
(författare)
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Post-translational fate of a mucin-like leukocyte sialoglycoprotein (CD43) aberrantly expressed in a colon carcinoma cell line.
- 1997
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 272:17, s. 11503-9
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes the biosynthesis of L-CanAg, a mucin-like glycoprotein which carries the carcinoma-associated carbohydrate epitope sialyl-Lewis a and is secreted by the colon adenocarcinoma cell line COLO 205. Recently, it has been shown that L-CanAg is a novel glycoform of CD43, a surface sialoglycoprotein normally found only on hematopoietic cells. Immunoprecipitation with alpha-GPEP18, a novel antiserum against the cytoplasmic domain of CD43, detected a transmembrane form of L-CanAg carrying sialyl-Lewis a. Cell surface biotinylation experiments demonstrated the presence of transmembrane L-CanAg at the plasma membrane and that COLO 205, unlike the leukocyte cell line HL-60, contained significant amounts of glycosylated intracellular CD43. Immunoprecipitation of phosphate-labeled COLO 205 cells revealed that membrane-bound L-CanAg, like leukocyte CD43, is a phosphoprotein. Interestingly, both surface- and phosphate-labeled L-CanAg were eluted earlier from a gel filtration column than their unlabeled counterparts, indicating that this method could separate membrane-bound L-CanAg from its soluble form. Immunoprecipitations of pulse-chase-labeled COLO 205 lysates fractionated by gel filtration showed that decrease in membrane-bound L-CanAg was concomitant with an increase in the intracellular soluble form. Together, these data indicate that transmembrane L-CanAg is fully glycosylated and phosphorylated before the extracellular domain is cleaved off and stored inside the cell before exocytosis.
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| 7. |
- 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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| 8. |
- Balciunas, Darius
(författare)
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Functional studies in yeast of cyclin C and the RNA polymerase II Mediator complex
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cyclin C belongs to a group of cyclins that are not cell cycle-regulated. It was first cloned from Drosophila and rat, but its role was not understood until the yeast cyclin C homologue Srb 11 was identified in several genetic screens for transcriptional repressors and subsequently was shown to be associated with the RNA polymerase II Mediator complex. The Mediator is a multisubunit complex that enables RNA polymerase II to respond to activators in vitro.In the work presented here, the yeast genes encoding cyclin C (Srb11/Gig3), its cyclin-dependent kinase (Srb10/Gig2), and a third associated protein (Srb8/Gig1) were identified in a genetic screen for negative regulators of the gluconeogenic genes. A further analysis of the cloned genes suggested that the encoded proteins function closely together.The Med1 subunit of the yeast Mediator complex was characterized. Evidence was found of a functional connection between Med1 and the cyclin C-dependent kinase. The expression of the GAL1 promoter is partly deregulated in cells lacking cyclin C, Med1, or another mediator subunit, Med2. This deregulated expression is seen also under derepressed non-inducing conditions, and is therefore not due to a failure of glucose repression.An analysis of the ability of different Mediator subunits to activate transcription when fused to a DNA binding domain indicated that Med1 and Srb7 are negatively regulated both by cyclin C and by the Sin4 subunit of the Mediator, but not by the Med2 or Gal11 subunits, even though Sin4, Med2 and Gal11 are a part of the same module within the Mediator.A screen was made for multicopy suppressors of disruptions in the SRB8, SRB10 and SRB11 genes. Since these disruptions lack selectable phenotypes in a wild type background, the failure of snf1 mig1 srb8/10/11 cells to grow on galactose was used to select suppressors. Four new genes were identified and named GISI-4. Evidence was obtained of a functional interaction between these genes and the RAS/cAMP pathway.
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| 9. |
- Björkqvist, Susan, 1967, et al.
(författare)
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Isoprene from expired air inside a private car
- 1997
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Ingår i: The science of the total environment. ; 207, s. 63-67
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Tidskriftsartikel (refereegranskat)abstract
- The concentration of isoprene inside a small-sized parked private car with one person was found to be of the order of 20 g/m3. Isoprene was then the major non-methane volatile hydrocarbon except in strongly traffic-polluted parking places. On driving, with intermediate fan ventilation, the isoprene levels were one order of magnitude lower. In the empty car, the concentrations were still much lower, proving that isoprene originates predominantly from expired air. Air samples were taken on triple-layer adsorbent cartridges and were analysed for volatile hydrocarbons by gas chromatography after thermal desorption. The analytical aluminium oxide column permitted simultaneous determination of a range of reported traffic-emitted hydrocarbons including the carcinogenic 1,3-butadiene and benzene.
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| 10. |
- Blomquist, H K, et al.
(författare)
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Glycerol kinase deficiency in two brothers with and without clinical manifestations.
- 1996
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Ingår i: Clinical genetics. - 0009-9163 .- 1399-0004. ; 50:5, s. 375-9
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Tidskriftsartikel (refereegranskat)abstract
- We report two brothers with glycerol kinase deficiency (GKD). The older brother had serious clinical symptoms, mental and growth retardation, abnormal skeleton, spontaneous fractures and premature loss of abnormal teeth. He and his mother had low serum phosphate levels. He had elevated serum and urine glycerol levels and GKD was found in cultured fibroblasts. Prenatal diagnosis was performed in the second pregnancy. Glycerol kinase activity was considered normal in a chorionic villus sample of the foetus. After birth, it was found that the boy had elevated serum and urine glycerol levels. Enzymatic analysis in cultured fibroblasts revealed that this boy also had GKD, in spite of having no expression of the disease. Chromosomal analyses in the parents and both boys were normal. Major rearrangements or deletions were not detected in molecular studies of DNA from the two brothers. The hybridisation pattern was normal and no allelic loss was observed.
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