| 1. |
- Davies, Julia, et al.
(författare)
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Respiratory tract mucins : structure and expression patterns
- 2002
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Ingår i: Novartis Foundation symposium. - Chichester, UK : John Wiley & Sons, Ltd. - 1528-2511 .- 1935-4657. ; 248, s. 76-93
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Goblet cells produce mainly MUC5AC, but also MUC5B and some MUC2 in apparently ‘irritated’ airways. MUC5B dominates in the submucosal glands although a little MUC5AC and MUC7 are usually present. MUC4 originates from the ciliated cells. After separation into a gel and a sol phase, lysozyme and lactoferrin are enriched in the salivary gel phase suggesting that mucus may act as a matrix for ‘protective’ proteins on the mucosal surface. A salivary MUC5B N-terminal fragment consistent with a cleavage event in the D’ domain was de-tected with antibodies against various N-terminal peptide sequences suggesting that assembly of MUC5B occurs through a mechanism similar to that of the von Willebrand factor. Identification of additional cleavage sites C-terminal to the D’ domain suggests that most of the N-terminal low-glycosylated part of MUC5B may be removed without affecting the oligomeric nature of the mucin. Possibly, the generation of mucins with different macromolecular properties through proteolytic ‘processing’ is one way of adapting the mucus polymer matrix to meet local physiological demands. Monomeric mucins that appear to turn over rapidly in the airway epithelium have been identified using radiolabelled mucin precursors. ‘Shedding’ of such mucins after microbe attachment may prevent colonization of epithelial surfaces.
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| 2. |
- Herrmann, Annkatrin, et al.
(författare)
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A high-density putative monomeric mucin is the major [(35)S]labelled macromolecular product of human colorectal mucins in organ culture.
- 2003
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Ingår i: Biochimie. - 1638-6183. ; 85:3-4, s. 381-390
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the biosynthesis of mucins in organ cultures of human colon using isopycnic density-gradient centrifugation following pulse labelling with [35S]sulphate and [3H]-D-glucosamine. A high-density [35S]sulphate labelled component, of larger size than MUC2 monomers, appeared in the tissue and also in the medium. It was not degraded by reduction, trypsin digestion, digestion with chondroitin ABC lyase or heparan sulphate III lyase, but was cleaved into smaller fragments following alkaline borohydride treatment and appears to be a monomeric, mucin-like molecule containing a protease-resistant domain with a larger hydrodynamic volume than MUC2 monomers. Although this macromolecule incorporated much more radiolabel than MUC2, it was not detected using chemical analysis and thus appears to be a component with a high metabolic turnover present in a very small amount. Most of the [3H]-D-glucosamine label was associated with low-density material that was well separated from MUC2, which was poorly labelled. Most of MUC2 was associated with the tissue as an ‘insoluble’ complex. The amount of MUC2 remained constant and its associated radiolabel increased only slightly with time. Analysis of the MUC2 subunits from the reduced ‘insoluble’ complex showed the typical reduction-insensitive oligomers and confirmed that the radiolabel was associated with this mucin. The large size of the [35S]-labelled putative monomeric mucin makes it difficult to separate it from reduced insoluble complex MUC2. As a result, many studies of intestinal mucin synthesis and secretion in the past have most likely been performed on ‘mixtures’ of this mucin and MUC2 and are thus not possible to interpret as the metabolic behaviour of oligomeric mucins.
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| 3. |
- Herrmann, Annkatrin
(författare)
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Intestinal mucins 'soluble and insoluble problems'
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Highly glycosylated glycoproteins – mucins – are the major constituents of the mucus layer and glycocalyx that cover mucosal surfaces. Twelve human mucin genes have been identified, and the mucins may be divided into those that are membraneassociated and those that are secreted. Among the latter, the oligomeric mucins are responsible for mucus formation. The various tissues have characteristic mucin ‘expression profiles’. Mucins from rat small intestine occurred as an ‘insoluble glycoprotein complex’. The complex was composed of disulphide-bonded subunits containing two highly glycosylated mucin domains. The latter were of different length but substituted with similar oligosaccharides. Most of the neutral and sialic acid-containing glycans were relatively short and ‘simple’ structures. The human colonic ‘insoluble glycoprotein complex’ was shown the be mainly composed of MUC2 and virtually all MUC2 was present in this form. In addition to disulphide bonds, a ‘novel’ reduction-insensitive bond that joined subunits was iden-tified. Proteolytic cleavage in the C-terminal end of MUC2 occurred, leaving a 120 kDa fragment attached to the complex through disulphide bonds. A high-molecular-mass, monomeric, mucin with a high turnover-rate and high density was identified in colonic tissue incubated with radiolabelled mucin precursors. Most of the radiolabel was incorporated into this mucin while MUC2 was only poorly labelled. The presence of the monomeric mucin may complicate interpretations of metabolic studies designed to examine MUC2. Changes in the glycosylation of MUC2 was identified in colorectal carcinoma, including variations in blood group A, Lea, Leb, as well as Tn, sialyl-Tn, sialyl-Lea, sialyl-Lex antigen expression. However, an increased expression of the sialyl-Tn was the only finding consistently observed in tumour tissue. The various glycoproteins that form the basis for mucosal protection are very large and complex structures that so far have been difficult to isolate and study. Consequently, the progress of this particular niche of ‘matrix biology’ – focused on the interface between the outside and the inside of the body – has been hampered by a ‘methodological burden’. The work described here adds significantly to the information on the structure and properties of some of the major players in this field and also provides novel methodological approaches. Such work is a prerequisite for understanding pathological conditions characterised by a deranged mucosal protection and thus for the development of future approaches for handling such conditions.
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| 4. |
- Pons, A, et al.
(författare)
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Sequential GC/MS analysis of sialic acids, monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives
- 2003
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42:27, s. 8342-8353
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Tidskriftsartikel (refereegranskat)abstract
- A GUMS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GUMS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GUMS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GUMS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.
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| 5. |
- Robbe-Masselot, Catherine, et al.
(författare)
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Expression of a Core 3 Disialyl-Le(x) Hexasaccharide in Human Colorectal Cancers: A Potential Marker of Malignant Transformation in Colon.
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907.
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Tidskriftsartikel (refereegranskat)abstract
- Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for many years but many of the studies of alterations in mucin carbohydrate have relied on histochemical or immunohistochemical methods, with little direct chemical analysis. In this study, we analyzed the O-glycosylation pattern of MUC2 glycoprotein isolated from colorectal carcinomas, transitional mucosa and resection margins from three patients with blood group A, B and O, respectively. After alkaline borohydride treatment, the released oligosaccharides were structurally characterized by nanoESI Q-TOF tandem mass spectrometry without prior fractionation or derivatization. As expected, we found an increased expression of sialyl-Tn antigen in the colonic cancer mucins. A more interesting feature was the increased expression of a core 3 sialyl-Le(x) hexasaccharide, NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc in tumor, which appeared to compete with its sulfo-Le(x) counterpart in normal tissue, SO3-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc. This antigen, whose structure was confirmed by NMR experiments, is based on a core 3 glycan and may be a potential marker for the malignant transformation of colonic cells. Unexpectedly, most of the glycans recovered in normal and carcinomas extracts were based on a sialylated core 3, GlcNAcbeta1-3(NeuAcalpha2-6)GalNAcol. Moreover, the pattern of glycosylation was very similar between mucins isolated from each sample, the main differences related to the level of expression of the major oligosaccharides. The data obtained in this investigation may have value for future screening studies on colorectal cancer.
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| 6. |
- Robbe-Masselot, C, et al.
(författare)
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Glycosylation of the two O-glycosylated domains of human MUC2 mucin in patients transposed with artificial urinary bladders constructed from proximal colonic tissue
- 2008
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 25:3, s. 213-224
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Tidskriftsartikel (refereegranskat)abstract
- Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly alpha2-6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.
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