| 1. |
- Thomsson, Kristina A, 1969, et al.
(författare)
-
Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced Fucalpha1-2 glycosyltransferase.
- 2002
-
Ingår i: The Biochemical journal. - 0264-6021. ; 367:Pt 3, s. 609-16
-
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.
|
|
| 2. |
- Thomsson, Kristina A, 1969
(författare)
-
Analytical approaches to the study of mucin glycosylation
- 2000
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mucins constitute the major protein component of the mucus layer, which covers the epithelial surfaces of the respiratory, the gastrointestinal and the genitourinary tracts. Mucins carry O-linked oligosaccharides which contribute to the molecule by 50-80 weight percent, and these can be highly diverse, with 50-150 different oligosaccharides found on a purified mucin population. Altered mucin glycosylation has been observed in mucus-related diseases, which implies the importance of the development of analytical methods and strategies for structural characterization and semiquantitative estimates of diverse mixtures of oligosaccharides.Oligosaccharides from mucin subpopulations from a patient with Cystic Fibrosis were characterized and compared regarding size, composition and sequence. Differences and similarities in the relative abundance of individual oligosaccharides were mapped using high temperature gas chromatography (GC), gas-chromatography/mass spectrometry (GC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) of subfractionated glycans. A similar approach was applied in a comparative study of the glycosylation on intestinal mucins from wild type and CFTR knock-out mice. Increased mucus production and an increased expression of the blood group H epitopes was found in the small intestine of CF mice. Increased mRNA levels for a fucosyltransferase Fut2 was identified by northern blot analysis on intestinal tissue sections from CF mice and compared to wild type mice, suggesting that this transferase could be responsible for the altered glycosylation.The two major mucin components in saliva, originally termed MG1 and MG2 were characterized and compared with respect to average oligosaccharide size, sequence, abundance and degree of diversity. 1H NMR spectroscopy, MALDI-TOF/MS and GC/MS of released oligosaccharides mixtures and subfractions of these showed that MG1 carried longer and more diverse glycans compared to MG2. 1H NMR spectroscopy of the glycans supported a higher diversity of peripheral fucose epitopes on MG1 compared to MG2, which mainly expressed Lex and sialyl-Lex blood group determinants. The two mucins shared few oligosaccharides in common, suggesting specific functions for each of these. Experiments confirming the binding of MG2 to L-selectin in in vitro studies proposed an involvement in the mediation of leukocyte interactions in the oral cavity.The use of liquid chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) for analyzing mixtures of nonderivatized sulfated mucin oligosaccharides was explored. The chromatographic separation of this subclass of analytes was compared on two columns, on a porous graphitized carbon column and an amino-bonded column. The analysis of a diverse mixture of sulfated oligosaccharides from porcine large intestine with negative ion LC-ESI/MS/MS allowed the identification of 28 different oligosaccharides during a single chromatographic analysis. Sufficient fragmentation was obtained by LC/MS/MS to distinguish between sequence isomers.
|
|
| 3. |
- Thomsson, Kristina A, 1969, et al.
(författare)
-
Sequencing of sulfated oligosaccharides from mucins by liquid chromatography and electrospray ionization tandem mass spectrometry.
- 2000
-
Ingår i: Analytical chemistry. - 0003-2700. ; 72:19, s. 4543-9
-
Tidskriftsartikel (refereegranskat)abstract
- As part of a strategy for profiling diverse mixtures of sulfated mucin-derived oligosaccharides, liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode has been explored. Two mixtures of sulfated oligosaccharide alditols from porcine stomach and large intestine were analyzed by straight phase chromatography using an amino-bonded column connected to a Q-TOF instrument. Nine sulfated mucin-derived oligosaccharide alditols from porcine stomach underwent extensive fragmentation allowing determination of their sequence. The fragmentation generated primary, secondary, and tertiary fragment ions informative for the elucidation of the saccharide sequence and localization of the sulfate group. From a single chromatographic analysis, the sequences of 28 different sulfated mucin oligosaccharide alditols purified from porcine large intestine were elucidated, revealing information concerning prominent core sequences and terminal blood group-type epitopes. Analysis of these two sulfated oligosaccharide mixtures demonstrated the usefulness of HPLC-ESI-MS/MS: the on-line separation of multiple isomeric suffated oligosaccharides as present in biological samples, informative fragmentation allowing the identification of the sequence of nonderivatized oligosaccharides, and a sensitivity sufficient for the analysis of quantities as obtained from natural sources.
|
|
| 4. |
- Thomsson, Kristina A, 1969, et al.
(författare)
-
The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse.
- 2002
-
Ingår i: Glycobiology. - 0959-6658 .- 1460-2423. ; 12:1, s. 1-14
-
Tidskriftsartikel (refereegranskat)abstract
- The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive beta-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Le(a), Le(x), Le(b), and Le(y) epitopes; (2) NeuAc was mainly linked alpha 2-3 to Gal or alpha 2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.
|
|
