| 1. |
- Bengtson, Per, et al.
(författare)
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Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:34, s. 31575-31582
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Tidskriftsartikel (refereegranskat)abstract
- The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLex and SLex-related epitopes. One individual showed lower levels of SLex expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg110 → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg110 is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLex and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.
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| 2. |
- Bengtson, Per, et al.
(författare)
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Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins
- 2002
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 169:7, s. 3940-3946
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Tidskriftsartikel (refereegranskat)abstract
- We recently identified several individuals carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding fucosyltransferase VII. This enzyme is involved in the biosynthesis of the sialyl Lewis x (Lex) epitope on human leukocytes, which has been identified as an important component of leukocyte ligands for E- and P-selectin. No enzyme activity was measurable in expression studies in COS-7 cells using the mutated FUT7 construct. One of the identified individuals carried this mutation homozygously. Flow cytometry analysis of polymorphonuclear leukocytes (PMN) from this individual showed a nearly complete absence of staining with mAbs directed against sialyl Lex and a diminished staining with an E-selectin IgG chimera. However, staining with P-selectin IgG chimera and Abs directed against P-selectin glycoprotein ligand-1 was not affected by the mutation. PMN from the homozygously mutated individual was further analyzed in an in vitro flow chamber assay. The number of rolling PMN and the rolling velocities on both E- and P-selectin were in the range of PMN from nonmutated individuals. FUT4 and FUT7 mRNA was quantified in PMN isolated from individuals carrying the FUT7 mutation. It was found that PMN from both FUT7 homozygously and heterozygously mutated individuals exhibited an elevated expression of FUT4 mRNA compared with PMN from FUT7 nonmutated individuals. The elevated expression of fucosyltransferase IV was reflected as an increased expression of the Lex and CD65s Ags on PMN from these individuals. The significance of the mutation was supported by transfection of BJAB cells.
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| 3. |
- Landberg, Eva, 1966-, et al.
(författare)
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Changes in Glycosylation of Human Bile-Salt-Stimulated Lipase during Lactation
- 2000
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 377:2, s. 246-254
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Tidskriftsartikel (refereegranskat)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption–ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.
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| 4. |
- Landberg, Eva, et al.
(författare)
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Changes in the relative amounts of α2-3 and α2-6 linked sialic acid on free and protein-bound milk oligosaccharides during lactation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Human milk contains large amounts of oligosaccharides, both in free and protein-bound forms. Sialic acid is a common constituent of milk oligosaccharides and is present α2-3 or α2-6 linked to galactose, α2-6 to N-acetyl glucosamine or α2-6 to N-acetyl galactosamine. High-performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to quantify four major sialylated milk oligosaccharides. The concentrations of the individual oligosaccharides were analyzed in milk from five donors, followed separately during six to nine months of lactation. The oligosaccharides containing sialic acid a2-6 linked to galactose ( 6-sialyl lactose and LSTc) decreased more than tenfold during lactation. In contrast, the concentration of 3-sialyl lactose (3-SL) containing sialic acid a2-3 linked to galactose remained constant during nine months of lactation. Disialyl lacto-Ntetraose (DSLNT) which contain sialic acid linked α2-3 to Gal and α2-6 to GlcNAc decreased approximately threefold during lactation. Lectin ELISA was used to analyze sialic acid on the secreted milk glycoprotein bile-salt-stimulated lipase (BSSL ). There was a gradual decrease in the binding of Sambucus Nigra lectin to BSSL, indicating decreased amount of α2-6 linked sialic acid during lactation. In contrast, binding of Maackia Amurensis lectin remained constant, indicating a constant expression of α2-3 linked sialic acid on BSSL during lactation. This suggests a shift from preferentially 6-linked to 3-linked sialic acid on free and protein-bound oligosaccharides during lactation. The shift may reflect changes in sialyltransferase activities and, on a higher level, changes in the population of mammary epithelial cells. This finding may be of importance for the development of a correct immune response to environmental challenges.
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| 5. |
- Landberg, Eva, et al.
(författare)
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Glycosylation of Bile-Salt-Stimulated Lipase from Human Milk : Comparison of Native and Recombinant Forms
- 1997
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 344:1, s. 94-102
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Tidskriftsartikel (refereegranskat)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19–26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types ofN-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short typeO-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.
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| 6. |
- Landberg, Eva, et al.
(författare)
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Quantitative changes of fucosylated human milk oligosaccharides during lactation in comparison to milk fucosyltransferase activity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Human milk contains 7-20 g/L of free oligosaccharides. The oligosaccharides show large variations in size and structure. It has been suggested that milk oligosaccharides can prevent pathogenic microorganisms from attaching to the gastrointestinal epithelium by blocking bacterial adhesins. However, the biological role of milk oligosaccharides is far from understood. In this study, the major fucosylated oligosaccharides in milk were followed during six to nine months of lactation. Individual oligosaccharides were separated and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The fucosylated oligosaccharides 2-fucosyl lactose, lacto-N-fucopentaose I and lacto-N-di-fucohexaose I showed decreasing concentrations in milk during lactation. The concentrations of lacto-difucotetraose, lacto-N-fucopentaose II and Ill remained constant, while 3-fucosyl lactose (3-FL) showed increasing concentrations during lactation. The increase of 3-FL was found for all individuals independent of secretor status, but did not correlate to milk fucosyltransferase activity when the product 3-FL was measured separately. Instead all individuals showed a decrease in fucosyltransferase activity during lactation, indicating that fucosyltransferase activity in milk does not reflect the biosynthetic activity in the mammary gland. This study shows that the composition of fucosylated oligosaccharides vary considerably during the first six months of lactation. This may reflect unique biological roles of certain oligosaccharides in the infant's adaptation to the environment.
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| 7. |
- Landberg, Eva, et al.
(författare)
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Temperature effects in high-performance anion-exchange chromatography of oligosaccharides
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 814:1-2, s. 97-104
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Tidskriftsartikel (refereegranskat)abstract
- High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection has been widely used for analysis of mono-, oligo- and polysaccharides. Many factors that affect separation of carbohydrates by HPAEC have been evaluated, however effect of temperature has not been carefully studied. In the present study, neutral and sialylated oligosaccharides from human milk and different types of N-linked oligosaccharides were analysed by HPAEC at temperatures ranging from 13 to 30°C. N-Acetyl neuraminic acid, galacturonic acid and stachyose were also analysed since they have been used as internal standards when analysing various oligosaccharides by HPAEC. All oligosaccharides showed decreased retention times with increased temperature. Even small differences in temperature (i.e. ±5°) resulted in considerable changes in retention times. In addition, individual oligosaccharides showed different relative changes in retention time with increased temperature. By changing the temperature, a switch in elution order of individual oligosaccharides were sometimes found. These results show that retention times relative to an internal standard cannot be used for oligosaccharide identification unless temperature is carefully controlled. Regulation of temperature is also a valuable tool in achieving optimal separation of oligosaccharides by HPAEC.
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| 8. |
- Liljeblad, Mathias, et al.
(författare)
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A Lectin Immunosensor Technique for Determination of α1-Acid Glycoprotein Fucosylation
- 2001
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 288:2, s. 216-224
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Tidskriftsartikel (refereegranskat)abstract
- The fucosylation of α1-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.
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| 9. |
- Liljeblad, Mathias, et al.
(författare)
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Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance
- 2000
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Ingår i: Glycoconjugate Journal. - 0282-0080 .- 1573-4986. ; 17:5, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.
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| 10. |
- Liljeblad, Mathias, et al.
(författare)
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Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique
- 2002
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Ingår i: Biosensors & bioelectronics. - 0956-5663 .- 1873-4235. ; 17:10, s. 883-891
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Tidskriftsartikel (refereegranskat)abstract
- A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze α1-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor β-1 (TGFβ1). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGFβ1 AGP fucosylation increased whereas AGP concentration decreased.
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