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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 α1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.
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  • 2
    ISSN: 1432-2013
    Keywords: Co-transport ; Nitrogen-sparing diet ; Proton-driven transport ; l-leucine hydroxy analogue ; l-2-hydroxy-4-methylpentanoic acid ; l-isoleucine hydroxy analogue ; l-2-hydroxy-3-methylpentanoic acid ; l-valine hydroxy analogue ; l-2-hydroxy-3-methylbutanoic acid ; l-lactate ; l-2-hydroxypropionic acid ; dl-methionine ; 2-hydroxy-4-methylthiobutanoic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Substitution of the α-amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogensparing diets for uremic patients. In this study, intestinal uptake of l-leucine HA (l-LeuHA) and l-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H+ or Na+ gradient stimulated uptake of both labelled substrates in a voltageclamped assay. The H+ gradient was the major driving force of uptake as compared with the Na+ gradient, and it led to a transient accumulation of both l-LeuHA and l-lactate. The proton ionophore carbonylcyanide p trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H+-gradient-driven uptake rates of both substrates, but was without effect on Na+-gradient-driven uptakes. The H+-gradient-driven l-LeuHA uptake was saturable (apparent Kt = 15.4 mM). α-HA of l-leucine, l-isoleucine, l-valine, d-leucine, d-valine or l-lactate inhibited the H+-gradient-driven l-LeuHA or l-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the l-or d-HA of branched-chain amino acids or with l-lactate stimulated tracer l-LeuHA and also tracer l-lactate uptakes in the presence of a H+ gradient. It is concluded that H+-gradient-driven transport of l- and d-stereoisomeric HA of branched-chain amino acids as well as of l-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na+gradient-driven l-lactate transport system in the rabbit intestinal brush-border membrane.
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  • 3
    ISSN: 1420-9071
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Since the pioneering work on structure and function of heteroglycans compiled in the classical books edited by A. Gottschalk in 19721, there have been several promising developments in glycoconjugate research, as reviewed in this article. In Part 1, contributed by A. Kobata, current knowledge on heteroglycan structures is presented and representative examples taken from higher organisms are given. Part 2, written by J. F. G. Vliegenthart and J. P. Kamerling, covers the most important achievements in methodology: procedures to obtain pure glycans and to analyze their structures. Part 3, contributed by J. Paulson, is devoted to biosynthesis of glycans now describable as pathways since several of the glycosyltransferases have been isolated and analyzed for specificity. In Part 4, contributed by E. Buddecke, current knowledge on functional roles of glycans is presented. It will become apparent that the prerequisite for valid work either in biosynthetic or functional context depends on solid structural information. This is particularly true whenever glycosyltransferase reaction products are being analyzed, or glycans involved in biological functions are investigated. Although in past years, a great deal of important knowledge has been gathered by use of crude glycosidase or glycosyltransferase activities (a notable example is found in reference 2), one may now postulate that glycans implicated in biological reactions should be thoroughly analyzed. This review may familiarize ‘newcomers’ with the field of glycoconjugate research with special emphasis on glycoprotein glycans. Glycolipids are not included in this article as they have recently been reviewed by S. I. Hakomori3. The reader is also referred to several excellent monographs4,5 and the Proceedings of the Glycoconjugate Symposia held biannually6–8.
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  • 4
    ISSN: 1573-7276
    Keywords: serum α1,3fucosyltransferase ; FUTVI ; colorectal cancer ; drainage vein ; anti-FUTVI
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have previously reported that the elevated activities of serum α1,3fucosyltransferase reverted to normal levels after curative removal of the tumors. To determine the origin of elevated serum α1,3fucosyltransferase, blood samples were obtained from both the drainage vein and the artery in patients with different stages of colorectal cancer at surgery. The enzyme levels in all samples from the drainage vein were found to be higher than the levels in the artery that fed the tumor. Hence, the origin of elevated α1,3fucosyltransferase in serum was thought to be the tumor rather than the liver that is the normal source of serum α1,3fucosyltransferase. When serum samples not only from colorectal cancer patients but also from patients with gastric, liver, lung, pancreas, bladder and esophagus cancer were treated with anti-FUTVI antibody, the measured activities of α1,3fucosyltransferase were markedly reduced. Further, secretion of α1,3fucosyltransferase from human colorectal carcinoma cells was also detected in the culture medium by Western immuno-blot analysis with anti-FUTVI antibody.
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  • 5
    ISSN: 1573-6776
    Keywords: fucosyltransferase ; N-acetylglucosaminyltransferase ; P-selectin ; PSGL-1 ; tricistronic vectors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Expression of recombinant glycoproteins carrying the sialyl-LewisX epitope requires host cells equipped with appropriate glycosyltransferases. Chinese hamster ovary cells transfected with tricistronic vectors and encoding the resistance marker gene, neoR, in the third cistron and core 2 N-acetylglucosaminyltransferase or α1,3-fucosyltransferase III or IV in either the first or second cistron, respectively, produced both enzyme activities in a constant ratio. A representative clone was transfected with PSGL-1 (P-selectin glycoprotien ligand 1) and conferred P-selectin-binding activity to PSGL-1.
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  • 6
    ISSN: 1573-4986
    Keywords: sialyl Lewis x ; enzymic synthesis ; N-acetylglucosaminyltransferase ; fucosyltransferase ; galactosyltransferase ; sialyltransferase ; recombinant glycosyltransferases ; P-selectin ; PSGL-1 ; β3-GalT, UDP-Gal:GalNAcα-R β-1,3-galactosyltransferase, β3-galactosyltransferase ; C2GnT, UDP-GlcNAc:Gal(β1-3)GalNAc(α1-R (GlcNAc to GalNAc) β-1,6-N-acetylglucosaminyltransferase, β6-N-acetylglucosaminyltransferase ; β4-GalT1, UDP-Gal:GlcNAc β-1,4-galactosyltransferase 1, β4-galactosyltransferase 1 ; ST3Gal3, CMP-Neu5Ac:Gal(β1-4)GlcNAc α-2,3-sialyltransferase 3, α3-sialyltransferase 3 ; α3-FucT6, GDP-Fuc:Gal(β1-4)GlcNAc (Fuc to GlcNAc) α-1,3-fucosyltransferase 6, α3-fucosyltransferase 6 ; Caco, sodium cacodylate (Na(CH3)2AsO2) - HCl buffer ; CIAP, Calf Intestinal Alkaline Phosphatase ; core 1, Gal(β1-3)GalNAc ; core 2, GlcNAc(β1-6)[Gal(β1-3)]GalNAc ; core 4, GlcNAc(β1-6)[GlcNAc(β1-3)]GalNAc ; core 6, GlcNAc(β1-6)GalNAc ; FPLC, Fast Protein Liquid Chromatography ; HPLC, High Performance Liquid Chromatography ; LacNAc, Gal(β1-4)GlcNAc ; lacto-N-biose, Gal(β1-3)GlcNAc ; MALDI TOF, Matrix Assisted Laser Desorption Ionisation Time of Flight ; MES, 2-(N-Morpholino)ethanesulfonic acid - NaOH buffer ; Me2SO, Dimethyl sulfoxide ; MLEV, Malcolm Levitt ; NMR, Nuclear Magnetic Resonance ; pNp, p-Nitrophenyl ; PSGL-1, P-Selectin Glycoprotein Ligand 1 ; ROESY, Rotating Frame Nuclear Overhauser Enhancement Spectroscopy ; sLex, Sialyl Lewis x ; TOCSY, Total Correlation Spectroscopy ; WEFT, Water Eliminated Fourier Transform
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-α-D-galactosaminide (GalNAc(α1-pNp as core substrate, the sLex-oligosaccharide Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(β1-3)GalNAc(α1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of β3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 β6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp in a yield of 〉85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk β4-galactosyltransferase 1 (EC 2.4.1.38) (yield of 〉85%), then sialylated using CMP-Neu5Ac and purified recombinant α3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human α3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 µmol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.
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  • 7
    ISSN: 1573-4986
    Keywords: glycosyltransferase ; assay ; high-throughput screening ; drug discovery
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37°C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well β-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 β1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewisx in Ο-glycans. A glycopolymer acceptor for β1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5–6 fold increase in throughput compared to the corresponding solution-phase assay.
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  • 8
    ISSN: 1573-4986
    Keywords: saccharomyces cerevisiae ; Pichia pastoris ; β1,4galactosyltransferase ; α2,6sialytransferase ; α2,3sialytransferase ; α1,3fucosyltransferase ; α1,2mannosyltransferase ; glycosylation engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur, P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include β1,4galactosyltransferase, α2,6sialyltransferase, α2,3sialyltransferase, α1,3fucosyltransferase III and VI and α1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.
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  • 9
    ISSN: 1573-4986
    Keywords: human T-lymphocytes ; Tn syndrome ; plant lectin ; sialoadhesin ; myelin-associated glycoprotein ; CD22
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Previously, β1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol 22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAcα1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Acα2,6GalNAcα1-O-R) or TF (Galβ1-3GalNAcα1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.
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  • 10
    ISSN: 1573-4986
    Keywords: Galactosyltransferase; B lymphocyte; Antibody; Rheumatoid arthritis; Agalactosyl IgG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract We have quantified the level of β4-galactosyltransferase protein in human B lymphocytes using an ELISA-based assay. Between 1-10ng of β4-galactosyltransferase was detected per mg total cellular protein, indicating that this enzyme constitutes 〈0.001% of B lymphocyte cellular protein. Akin to previous studies, individuals with rheumatoid arthritis exhibited reduced lymphocytic galactosyltransferase enzyme activity compared with normal controls when using ovalbumin as the acceptor substrate. The levels of enzyme protein present in B lymphocytes from patients with rheumatoid arthritis was, however, not reduced suggesting that the B lymphocyte galactosyltransferase catalytic activity may be regulated post-translationally.
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