- Sialic acid metabolic engineering of breast cancer cells interferes with adhesion and migration
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Breast cancer is the most frequent cancer diagnosed in women and the second most common cancer-causing death worldwide. The major problem around the management of breast cancer is its high heterogeneity and the development of therapeutic resistance. Therefore, understanding the fundamental breast cancer biology is crucial for better diagnosis and therapy. Protein sialylation is a key posttranslational modification of glycoproteins, which is also involved in tumor progression and metastasis. Increased expression of sialic acids (Sia) can interfere in receptor-ligand interactions and might protect tumor cells from the immune system. Furthermore, Sia content on the cell membrane plays a role in cancer resistance towards chemo- and radiation therapy. In this study, we glycoengineered MCF-7 breast cancer cells using a series of non-natural Sia precursors, which are prolonged in their acyl side chain. We observed a significant reduction in the natural Sia (N-Acetylneuraminic acid) expression after cultivation of MCF-7 cells with these Sia precursors. In addition, the expression of polySia, a unique glycosylation of the neural cell adhesion molecule NCAM, which interferes with cell adhesion, was decreased. We conclude that sialic acid engineering i) opens up novel opportunities to study the biological role of Sia in breast cancer and ii) provides a toolbox to examine the sialic acid-dependent complex cellular alterations in breast cancer cell biology.
- Gnanapragassam, Vinayaga Srinivasan,Horstkorte, Rüdiger,Nagasundaram, Manimozhi
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- Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor
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The synthesis of N-acetylneuraminic acid (Neu5Ac) derivatives is drawing more and more attention in glycobiology research because of the important role of sialic acids in e. g. cancer, bacterial, and healthy cells. Chemical preparation of these carbohydrates typically relies on multistep synthetic procedures leading to low overall yields. Herein we report a continuous flow process involving N-acetylneuraminate lyase (NAL) immobilized on Immobead 150P (Immobead-NAL) to prepare Neu5Ac derivatives. Batch experiments with Immobead-NAL showed equal activity as the native enzyme. Moreover, by using a fivefold excess of either N-acetyl-D-mannosamine (ManNAc) or pyruvate the conversion and isolated yield of Neu5Ac were significantly improved. To further increase the efficiency of the process, a flow setup was designed providing a chemoenzymatic entry into a series of N-functionalized Neu5Ac derivatives in conversions of 48–82%, and showing excellent stability over 1 week of continuous use. (Figure presented.).
- Bloemendal, Victor R. L. J.,Moons, Sam J.,Heming, Jurriaan J. A.,Chayoua, Mohamed,Niesink, Olaf,van Hest, Jan C. M.,Boltje, Thomas J.,Rutjes, Floris P. J. T.
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p. 2443 - 2447
(2019/05/17)
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- Highly efficient and selective biocatalytic production of glucosamine from chitin
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N-Acetyl glucosamine (GlcNAc) is one of the most abundant biomolecules on Earth and is cheaply available from chitin, a major component of crustaceans. The key step in the conversion of GlcNAc to high-value products is the de-N-acetylation to glucosamine, in itself a valuable dietary supplement that is produced at over 29:000 tons scale per annum by chemical hydrolysis, a process that requires harsh reaction conditions and leads to side products requiring separation. Here, we report for the first time the isolation and characterisation of an enzyme, a deacetylase from Cyclobacterium marinum that is able to catalyse the highly selective quantitative hydrolysis of GlcNAc to glucosamine under mild reaction conditions. This enzyme is small (38 kDa), is easily obtainable by heterologous expression in E. coli, has high turnover rates (kcat=61 s-1), tolerates high substrate concentrations (over 100 g L-1) and can be repeatedly re-used as an immobilised catalyst. When coupled with chitinase, the high selectivity of the enzyme for GlcNAc over other biomolecules allowed one-pot extraction of glucosamine from crude solid mushroom fractions containing chitin, thus allowing for alternative production of glucosamine from non-animal sources, of benefit to consumers with crustacean allergies and vegan diets. We suggest that the deacetylase fills an important gap in the sustainable exploitation of GlcNAc and chitin.
- Lv,Laborda,Huang,Cai,Wang,Lu,Doherty,Liu,Flitsch,Voglmeir
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supporting information
p. 527 - 535
(2017/08/15)
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- Synthesis and characterization of N-acyl-tetra-O-acyl glucosamine derivatives
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Novel 1,3,4,6-tetra-O-acyl-N-acyl-d-glucosamine derivatives were synthesized from glucosamine hydrochloride (GlcN·HCl) by the acylation with pyridine as a catalyst. A derivative of tetra-O-acetyl glucosamine contained ketoprofen, a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects, was first synthesized. In analysis of the NMR spectra, the ratio of α:β-anomer showed that penta-acyl-d- glucosamine derivatives and N-acetylated glucosamines containing O-acyl groups have been only the α-anomer. Meanwhile, both the intermediates and the glucoconjugate compound of ketoprofen have only the β-anomer.
- Dang, Chi-Hien,Nguyen, Cong-Hao,Nguyen, Thanh-Danh,Im, Chan
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p. 6239 - 6245
(2014/01/23)
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- AMPHETAMINE PRODRUGS
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The present invention relates to amphetamine prodrugs which provide colonic release of amphetamine.
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Paragraph 287; 288; 289
(2014/01/17)
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- Modified GM3 gangliosides produced by metabolic oligosaccharide engineering
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Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.
- Whitman, Chad M.,Yang, Fan,Kohler, Jennifer J.
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supporting information; experimental part
p. 5006 - 5010
(2011/10/09)
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- Fluorination of mammalian cell surfaces via the sialic acid biosynthetic pathway
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Metabolic oligosaccharide engineering has been employed to introduce fluorine-containing groups onto mammalian cell surfaces. Incubation of HeLa, Jurkat, and HL60 cells in culture with fluorinated sialic acid and mannosamine analogues resulted in cell-sur
- Dafik, Laila,d'Alarcao, Marc,Kumar, Krishna
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scheme or table
p. 5945 - 5947
(2009/06/25)
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- N-butyryl glucosamine increases matrix gene expression by chondrocytes
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Proteoglycan synthesis is dependent on N-acetyl glucosamine (GlcNAc) produced by the hexosamine biosynthetic pathway or obtained exogenously. Although used therapeutically to relieve symptoms of osteoarthritis, the actions of glucosamine and its analogs on cartilage are poorly understood. The purpose of this study was to determine the effects on chondrocytes of N-acylated-glucosamine analogs bearing alkyl chains of different lengths. Chondrocytes isolated from neonatal rat femoral condyles were cultured in the presence of glucosamine analogs. GlcNAc, N-proprionyl glucosamine (GlcNPro), or N-butyryl glucosamine (GlcNBu) did not alter cell number, lactate dehydrogenase release, or metabolic acid production, consistent with lack of cytotoxicity. Treatment of chondrocyte cultures with GlcNBu for 6 days significantly increased levels of type II collagen and aggrecan mRNA as determined by Northern blot analysis. In contrast, GlcNAc and GlcNPro had no significant effect. A significant increase in type II collagen mRNA was induced by GlcNBu within 3 days. GlcNBu did not alter stability of type II collagen mRNA, suggesting it acts on gene transcription. We have previously shown that tumor necrosis factor-α (TNFα) decreases levels of type II collagen mRNA. However, chondrocytes pretreated with GlcNBu maintained type II collagen mRNA at control levels in the presence of TNFα. These results establish that the N-butyrylated analog of glucosamine but not GlcNAc promotes matrix gene expression by chondrocytes. Thus, GlcNBu has the potential for use as a chondro-protective agent in osteoarthritis.
- Poustie, Mark W.,Carran, John,McEleney, Kevin,Dixon, S. Jeffrey,Anastassiades, Tassos P.,Bernier, Suzanne M.
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p. 610 - 616
(2007/10/03)
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- Biological properties of N-acyl and N-haloacetyl neuraminic acids: Processing by enzymes of sialic acid metabolism, and interaction with influenza virus
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Several unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepared enzymatically using immobilised sialic acid aldolase. N-Trifluoroacetyl-, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of association of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (Kiapp 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland. Copyright
- Humphrey, Andrew J,Fremann, Claire,Critchley, Peter,Malykh, Yanina,Schauer, Roland,Bugg, Timothy D.H
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p. 3175 - 3185
(2007/10/03)
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- 2-DEOXY-1,3,4,5,6-PENTA-O-METHYL-2-(N-METHYLACETAMIDO)-D-GLUCITOL AND DERIVATIVES UNDERGO C-METHYLATION AT THE N-METHYLACETAMIDO GROUP ON REPEATED HAKOMORI METHYLATION
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After Hakomori methylation of 2-acetamido-2-deoxy-D-glucitol, the expected 2-deoxy-1,3,4,5,6-penta-O-methyl-2-(N-methylacetamido)-D-glucitol (3) was identified by g.l.c.-m.s. as the major product, and two minor products, 2-acetamido-2-deoxy-1,3,4,5,6-penta-O-methyl-D-glucitol (2) and 2-deoxy-1,3,4,5,6-penta-O-methyl-2-(N-methylpropionamido)-D-glucitol (4), were present.The proportions and yields of these products were dependent on the reagent (sodium or potassium hydride) used for the preparation of the methylsulfinylmethanide.On Hakomori methylation of 2 and 3, the N-methylpropionamido (4), N-methylisobutyramido, and traces of the N-methylpivalamido derivatives of 2-deoxy-1,3,4,5,6-penta-O-methyl-D-glucitol were formed.Using trideuteriomethyl iodide for methylation (e.g., of 3), it was found by g.l.c.-m.s. that the newly introduced methyl group(s) were located at the β-carbon of the N-methylacetamido group.Analogous results were obtained with 2-deoxy-4-O--1,3,5,6-tetra-O-methyl-2-(N-methylacetamido)-D-glucitol.
- Zaehringer, Ulrich,Rietschel, Ernst Theodor
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