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Ningguo Gao - One of the best experts on this subject based on the ideXlab platform.
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Letter to the Glycoforum: Improved protocols for preparing lipid-linked and related saccharides for Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).
Glycobiology, 2013Co-Authors: Ningguo Gao, Justin Holmes, Mark A. LehrmanAbstract:Our laboratory has used Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) to analyze many saccharides related to the dolichol pathway (Gao and Lehrman, 2002, 2003; Lehrman, 2007). This includes the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol, LLO intermediates, mannose-P-dolichol, glucose-P-dolichol, N-glycans, free glycans, sugar mono- and bis-phosphates, and nucleotide-sugars. Some examples of the variety of saccharides that can be studied are given in (Gao and Lehrman, 2002; Gao et al., 2011; Gao et al., 2008). In addition to these studies with mammalian cultured cells and tissues, we used FACE to identify saccharides from an assortment of sources including Danio rerio (Cline et al., 2012), Saccharomyces cerevisiae (Rush et al., 2009), Trypanosoma brucei (Jelk et al., 2013), and Arabidopsis thaliana (Amtmann, A., unpublished). Since publication of a comprehensive method (Gao and Lehrman, 2006) we identified problematic steps during the preparation of samples, prior to gel Electrophoresis. Here, as Supplemental Information, we provide a revised protocol (in lab bench-ready format) for preparing dolichol pathway samples for FACE. One vexing problem in particular is solved with this new protocol. Previously, it was necessary at different stages to dry proteinacious cellular residues containing saccharides of interest. Great care was necessary to avoid overdrying, to prevent these residues from forming tough unworkable aggregates refractory to extraction. The extractions were further hampered by variable losses of residue at each step. These and other technical problems have now been solved with a single-step extraction method2 in which three distinct phases are generated (each enriched with a different group of saccharides), and by eliminating the need to dry proteinacious cellular residues. Various members of our group have successfully used different aspects of this new protocol, which we conclude is a significant improvement over our earlier methods. We thank Dan Beriault for very helpful comments on the text. Supported by NIH grant GM038545.
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Non-radioactive analysis of lipid-linked oligosaccharide compositions by Fluorophore-Assisted Carbohydrate Electrophoresis.
Methods in enzymology, 2006Co-Authors: Ningguo Gao, Mark A. LehrmanAbstract:Abstract Lipid‐linked oligosaccharides (LLOs) are the donors of glycans that modify newly synthesized proteins in the endoplasmic reticulum (ER) of eukaryotes, resulting in formation of N‐linked glycoproteins. The vast majority of LLO analyses have relied on metabolic labeling with radioactive sugar precursors, but these approaches have technical limitations resulting in many important questions about LLO synthesis being left unanswered. Here we describe the application of a facile non‐radioactive technique, fluorophore‐assisted Carbohydrate Electrophoresis (FACE), which circumvents these limitations. With FACE, steady‐state LLO compositions can be determined quantitatively from cell cultures and animal tissues. We also present FACE methods for analysis of phosphosugars and nucleotide sugars, which are metabolic precursors of LLOs.
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Application of Fluorophore-Assisted Carbohydrate Electrophoresis for the study of the dolichol pyrophosphate-linked oligosaccharides pathway in cell cultures and animal tissues.
Methods in molecular biology (Clifton N.J.), 2006Co-Authors: Ningguo GaoAbstract:Defects in the synthesis of dolichol-linked oligosaccharide (or lipid-linked oligosaccharide [LLO]) cause severe, multisystem human diseases called type 1 congenital disorders of glycosylation (CDG type 1). LLOs are also involved in another disease, neuronal ceroid lipofuscinosis. Because of the low abundance of LLOs, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine, and therefore have been limited almost entirely to cell cultures and tissue slices. A procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO pathway analysis based on Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). It is feasible to analyze almost any component in the LLO pathway with the application FACE, from sugar precursors to mature LLO (Glc3Man9GlcNAc2-P-P-dolichol).
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Analysis of glycosylation in CDG-Ia fibroblasts by Fluorophore-Assisted Carbohydrate Electrophoresis: implications for extracellular glucose and intracellular mannose 6-phosphate.
The Journal of biological chemistry, 2005Co-Authors: Ningguo Gao, Jie Shang, Mark A. LehrmanAbstract:Abstract Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, Fluorophore-Assisted Carbohydrate Electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc2-P-P-dolichol, without hypoglycosylation, CDG-Ia fibroblasts grown with physiological glucose. This suggested a “missing link” to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc3Man9GlcNAc2-P-P-dolichol. This led to futile cycling the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.
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Fluorophore-Assisted Carbohydrate Electrophoresis: a sensitive and accurate method for the direct analysis of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues.
Methods (San Diego Calif.), 2005Co-Authors: Ningguo GaoAbstract:Lipid-linked oligosaccharides (LLOs) such as Glc3Man9GlcNAc2-P-P-dolichol are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I Congenital Disorders of Glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine. In this article, a procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO analysis based on Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). First, LLOs are extracted and partially purified. Next, oligosaccharides released from LLOs are labeled with negatively charged fluorophores: 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) or 7-amino-1,3-naphthalenedisulfonic acid (ANDS). A specialized form of polyacrylamide gel Electrophoresis is then used to resolve and measure ANTS or ANDS labeled oligosaccharides. Finally, the resolved oligosaccharides are detected and quantified by fluorescence imagers using CCD cameras.
Mark A. Lehrman - One of the best experts on this subject based on the ideXlab platform.
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Letter to the Glycoforum: Improved protocols for preparing lipid-linked and related saccharides for Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).
Glycobiology, 2013Co-Authors: Ningguo Gao, Justin Holmes, Mark A. LehrmanAbstract:Our laboratory has used Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) to analyze many saccharides related to the dolichol pathway (Gao and Lehrman, 2002, 2003; Lehrman, 2007). This includes the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol, LLO intermediates, mannose-P-dolichol, glucose-P-dolichol, N-glycans, free glycans, sugar mono- and bis-phosphates, and nucleotide-sugars. Some examples of the variety of saccharides that can be studied are given in (Gao and Lehrman, 2002; Gao et al., 2011; Gao et al., 2008). In addition to these studies with mammalian cultured cells and tissues, we used FACE to identify saccharides from an assortment of sources including Danio rerio (Cline et al., 2012), Saccharomyces cerevisiae (Rush et al., 2009), Trypanosoma brucei (Jelk et al., 2013), and Arabidopsis thaliana (Amtmann, A., unpublished). Since publication of a comprehensive method (Gao and Lehrman, 2006) we identified problematic steps during the preparation of samples, prior to gel Electrophoresis. Here, as Supplemental Information, we provide a revised protocol (in lab bench-ready format) for preparing dolichol pathway samples for FACE. One vexing problem in particular is solved with this new protocol. Previously, it was necessary at different stages to dry proteinacious cellular residues containing saccharides of interest. Great care was necessary to avoid overdrying, to prevent these residues from forming tough unworkable aggregates refractory to extraction. The extractions were further hampered by variable losses of residue at each step. These and other technical problems have now been solved with a single-step extraction method2 in which three distinct phases are generated (each enriched with a different group of saccharides), and by eliminating the need to dry proteinacious cellular residues. Various members of our group have successfully used different aspects of this new protocol, which we conclude is a significant improvement over our earlier methods. We thank Dan Beriault for very helpful comments on the text. Supported by NIH grant GM038545.
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Non-radioactive analysis of lipid-linked oligosaccharide compositions by Fluorophore-Assisted Carbohydrate Electrophoresis.
Methods in enzymology, 2006Co-Authors: Ningguo Gao, Mark A. LehrmanAbstract:Abstract Lipid‐linked oligosaccharides (LLOs) are the donors of glycans that modify newly synthesized proteins in the endoplasmic reticulum (ER) of eukaryotes, resulting in formation of N‐linked glycoproteins. The vast majority of LLO analyses have relied on metabolic labeling with radioactive sugar precursors, but these approaches have technical limitations resulting in many important questions about LLO synthesis being left unanswered. Here we describe the application of a facile non‐radioactive technique, fluorophore‐assisted Carbohydrate Electrophoresis (FACE), which circumvents these limitations. With FACE, steady‐state LLO compositions can be determined quantitatively from cell cultures and animal tissues. We also present FACE methods for analysis of phosphosugars and nucleotide sugars, which are metabolic precursors of LLOs.
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Analysis of glycosylation in CDG-Ia fibroblasts by Fluorophore-Assisted Carbohydrate Electrophoresis: implications for extracellular glucose and intracellular mannose 6-phosphate.
The Journal of biological chemistry, 2005Co-Authors: Ningguo Gao, Jie Shang, Mark A. LehrmanAbstract:Abstract Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, Fluorophore-Assisted Carbohydrate Electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc2-P-P-dolichol, without hypoglycosylation, CDG-Ia fibroblasts grown with physiological glucose. This suggested a “missing link” to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc3Man9GlcNAc2-P-P-dolichol. This led to futile cycling the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.
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Alternative sources of reagents and supplies for Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)
Glycobiology, 2002Co-Authors: Ningguo Gao, Mark A. LehrmanAbstract:Ningguo Gao and Mark A. Lehrman Dept. Pharmacology UT-Southwestern Medical Center 5323 Harry Hines Boulevard Dallas, TX 75390-9041 # Contact information: (214)-648-2323; mark.lehrman@utsouthwestern.edu Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) is a simple, sensitive, and versatile method for analysis of both monosaccharides and oligosaccharides, and has become an important method in Glycobiology. Recently, the major provider of reagents and supplies for FACE has ceased production and shipping. To assist those who have relied upon the major provider for FACE reagents and supplies, the Editors have asked us to summarize alternative commercial sources and procedures that we have used (Gao and Lehrman, 2002a; Gao and Lehrman, 2002b). Our procedures are modified from those published previously (Jackson, P., 1994; Starr, C. M. et al., 1996).
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Analyses of dolichol pyrophosphate–linked oligosaccharides in cell cultures and tissues by Fluorophore-Assisted Carbohydrate Electrophoresis
Glycobiology, 2002Co-Authors: Ningguo Gao, Mark A. LehrmanAbstract:Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.
Yves Briers - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided Fluorophore-Assisted Carbohydrate Electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of Carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided Fluorophore-Assisted Carbohydrate Electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Analysis of the substrate specificity of -L-arabinofuranosidases by DNA sequencer-aided Fluorophore-Assisted Carbohydrate Electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of Carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided Fluorophore-Assisted Carbohydrate Electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
Isao Kusakabe - One of the best experts on this subject based on the ideXlab platform.
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Preparation of (1→4)-β-d-xylooligosaccharides from an acid hydrolysate of cotton-seed xylan: suitability of cotton-seed xylan as a starting material for the preparation of (1→4)-β-d-xylooligosaccharides
Carbohydrate research, 2002Co-Authors: Hyeon Jin Sun, Shigeki Yoshida, Nyun Ho Park, Isao KusakabeAbstract:Cotton-seed residual cake, which is a byproduct of the process of oil extraction from the seed, was delignified with sodium hypochlorite (1% available chlorine). Xylan was then prepared from the delignified wet material by alkali extraction with 15% sodium hydroxide. The cotton-seed xylan contained 64.7% xylose and 9.4% uronic acid. The xylan was hydrolyzed with 0.125 M sulfuric acid at 90 °C for 15 min. The resultant hydrolysis products were separated by gel-permeation chromatography on BioGel P-4 and Toyopearl HW-40F columns connected in series, with water as an eluate. Xylose and xylooligosaccharides with a degree of polymerization ranging from DP 2 to 15 were separated under such conditions, and each xylooligosaccharide-containing peak fraction afforded a single band on Fluorophore-Assisted Carbohydrate Electrophoresis. These results suggest that cotton-seed xylan is suitable for the preparation of xylose and xylooligosaccharides.
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Effects of Unsaturated Uronic Acid Residues at Non-reducing End on Bond Cleavage Frequency of Poly(1,4-α-L-guluronide) Lyase from Enterobacter cloacae M-1.
Bioscience biotechnology and biochemistry, 1998Co-Authors: Tomoko Shimokawa, Shigeki Yoshida, Isao KusakabeAbstract:The mode of action of poly(1,4-α-L-guluronide) lyase from Enterobacter cloacae M-1 on unsaturated oligoguluronic acids was studied using Fluorophore-Assisted Carbohydrate Electrophoresis. The polyguluronate lyase degraded unsaturated penta-, hexa-, and heptaguluronic acids, but not unsaturated oligoguluronic acids with DPs less than 4. On comparison with the aspect of enzymatic degradation of unsaturated oligoguluronic acid and saturated oligoguluronic acid having the same DP, the former was degraded faster than the latter, and also the cleavage pattern of the polyguluronate lyase on unsaturated oligoguluronic acids was considerably different from that on saturated oligoguluronic acids. From the results described above, we suggest that the affinity of the first subsite from the non-reducing end side of the enzyme to Δ residues is lower than that to GulA residues.
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Some properties and action mode of (1-->4)-alpha-L-guluronan lyase from Enterobacter cloacae M-1.
Carbohydrate research, 1997Co-Authors: Tomoko Shimokawa, Shigeki Yoshida, Isao Kusakabe, Toshio Takeuchi, Katsumi Murata, Hideyuki KobayashiAbstract:An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2–9) generated with the enzyme was investigated by Fluorophore-Assisted Carbohydrate Electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having a dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.
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Isolation and quantification of alginate-derived oligouronic acids by Fluorophore-Assisted Carbohydrate Electrophoresis
Carbohydrate Research, 1997Co-Authors: Tomoko Shimokawa, Shigeki Yoshida, Isao Kusakabe, Toshio Takeuchi, Katsumi MurataAbstract:Abstract Alginate-derived oligouronic acids were analyzed by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). Oligoguluronic acids and oligomannuronic acids were labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and were separated by polyacrylamide gel Electrophoresis. The mobilities of oligoguluronic acids and oligomannuronic acids were much higher than those of maltooligosaccharides, and the best resolution of oligouronic acids having DPs lower than 10 was achieved on 40% polyacrylamide gel. Over the range 0.25 to 4 nmol, the fluorescence intensity of an ANTS-labeled oligouronic acid band could be correlated linearly to the quantity of the oligouronic acid. The distribution of DPs of polyguluronic acid and polymannuronic acid also were estimated by using FACE.
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Preparation of Two Series of Oligo-guluronic Acids from Sodium Alginate by Acid Hydrolysis and Enzymatic Degradation
Bioscience Biotechnology and Biochemistry, 1996Co-Authors: Tomoko Shimokawa, Shigeki Yoshida, Toshio Takeuchi, Katsumi Murata, Tadashi Ishii, Isao KusakabeAbstract:The objective of this study was to prepare two series of authentic oligo-guluronic acids from sodium alginate. Oligo-guluronic acids (DP = 1–9) were prepared from an acid hydrolysate of poly-guluronic acid by successive chromatographies of Bio-Gel P-6 and Q Sepharose Fast Flow. Oligo-guluronic acids having 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid residues at the non-reducing end (DP = 2–7) were prepared from the enzymatic degradation products of the poly-guluronic acid in the same manner. Each of the isolated oligo-guluronic acids gave a single band on Fluorophore-Assisted Carbohydrate Electrophoresis. These results suggest that successive chromatographies used in this study are well suited for the preparation of alginate-derived oligouronic acids.
John M. Whitelock - One of the best experts on this subject based on the ideXlab platform.
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Small-scale enzymatic digestion of glycoproteins and proteoglycans for analysis of oligosaccharides by LC-MS and FACE gel Electrophoresis.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: Ruby P. Estrella, John M. Whitelock, Rebecca H. Roubin, Nicolle H. Packer, Niclas G. KarlssonAbstract:Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel Electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.
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Characterization and purification of glycosaminoglycans from crude biological samples.
Journal of agricultural and food chemistry, 2007Co-Authors: Neil P. Davies, Rebecca H. Roubin, John M. WhitelockAbstract:Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using Fluorophore-Assisted Carbohydrate Electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common s...
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Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan.
FEBS letters, 2005Co-Authors: Sarah M. Knox, Amanda J. Fosang, James Melrose, John M. WhitelockAbstract:Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and Fluorophore-Assisted Carbohydrate Electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.