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Ole K Greinertollersrud - One of the best experts on this subject based on the ideXlab platform.
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the non classical n glycan processing pathway of bovine brain ecto nucleotide phosphodiesterase pyrophosphatase 6 enpp6 is brain specific and not due to mannose 6 phosphorylation
Neurochemical Research, 2014Co-Authors: Ole K GreinertollersrudAbstract:Ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-Glycans in each monomer. In brain s-eNPP6 the N-Glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-Glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.
Ole K. Greiner-tollersrud - One of the best experts on this subject based on the ideXlab platform.
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The non-classical N-Glycan processing pathway of bovine brain ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is brain specific and not due to mannose-6-phosphorylation
Neurochemical Research, 2014Co-Authors: Ole K. Greiner-tollersrudAbstract:Ecto -nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-Glycans in each monomer. In brain s-eNPP6 the N-Glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-Glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.
Manfred Wuhrer - One of the best experts on this subject based on the ideXlab platform.
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Reversed-phase separation methods for glycan analysis
Analytical and Bioanalytical Chemistry, 2017Co-Authors: Gerda C. M. Vreeker, Manfred WuhrerAbstract:Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical Abstract Glycan isomer separation by reversed phase liquid chromatography
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Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS
Analytical Chemistry, 2016Co-Authors: Archana Shubhakar, Radoslaw P. Kozak, Karli R. Reiding, Daniel I. R. Spencer, Daryl L. Fernandes, Louise Royle, Manfred WuhrerAbstract:Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-Glycan release, enrichment of N-Glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.
Shin-ichiro Nishimura - One of the best experts on this subject based on the ideXlab platform.
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Serum N-Glycan profiling is a potential biomarker for castration-resistant prostate cancer.
Scientific Reports, 2019Co-Authors: Teppei Matsumoto, Shingo Hatakeyama, Tohru Yoneyama, Yuki Tobisawa, Yusuke Ishibashi, Hayato Yamamoto, Takahiro Yoneyama, Yasuhiro Hashimoto, Shin-ichiro NishimuraAbstract:We investigated the diagnostic and prognostic potential of serum N-Glycan profiling for castration-resistant prostate cancer (CRPC). We retrospectively investigated serum N-Glycan structural analysis by glycoblotting for 287 patients with benign prostatic hyperplasia (BPH), 289 patients with newly diagnosed prostate cancer (PC), 57 patients with PC treated with androgen-deprivation therapy without disease progression (PC-ADT), and 60 patients with CRPC. N-Glycan profiling was compared between the non-CRPC (BPH, newly diagnosed PC and PC-ADT) and CRPC patients. We obtained the quantitative score for CRPC (CRPC N-Glycan score) by discriminant analysis based on the combination of 9 N-Glycans that were significantly associated with CRPC. The median CRPC N-Glycan score was found to be significantly greater in CRPC patients than in non-CRPC patients. The CRPC N-Glycan score could classify CRPC patients with sensitivity, specificity, and area under the curve of 87%, 69%, and 0.88, respectively. The CRPC N-Glycan score >1.7 points was significantly associated with poor prognosis in patients with CRPC. The glycoprotein analysis showed that not immunoglobulins but α-1-acid glycoprotein (AGP) were a potential candidate for the carrier protein of N-Glycans. The overexpression of specific N-Glycans may be associated with their castration-resistant status and be a potential biomarker for CRPC.
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simple separation of isomeric sialylated n glycopeptides by a zwitterionic type of hydrophilic interaction chromatography
Journal of Separation Science, 2006Co-Authors: Yasuhiro Takegawa, Takuro Keira, Hiroaki Nakagawa, Kisaburo Deguchi, Shin-ichiro NishimuraAbstract:Asparagine-linked oligosaccharides (N-Glycans) usually show structural heterogeneity, especially in proteins with sialylated N-Glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-Glycan isomers as well as for the previously reported neutral N-Glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-Glycans derivatized with 2-aminopyridine (PA N-Glycans) demonstrate that the retentions of the N-Glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-Glycan isomers.
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Simple separation of isometric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography
Journal of Separation Science, 2006Co-Authors: Yasuhiro Takegawa, Hiroki Ito, Takuro Keira, Hiroaki Nakagawa, Kisaburo Deguchi, Shin-ichiro NishimuraAbstract:Asparagine-linked oligosaccharides (N-Glycans) usually show structural heterogeneity, especially in proteins with sialylated N-Glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-Glycan isomers as well as for the previously reported neutral N-Glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-Glycans derivatized with 2-aminopyridine (PA N-Glycans) demonstrate that the retentions of the N-Glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-Glycan isomers.
Shunji Natsuka - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of n glycans of the planarian dugesia japonica
FEBS Journal, 2011Co-Authors: Shunji Natsuka, Yukiko Hirohata, Shinichi Nakakita, Wataru Sumiyoshi, Sumihiro HaseAbstract:To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-Glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-Glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-Glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-Glycans, it was shown that the N-Glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-Glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.
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Structural analysis of N‐glycans of the planarian Dugesia japonica
FEBS Journal, 2011Co-Authors: Shunji Natsuka, Yukiko Hirohata, Shinichi Nakakita, Wataru Sumiyoshi, Sumihiro HaseAbstract:To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-Glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-Glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-Glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-Glycans, it was shown that the N-Glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-Glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.
