Heterogeneity of the bovine κ-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry
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Carbohydrate-deficient glycoprotein syndromes (CDGS) type I are a group of genetic diseases characterized by a deficiency of N-linked protein glycosylation in the endoplasmic reticulum. The majority of these CDGS patients have phosphomannomutase (PMM) deficiency (type A). This enzyme is required for the synthesis of GDP-mannose, one of the substrates in the biosynthesis of the dolichol-linked oligosaccharide Glc3Man9GlcNAc2. This oligosaccharide serves as the donor substrate in the N-linked glycosylation process. We report on the biochemical characterization of a novel CDGS type I in fibroblasts of four related patients with normal PMM activity but a strongly reduced ability to synthesize glucosylated dolichol-linked oligosaccharide leading to accumulation of dolichol-linked Man9GlcNAc2. This deficiency in the synthesis of dolichol-linked Glc3Man9GlcNAc2 oligosaccharide explains the hypoglycosylation of serum proteins in these patients, because nonglucosylated oligosaccharides are suboptimal substrates in the protein glycosylation process, catalyzed by the oligosaccharyltransferase complex. Accordingly, the efficiency of N-linked protein glycosylation was found to be reduced in fibroblasts from these patients.
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Oligosaccharide
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Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.
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N-linked glycosylation
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Glycosylation is one of the most prevalent post-translational modifications found on human proteins. There is a great interest in developing new methodologies for the synthesis of glycoproteins, both to elucidate the functions of glycans on proteins and to exploit the beneficial properties they can confer on biotherapeutics. Here, research was carried out investigating enzymatic techniques to achieve the formation of homogenous glycoproteins. EndoS was the first enzyme investigated. EndoS natural activity is for the hydrolysis of glycans specifically from IgG, yet there has been research into its use for the opposite reaction and for glycan extension reactions. Here, a number of EndoS mutants were formed and investigated for this reaction, with a number of novel constructs giving a moderate yield of the glycosylated product. Structural investigations of EndoS were also conducted. In addition, research was conducted into the use of PglB in vitro for the direct glycosylation of an asparagine reside on a peptide with GlcNAc. Studies here demonstrate that it is possible to use far simpler glycan donor substrates with PglB. Additional studies showed that it was possible to conduct further enzymatic glycosylation reactions with GalT and EndoA after the initial PglB reaction. Further research was undertaken with EndoA, with it being desired to investigate whether it could catalyse the formation of a thioglycosidic linkage between two glycans, with the thiol bearing glycan acceptor to be attached to a peptide using PglB. After the successful synthesis of all the substrates, disappointingly, neither of the two enzymatic reactions were successful. Finally, the use of a glycal donor in an EndoA catalysed glycosylation was investigated. Unfortunately, no consumption of the glycan was observed in this reaction.
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Glycosylation changes are often described in different physiological processes and pathological conditions and as a lifestyle effect. Alterations in cancer cells metabolism result in the production of altered glycan structures, which are being recognized by the immune system that result in generation of novel anti-glycan antibodies. Presence and screening of antibodies was performed using glycan arrays. Immunoglobulin G (IgG) glycosylation was performed using plasma purification and glycan chromatography. Results show significant differences between healthy individuals and those with cancer. Antibody binding to 24/48 glycans give positive results with great statistical significance between studied sample groups. IgG glycosylation analysis shows that 10/23 glycan chromatographic peaks are changed in colorectal cancer and that galactosylation is one of the main indicators of appearance and progression of the disease. Understanding of IgG glycosylation changes and protein-glycan interactions, and the fact they are effective in numerous diseases are important for understanding biology of cancer cells.
Glycome
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Abstract Human genetic diseases that affect N‐glycosylation result from the defective synthesis of the N‐linked sugar moiety (glycan) of glycoproteins. The role of glycans for proper protein folding and biological functions is illustrated in the variety and severity of clinical manifestations shared by congenital disorders of glycosylation (CDG). This family of inherited metabolic disorders includes defects in the assembly of the oligosaccharide precursor that lead to an under‐occupancy of N‐glycosylation sites (CDG‐I), and defects of glycan remodeling (CDG‐II). Mass spectrometry constitutes a key tool for characterization of CDG‐I defects by mass resolution of native protein glycoforms that differ for glycosylation‐site occupancy. Glycan MS analyses in CDG‐II is mandatory to detect whenever possible a repertoire of structures to pinpoint candidate enzymes and genes responsible for the abnormal N‐glycan synthesis. In this manuscript, we review the MS applications in the area of CDG and related disorders with a special emphasis on those techniques that have been already applied or might become functional for diagnosis, characterization, and treatment monitoring in some specific conditions. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:517–542, 2009
N-linked glycosylation
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Native electrospray ionization was known to preserve the protein structure in solution, which overcame the uncontrollable acidification of droplets during transfer from solution into the gas phase in conventional electrospray ionization. However, detailed experimental studies on when and how could native electrospray ionization minimize structural perturbations remain quite unclear. Herein, we conducted molecular dynamics simulations to investigate the protein structure evolution during electrospray ionization. At a neutral droplet pH, the protein structure in solution could be retained after evaporation, which was in accordance with previous reports. As the droplet pH deviated from neutral, we have found that the compact protein structure would not unfold until the last 10 ns prior to the final desolvation, which demonstrated that the role of native electrospray ionization in preserving the protein structure was mainly reflected on the final evaporation stages. The present study might provide new insights into studying the microscopic biomolecular events occurring during the liquid–gas interface transition and their influence on solution–structure retention.
Extractive electrospray ionization
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This chapter contains sections titled: Introduction – Glycosylated Proteins Basic Building Blocks of Glycosylation in Human Cells Formation of Complex Glycan Structures Glycan Synthesis is Catalyzed by Enzymes of Glycosylation – the "Glycozymes" Protein Glycosylation – Relationship Between N-linked and O-linked Glycoproteins N-linked Glycoproteins O-linked Glycoproteins O- and N-linked Glycan Chain Extension and Commonly Occurring Glycan Motifs Analytical Methodologies Developed to Detect and Characterize Glycosylation Conclusion References
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N-linked glycosylation
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