Diversity in structure and functions of antibody sialylation in the Fc

Raju, T. Shantha; Lang, Stanley

doi:10.1016/j.copbio.2014.06.014

Cited by 39 publications

(30 citation statements)

References 45 publications

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“…Then the samples were digested with 20 g of trypsin in 100 mM Tris-HCl, pH 8.2 containing 1 mM CaCl 2 at 37°C overnight. The trypsin-digested samples were desalted by a reverse phase cartridge (Sep-Pak C 18 , Restek) and dried by lyophilization. The dried samples were resuspended in 50 mM sodium citrate buffer, pH 5.0.…”

Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

“…The typical transglycosylation reaction at an analytical level of mass spectrometry was performed with a reaction mixture composed of 20 g of sialylglycopeptide, 320 g of 13 C 6 -labeled glucose, and 1 milliunit of endo-␤-N-acetylglucosaminidase M in a total volume of 30 l of 100 mM potassium phosphate buffer, pH 6.0. The released isotopically labeled N-linked glycan was purified from the peptides by a reverse phase cartridge (Sep-Pak C 18 ) with a 5% acetic acid elution solution and dried down for use as an internal standard. The resulting internal standard was used to improve the precision of the relative quantitative analysis for the N-linked glycans from the plants.…”

Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

“…100 nmol of glycopeptide in 20 -50 l of sodium citrate buffer without BSA were incubated with 0.2-0.5 milliunit of peptide-Nglycosidase A (Calbiochem) for 18 h at 37°C. The released N-linked glycans were purified from peptides by reverse phase cartridge (Sep-Pak C 18 ) with a 5% acetic acid elution solution. The samples were dried by lyophilization, and the novel internal standard was added in a constant amount to each sample to improve the precision of quantitative analysis before permethylation.…”

Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

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Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

Yoo

Seo

et al. 2015

Journal of Biological Chemistry

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Background:The largest N-glycan in plants is the paucimannosidic N-glycan with Man 3 XylFuc(GlcNAc) 2 structure. Results: A sophisticated mechanism producing the largest N-glycan in plants is proposed. Conclusion: Limited addition of the 6-arm GlcNAc to the common N-glycan acceptor (GlcNAcMan 3 (GlcNAc) 2 ) facilitates formation of the largest N-glycan in plants.Significance: This sophisticated mechanism expands our knowledge of the energy-efficient N-glycan processing in plants.

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Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

Section: Preparation Of An Isotopically Labeled N-linked Glycan As Anmentioning

confidence: 99%

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Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

Yoo

Seo

et al. 2015

Journal of Biological Chemistry

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“…Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4,5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway.…”

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confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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“…Moreover, sialylation of antibodies is thought to play an important role for anti-inflammatory properties [66]. However, the potential use of sialylated IgGs in this context is still controversial because the used preparations to perform studies are often heterogeneous in terms of glycosylation [67].…”

Section: Optimization Of Pharmacokinetics and Biological Activity By mentioning

confidence: 99%

Using glyco-engineering to produce therapeutic proteins

Dicker

Strasser

2015

Expert Opinion on Biological Therapy

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Glyco-engineering of expression platforms is increasingly recognized as an important strategy to improve biopharmaceuticals. A better understanding and control of the factors leading to glycan heterogeneity will allow simplified production of recombinant glycoprotein therapeutics with less variation in terms of glycosylation. Further technological advances will have a major impact on manufacturing processes and may provide a completely new class of glycoprotein therapeutics with customized functions.

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Diversity in structure and functions of antibody sialylation in the Fc

Cited by 39 publications

References 45 publications

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Using glyco-engineering to produce therapeutic proteins

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