Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system

Kato, Koichi; Yamaguchi, Yoshiki; Arata, Yoji

doi:10.1016/j.pnmrs.2010.03.001

Cited by 56 publications

(31 citation statements)

References 94 publications

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“…In addition to high glycoprotein yield, the utility of stable isotope-labeling techniques is key to the NMR analysis of glycoproteins. Several review articles [40][41][42][43] have outlined methodologies and applications of uniform and selective isotope labeling of glycoproteins, based on metabolic labeling within eukaryotic cells or enzymatic attachment of isotope-labeled monosaccharides in vitro.…”

Section: Production Of Isotopically Labeled Glycoproteins For Nmr Anamentioning

confidence: 99%

Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms

Satoh

Kato

2014

Current Opinion in Structural Biology

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Because of the complexity, heterogeneity, and flexibility of the glycans, the structural analysis of glycoproteins has been eschewed until recently, with a few prominent exceptions. This aversion may have branded structural biologists as glycophobics. However, recent technological advancements in glycoprotein expression systems, employing genetically engineered production vehicles derived from mammalian, insect, yeast, and even bacterial cells, have yielded encouraging breakthroughs. The major advance is the active control of glycoform expression of target glycoproteins based on the genetic manipulation of glycan biogenetic pathways, which was previously overlooked, abolished, or considered unmanageable. Moreover, synthetic and/or chemoenzymatic approaches now enable the preparation of glycoproteins with uniform glycoforms designed in a tailored fashion.

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Section: Production Of Isotopically Labeled Glycoproteins For Nmr Anamentioning

confidence: 99%

Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms

Satoh

Kato

2014

Current Opinion in Structural Biology

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“…NMR fails to solve complete, all-atom structures with large molecules due to insufficient resolution of each peak and because of signal sapping line broadening effects that scale with molecule mass (Cavanagh et al, 2007; Tugarinov et al, 2004). However, targeted studies using selective labeling can reveal molecular features not visible through any other technique (Kato et al, 2010; Religa et al, 2010; Tugarinov et al, 2006). By combining the best aspects of solution and solid-state techniques we can characterize relationships between structure and activity with high precision.…”

Section: Introductionmentioning

confidence: 99%

The Structural Role of Antibody N-Glycosylation in Receptor Interactions

2015

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Asparagine(N)297-linked glycosylation of IgG Fc is required for binding to FcγRIIa, IIb and IIIa though it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc indicating N-glycosylation does not maintain relative Fc Cγ2/Cγ3 domain orientation. However, the conformation of the C'E loop, which contains N297, was significantly perturbed in the aglycosylated Fc variant. The conformation of the C'E loop as measured with a range of Fc variants shows a strong correlation with FcγRIIIa affinity. These results indicate the primary role of the IgG1 Fc N-glycan is to stabilize the C'E loop through intramolecular interactions between carbohydrate and amino acid residues and preorganize the FcγRIIIa interface for optimal binding affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other antibodies including IgG2-4, IgD, IgE and IgM.

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“…However, making unambiguous assignments of the chemical shifts needed for 3 D structure determination can be challenging due to the abundance of overlapping 1 H and, to a lesser degree, 13 C resonances. Overlapping signals can be resolved by conducting 13 C‐edited or 13 C‐coupled NMR experiments, but limitations in sensitivity generally mean that uniform isotopic labeling is required . In contrast to proteins, the lack of templated biosynthesis of and the presence of heterogeneity in naturally occurring glycans has limited the production of uniformly isotopically labeled sugars to those present on the outer membrane of Escherichia coli or to oligomannosides produced in an engineered yeast strain .…”

Section: Introductionmentioning

confidence: 99%

Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

et al. 2017

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Man9GlcNAc2 (Man-9) present at the surface of HIV constitutes the binding sites of several HIV neutralizing agents and mammalian lectin DC-SIGN that is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in free state and when bound by an HIV entry inhibitor protein and define the minimum epitopes of two HIV binding proteins using NMR spectroscopy. In this regard we developed a robust expression system for production of 13C,15N-labeled glycans in mammalian cells that facilitated the implementation of 3D 13C-edited spectra to deconvolute spectral overlap allowing for the solution structure determination of Man-9. The studies reveal that Man-9 interacts with HIV-binding proteins via distinct binding epitopes and adopts divergent conformations in the bound state. In combination with molecular dynamics we find that these receptor-bound conformations are sampled by Man-9 in the free state suggesting a mechanism for diverse recognition.

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Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system

Cited by 56 publications

References 94 publications

Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms

Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms

The Structural Role of Antibody N-Glycosylation in Receptor Interactions

Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

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