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

Satoh, Tadashi; Kato, Koichi

doi:10.1016/j.sbi.2014.03.008

Cited by 20 publications

(6 citation statements)

References 75 publications

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“…In general, carbohydrate chains are conformationally dynamic compared to polypeptide chains and, therefore, yield ambiguous electron densities in the crystal structures of glycoproteins [48,49]. However, this is not true for the N-glycans of IgG-Fc because they are packed between the two C H 2 domains and, therefore, are restricted in terms of internal motion, which has been confirmed by the previous and present MD simulations [24].…”

Section: Resultssupporting

confidence: 54%

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

et al. 2019

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The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.

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Section: Resultssupporting

confidence: 54%

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

et al. 2019

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“…[1,2] The determinants recognised by lectins often consist of di-to tetrasaccharides with an onreducing terminal residue. [3][4][5][6] Thus, crystallographic data have revealed the structural basis of the oligosaccharide-binding specificities of lectins. [3][4][5][6] Thus, crystallographic data have revealed the structural basis of the oligosaccharide-binding specificities of lectins.…”

Section: Introductionmentioning

confidence: 99%

“…[3][4][5] The details of carbohydrate-proteini nteractions have been elucidated by X-ray crystallography,w hich can delineate the conformation of an oligosaccharide ligand accommodated in the carbohydratebinding pocket of lectin and its intermolecular atomicc ontacts. [3][4][5][6] Thus, crystallographic data have revealed the structural basis of the oligosaccharide-binding specificities of lectins. However,i ts hould be noted that oligosaccharides exhibit considerable flexibility of their glycosidic linkages, [7] thus giving rise to dynamic conformationale nsembles in solution.…”

Section: Introductionmentioning

confidence: 99%

Conformational Analysis of a High‐Mannose‐Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR‐Validated Molecular Dynamics Simulation

et al. 2017

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Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism.

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“…Through the development and improvement of hardware and software, along with sample preparation methodologies that include sophisticated stable isotopelabeling methods, they have successfully determined the atomic coordinates of proteins [9]. Carbohydrate NMR spectroscopy remains less mature in comparison with protein NMR studies [10]. In addition to the difficulties of sample preparation, carbohydrate NMR spectroscopists often suffer from the overlap of peaks due to the lack of variability of local chemical structures, as well as an insufficiency of conformational restraints provided by NOE data due to low proton density, compared with proteins.…”

Section: Introductionmentioning

confidence: 99%

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

Kato

Yamaguchi

2015

Glycoconj J

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Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques have recently been applied to a wide variety of biomolecular systems, using sophisticated immobilization methods to attach paramagnetic probes, such as spin labels and lanthanide-chelating groups, at specific sites of the target biomolecules. This is also true in the field of carbohydrate NMR spectroscopy. NMR analysis of oligosaccharides is often precluded by peak overlap resulting from the lack of variability of local chemical structures, by the insufficiency of conformational restraints from nuclear Overhauser effect (NOE) data due to low proton density, and moreover, by the inherently flexible nature of carbohydrate chains. Paramagnetic probes attached to the reducing ends of oligosaccharides cause paramagnetic relaxation enhancements (PREs) and/or pseudocontact shifts (PCSs) resolve the peak overlap problem. These spectral perturbations can be sources of long-range atomic distance information, which complements the local conformational information derived from J couplings and NOEs. Furthermore, paramagnetic NMR approaches, in conjunction with computational methods, have opened up possibilities for the description of dynamic conformational ensembles of oligosaccharides in solution. Several applications of paramagnetic NMR techniques are presented to demonstrate their utility for characterizing the conformational dynamics of oligosaccharides and for probing the carbohydrate-recognition modes of proteins. These techniques can be applied to the characterization of transient, non-stoichiometric interactions and will contribute to the visualization of dynamic biomolecular processes involving sugar chains.

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

Cited by 20 publications

References 75 publications

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

Conformational Analysis of a High‐Mannose‐Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR‐Validated Molecular Dynamics Simulation

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

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