Technical Basis for Nuclear Magnetic Resonance Approach for Glycoproteins

Kato, Koichi; Yanaka, Saeko; Yagi, Hirokazu

doi:10.1007/978-981-10-5966-7_15

Cited by 10 publications

(3 citation statements)

References 94 publications

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“…Oligosaccharide structures have dynamic properties in solution and, therefore, have been described as conformational ensembles. − It has been supposed that lectin selects and captures specific conformational species of oligosaccharides among the ensemble, thereby promoting biological functions. , Upon their interactions, the oligosaccharides may further undergo conformational deformation to fit into the binding pocket of lectin. ,, Therefore, to gain quantitative insights into the energetics of carbohydrate recognition by lectins, detailed structural information is needed concerning their target oligosaccharides in both the precomplexed and lectin-complexed states. ,− Moreover, such knowledge is crucial to design artificial oligosaccharides with improved functionality.…”

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confidence: 99%

Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity

et al. 2019

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We developed an approach to improve the lectinbinding affinity of an oligosaccharide by remodeling its conformational space in the precomplexed state. To develop this approach, we used a Lewis X-containing oligosaccharide interacting with RSL as a model system. Using an experimentally validated molecular dynamics simulation, we designed a Lewis X analogue with an increased population of conformational species that were originally very minor but exclusively accessible to the target lectin without steric hindrance by modifying the nonreducing terminal galactose, which does not directly contact the lectin in the complex. This Lewis X mimetic showed 17 times higher affinity for the lectin than the native counterpart. Our approach, complementing the lectinbound-state optimizations, offers an alternative strategy to create high-affinity oligosaccharides by increasing populations of onpathway metastable conformers.

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confidence: 99%

Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity

et al. 2019

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“…NMR can be a valuable tool for detecting weak interactions while dealing with heterogeneous systems. For selective observation of antibody NMR signals, we have established stable-isotope-labeling techniques of IgG glycoproteins using various eukaryotic expression systems ( 10 , 14 , 25 , 26 ). In the present study, we prepared uniformly 15 N-labeled rituximab, which was cleaved into Fab and Fc fragments for HSQC spectral measurements, to assess their interactions with HSA or polyclonal IgG-Fab.…”

Section: Resultsmentioning

confidence: 99%

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins

et al. 2023

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Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum–protein interactions in the design and application of antibody-based drugs with increased efficacy and safety.

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“…For the NMR measurements, the fucosyl Fc fragment was cleaved from IgG1 metabolically labeled with [CO, α, β, γ, ε1, ε2- 13 C 6 ; β2, δ1, δ2- 2 H 3 ; 15 N] tyrosine (Taiyo Nippon Sanso, Tokyo, Japan). The metabolic isotope labeling, proteolytic digestion, and purification of the Fc fragments were performed according to previously reported protocols [13,38].…”

Section: Methodsmentioning

confidence: 99%

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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Technical Basis for Nuclear Magnetic Resonance Approach for Glycoproteins

Cited by 10 publications

References 94 publications

Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity

Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins

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

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