On August 5, 2025, a research team led by Koichi Kato from the National Institute of Natural Sciences in Japan published an article in PNAS entitled "Exploring glycoform-dependent dynamic modulations in human immunoglobulin G via computational and experimental approaches." This study, combining isotope-labeled NMR with molecular dynamics simulations, systematically revealed how IgG1-Fc glycoforms (galactosylation and fucosylation) regulate structural dynamics and effector functions in different but synergistic ways. The results showed that galactosylation acts like an "anchor and wedge," stabilizing the global Fc conformation and reducing entropy loss during binding; while defucosylation mainly enhances binding to FcγRIIIa by releasing the key residue Tyr296. The combination of both significantly improves antibody-dependent cellular cytotoxicity (ADCC) and complement activity, providing new insights for antibody drug optimization.
This article integrates computational and experimental methods to investigate the impact of glycoform alterations on the dynamic structure of the IgG1 Fc region. Four distinct IgG1-Fc glycoforms were obtained through a combination of Cell Engineering and in vitro enzymatic reactions, exhibiting differences in core fucosylation and terminal galactosylation. Using stable isotope-labeled NMR spectroscopy, simultaneous observation of glycan and protein signals revealed that galactosylation induces chemical shift perturbations extending from the glycan-protein interface to the CH2-CH3 region boundary. Molecular dynamics simulations showed that the absence of galactose enhances the mobility of the glycans and the CH2 region, broadening the conformational space of the Fc quaternary structure. This increased flexibility may lead to greater entropy loss upon binding with effector molecules, as these effector molecules confine the Fc to an asymmetric conformation.
In contrast, fucosylation has a more localized effect, primarily impacting the dynamics of residues involved in Fc γRIIIa binding. The authors' research reveals, at the atomic level, the distinct yet synergistic mechanisms by which galactosylation and fucosylation regulate IgG1-Fc dynamics and effector function, providing crucial information for the optimization of therapeutic antibodies.
Antibodies (IgG) are multifunctional proteins with Glycans that play a central role in immune defense and biopharmaceuticals. The Fab region recognizes antigens, while the Fc region triggers effector functions such as ADCC and CDC by binding to Fcγ receptors (FcγRs) and complement C1q. The Asn297 glycan located in the CH2 domain is crucial for Fc function. IgG glycan types vary significantly among individuals and in different disease states.
Core fucosylation reduces the affinity of IgG1 for FcγRIIIa on NK cells, thereby inhibiting ADCC; conversely, defucosylation enhances ADCC, a property already applied in the development of therapeutic antibodies. Furthermore, serum IgG1 in severe COVID-19 patients often exhibits a high proportion of defucosylation.
The galactosylation level of the Fc glycan increases during pregnancy but decreases in inflammatory diseases such as rheumatoid arthritis. Previous studies have shown that galactosylation enhances complement activation and ADCC, and its mechanism is related to increased binding to FcγR and C1q. The terminal galactose on the Manα1-6 branch plays a particularly crucial role. Notably, the combination of galactosylation and defucosylation synergistically and significantly enhances FcγRIIIa-mediated ADCC. Therefore, glycan structure optimization has become an important consideration in the development and production of antibody drugs.
Glycan chains on glycoproteins are highly flexible; therefore, combining experimental techniques (such as NMR) with computational methods (such as molecular dynamics simulations) is an effective means of elucidating their complex structures. This study compared four different glycoforms of human IgG1-Fc, prepared using cell engineering and in vitro enzymatic reactions, and performed detailed Structural Analysis using glycan-derived NMR signals. Simultaneously, the experimental data were combined with molecular dynamics simulation results to explore the dynamic interaction network within the Fc molecule.
The research team constructed four IgG1-Fc glycoforms with purification levels >95%:
Through cell engineering (CHO cell expression, Fut8 gene knockout, co-expression of galactosyltransferase); in vitro enzymatic modification (galactosyltransferase + UDP-Gal, galactosidase); isotope-labeled NMR (observation of glycan and protein signals); and long-term molecular dynamics simulations (2.56 μs trajectory, construction of conformational ensemble model), this series of combined techniques allowed them to systematically analyze the impact of glycoform changes on Fc structural dynamics at the atomic level.
NMR results show that removing the Manα1-6 terminal G6′ causes a series of chemical shift perturbations that diffuse from the glycan-protein interface to the CH2–CH3 boundary.
MD simulations indicate that degalactosylation enhances glycan motility, increases the flexibility of the CH2 domain, and significantly widens the conformational space of the Fc quaternary structure.
This means that galactose acts as an "anchor" to stabilize the glycan chain and also acts as a "wedge" to restrict the CH2–CH3 wobble, reducing the entropy penalty during effector molecule binding.
Fig. 1 Galactosylation suppresses conformational fluctuations in the Fc region, enhancing interactions with effector molecules. (Yanaka, et al. 2025)
Fucose orients the Tyr296 residue in a direction unfavorable to FcγRIIIa binding, causing steric conflict with the FcγRIIIa N162 glycan chain. After defucosylation, Tyr296 gains greater conformational freedom, while eliminating the conflict and significantly improving FcγRIIIa binding.
Unlike galactosylation, fucosylation has a localized effect, primarily acting on key residues.
Galactosylation: Globally stabilizes Fc, enhancing complement activation.
Defucosylation: Optimizes local conformation, enhancing FcγRIIIa binding.
Combination of both: Significantly enhances ADCC, with complementary mechanisms.
This study uses the term "molecular meridians" to describe the propagation pathway of galactosylation, influencing distal domains from the glycan ends, transmitting signals like meridians. This long-range regulation provides new insights for antibody glycan engineering: future research could explore amino acid modifications at these meridian nodes to optimize antibody function using allosteric effects; it also reminds us that in developing next-generation antibody drugs, we must not only focus on local binding pockets but also consider the entire dynamic energy landscape.
Overall, this study reveals at the atomic level the different but synergistic mechanisms by which galactosylation and fucosylation regulate the structure and function of IgG1-Fc, providing a solid theoretical foundation for the optimization of therapeutic antibodies. In the future, combining experimental and computational methods will play an increasingly important role in antibody drug design.
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