Dataset Information

Molecular Basis of Broad Spectrum N-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2.

ABSTRACT: Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-?-N-acetylglucosaminidase, EndoS2, which deglycosylates the conserved N-linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these schemes because it accommodates a broad range of N-glycans, including high-mannose, complex, and hybrid types; however, its mechanism of substrate recognition is poorly understood. We present crystal structures of EndoS2 alone and bound to complex and high-mannose glycans; the broad N-glycan specificity is governed by critical loops that shape the binding site of EndoS2. Furthermore, hydrolytic experiments, domain-swap chimeras, and hydrogen-deuterium exchange mass spectrometry reveal the importance of the carbohydrate-binding module in the mechanism of IgG recognition by EndoS2, providing insights into engineering enzymes to catalyze customizable glycosylation reactions.

SUBMITTER: Klontz EH

PROVIDER: S-EPMC6439443 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Publications

Molecular Basis of Broad Spectrum N-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2.

Klontz Erik H EH Trastoy Beatriz B Deredge Daniel D Fields James K JK Li Chao C Orwenyo Jared J Marina Alberto A Beadenkopf Robert R Günther Sebastian S Flores Jair J Wintrode Patrick L PL Wang Lai-Xi LX Guerin Marcelo E ME Sundberg Eric J EJ

ACS central science 20190206 3

Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-β-N-acetylglucosaminidase, EndoS2, which deglycosylates the conserved N-linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these s ...[more]

PMID: 30937380

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Project description:Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.

Dataset Information

Molecular Basis of Broad Spectrum N-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2.

Publications

Molecular Basis of Broad Spectrum <i>N</i>-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2.

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