Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases

Abstract

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.

The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition

“…The EndoS2-IgG1 Fc crystal structure demonstrates how the enzyme contacts IgG1 Fc with its GH and CBM domains, as also elucidated from hydrogen-deuterium exchange of an EndoS2-rituximab complex ( 51 ) and similarly observed for the related EndoS complex ( 55 , 56 ). However, upon binding to the Fc, EndoS2 undergoes a domain rearrangement, with the relative orientation of GH, LRR, and hIg domains changing ( Figs.…”

“…EndoS and EndoS2 are multi-domain enzymes, comprised of a catalytic glycosyl hydrolase (GH) domain, a leucine-rich repeat (LRR) domain, a hybrid immunoglobulin fold (hIg) domain, and the so-called carbohydrate-binding module (CBM) ( 50 , 52 , 53 , 54 ); EndoS additionally has N- and C-terminal 3-helix bundles ( 50 , 53 ). Recent structural studies revealed that the functional role of the CBM in EndoS was not to bind carbohydrates but rather to specify peptide binding to the Fc surface ( 55 , 56 ). The structural basis for IgG recognition by EndoS2 is less clear, although hydrogen-deuterium exchange data show a role for both GH and CBM domains in Fc binding ( 51 ), which indicates a similar mode of Fc recognition to that observed for EndoS ( 55 , 56 ).…”

“…Recent structural studies revealed that the functional role of the CBM in EndoS was not to bind carbohydrates but rather to specify peptide binding to the Fc surface ( 55 , 56 ). The structural basis for IgG recognition by EndoS2 is less clear, although hydrogen-deuterium exchange data show a role for both GH and CBM domains in Fc binding ( 51 ), which indicates a similar mode of Fc recognition to that observed for EndoS ( 55 , 56 ).…”

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers

“…Rituximab, an anti-CD20 chimeric monoclonal antibody, targets B cells and is often used as a therapeutic for B cell lymphomas ( 37 ). After purification by protein A affinity chromatography ( 38 ) and treatment with the IgG-specific endoglycosidase EndoS2 ( 39 , 40 ) to remove heterogeneous glycosylation at residue Asn297, three potential products with unique masses that are easily detectable by liquid chromatography–mass spectrometry (LC-MS) can result from such a coexpression, including an intact Fc (~50 kDa), an intact IgG (~150 kDa), and a monovalent IgG molecule (Fab 1 Fc; ~100 kDa) ( Fig. 2A ).…”

Structure-Based High-Efficiency Homogeneous Antibody Platform by Endoglycosidase Sz Provides Insights into Its Transglycosylation Mechanism

“…The bound G2S2-oxazoline is carried by EndoSz-D234M to the target, Asn-GlcNAc (+1). How EndoSz-D234M targets Asn-GlcNAc (+1) remains unknown, which is presumably mediated by the β-sandwich domain as which EndoS had identified interactions with the CH2-CH3 joint of Fc . According to the structural analysis of the EndoSz-D234M/G2S2-oxazoline complex (Figure b), Gln304 and Asp280 might play important roles in accommodating NAG (+1), especially for the acetamido group.…”