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

Abstract

Bacterial pathogens have evolved diverse and intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes secretes two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-linked 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 analysis, 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.

Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate

“…6c). In addition, we obtained a native stable complex between BT1285i and Fc HM at low protein concentration (0.05 mg mL -1 ) that we visualized by negative-stain transmission electron microscopy (NS-TEM), which did not require chemical crosslinking to be stabilized as previously reported for the EndoS-Fc complex 57 , due to the high affinity of BT1285 for HM glycans on Fc. By NS-TEM analysis of the BT1285i:Fc-HM complex we generated a three-lobed ~19 Å structure, in which one molecule of BT1285 is positioned to attack the Asn297-linked glycan on each Fc protomer and making contact just with the HM N-glycan on N297, which also supported the 2:1 BT1285i:Fc-HM stoichiometry (Fig.…”

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).…”

Bespoke conformation and antibody recognition distinguishes the streptococcal immune evasion factors EndoS and EndoS2

“…Crystallographic studies of enzymes in complex with their glycan substrate have provided insight into the mechanism of action of endoglycosidases [11,12,[57][58][59]. Our recent structure of EndoS in complex with its IgG1 Fc substrate [55] and a cryo-electron microscopy study [56] provided further insight. Here, we present the crystal structure of EndoS2 in complex with IgG1 Fc substrate at a resolution of 3.0 Å (Table 1), with N-linked glycans modelled using the carbohydrate module in Coot [60] and validated using Privateer [61], as shown in Figure 1.…”

“…These endoglycosidases are multi-domain enzymes, with the catalytic glycosidase domain and the so-called carbohydrate binding module (CBM) being of particular interest [12,[52][53][54]. Recent structural studies revealed that the functional role of the CBM in EndoS is not to bind carbohydrate, but rather to specify peptide binding on the Fc surface [55,56], which provided a structural rationale for the abrogated enzymatic activity of EndoS lacking the CBM [52,53], further explaining the inability of EndoS to cleave glycans from denatured IgG [6]. Similarly, it is known for that the CBM of EndoS2 is essential for activity.…”