The mechanistic basis for interprotomer deglycosylation of antibodies by corynebacterial IgG-specific endoglycosidases

Article https://doi.org/10.1038/s41467-025-60986-w The mechanistic basis for interprotomer deglycosylation of antibodies by corynebacterial IgG-specific endoglycosidases Received: 6 February 2025 1234567890():,; 1234567890():,; Accepted: 10 June 2025 Check for updates Diego E. Sastre 1 , Stylianos Bournazos 2, Maros Huliciak 1, Barbara Ann C. Grace2, E. Josephine Boder2, Jonathan Du 1,7, Nazneen Sultana1,8, Tala Azzam 1, Trenton J. Brown1, Maria W. Flowers1, Pete Lollar3, Ting Xu1, Tatiana A. Chernova 1, Alasdair D. Keith 1, Meredith Keen1, Abigail Saltzman1, Ana Martinez Gascueña4, Beatriz Trastoy 4,5, Marcelo E. Guerin 6, Filipp Frank 1, Eric A. Ortlund 1, Jeffrey V. Ravetch 2 & Eric J. Sundberg 1 Corynebacterium diphtheriae clade species secrete single-domain endo-β-Nacetylglucosaminidases (ENGases) that specifically bind to human IgG antibodies and hydrolyze their N297-linked glycans. Here, we define the molecular mechanisms of IgG-specific deglycosylation for the entire family of corynebacterial IgG-specific ENGases, including but not limited to CU43 and CM49. By solving the crystal structure of CU43 in a 1:1 complex with the IgG1 Fc region, combined with targeted and saturation mutagenesis analysis and activity measurements using engineered antibodies, we establish an interprotomeric mechanism of recognition and deglycosylation of IgG antibodies. Using in silico modeling, small-angle X-ray scattering and saturation mutagenesis we determine that CM49 uses a unique binding site on the Fc region, to process N297-linked glycans. Moreover, we demonstrate that CU43 treatment is highly effective in abrogating Fc effector functions in humanized mouse models, while preserving the neutralizing capacity of anti-influenza IgG antibodies, thereby conferring protection against lethal influenza challenge. Nearly all Endo-β-N-acetylglucosaminidases (endoglycosidases, ENGases) recognize and hydrolyze the β−1,4 linkage between the first two N-acetylglucosamine (GlcNAc) saccharides of asparagine-linked glycans on their glycoprotein substrates (EC 3.2.1.96) strictly through glycan-specific mechanisms1–4. However, there exist rare ENGases that hydrolyze only the N297-linked glycan on the fragment crystallizable (Fc) region of IgG antibodies5. This glycan serves as the major molecular determinant for Fc γ receptor (FcγR) binding and its presence and chemical composition dictate antibody-mediated effector functions6,7. Consequently, the activity of IgG-specific ENGases 1 Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA. 2Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY, USA. 3Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. 4Structural Glycoinmunology Laboratory, Biobizkaia Health Research Institute, Barakaldo, Bizkaia, Spain. 5Ikerbasque, Basque Foundation for Science, Bilbao, Spain. 6Structural Glycobiology Laboratory, Department of Structural and Molecular Biology; Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona Science Park, c/Baldiri Reixac 48, Tower R, Barcelona, Catalonia, Spain. 7Present address: Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, Australia. 8Present address: Structural Biochemistry Unit, National Institute of Dental and Craniofacial Research (NIDCR)/National Institute of Health, e-mail: ; Bethesda, MD, USA. Nature Communications | (2025)16:6147 1 Article suppresses the immune response and, thus, some pathogenic bacteria secrete these IgG-specific ENGases to evade the host immune response, enhancing bacterial survival and virulence8. We previously defined the structural determinants governing N-glycan specificities of the streptococcal multi-domain IgG-specific ENGases EndoS, which hydrolyzes only biantennary complex-type N-glycans, and EndoS2, which hydrolyzes complex-type, hybrid and high-mannose N-glycans9–11. We and others have also described the molecular mechanism of recognition and specificity by EndoS and EndoS2 for IgG antibodies, revealing how these enzymes exclusively process the N-glycan on the Fc region of IgG. These studies also highlighted the requirement of a non-enzymatic β-sandwich domain to create an anchor point on the IgG substrate by forming a proteinprotein interaction10–14. Corynebacterium pseudotuberculosis secretes CP40, a 40 kDa protein identified as a protective antigen against ovine caseous lymphadenitis that was erroneously described as a serine protease15 but subsequently characterized as an ENGase capable of hydrolyzing N-glycans on human IgG antibodies16. More recently, we identified a family of single-domain ENGases secreted by different pathogenic corynebacterial species that exhibit strict specificity for the N297linked glycan on human IgG antibodies, including CP40. We demonstrated potent in vivo efficacy of a prototypical member of this IgG-specific ENGase family, known as CU43, in mitigating diverse pathologies that rely on IgG-mediated effector functions, including the prevention of CD4 + T cell and B cell depletion, autoimmune hemolytic anemia, and antibody-dependent enhancement in severe dengue disease17. However, the precise molecular mechanism by which corynebacterial IgG-specific ENGases hydrolyze N-glycans exclusively on IgG antibody substrates is yet unknown. In this work, we unveil the catalytic mechanisms of the entire family of IgG-specific ENGases secreted by pathogenic corynebacterial species with strict specificity for deglycosylation of human IgG antibodies. We show that these ENGases can be further classified into subfamilies, each of which exhibits a distinct molecular mechanism of action. Additionally, we show that CU43 deglycosylates IgG antibodies through an inter-protomer mechanism by binding to one Fc protomer to hydrolyze the N297-linked glycan on the opposite Fc protomer, contrasting the intra-protomer deglycosylation mechanism employed by the Streptococcal IgG-specific ENGases. Finally, we demonstrate in a humanized mouse model of lethal influenza infection that IgG deglycosylation by CU43 preserves the protective neutralization capacity of an anti-flu broadly neutralizing antibody, unlike the IgG degrader therapeutic Efgartigimod. Our insights into the molecular mechanisms by which corynebacterial IgG-specific ENGases hydrolyze Fc glycans will guide their development as therapeutics for antibody-mediated diseases in humans. Results CU43 forms a complex with the Fc region with 1:1 stoichiometry We previously showed that the single-domain IgG-specific ENGase CU43 (Fig. 1a) is able to interact specifically with both protein and glycan components of the Fc region human IgG antibodies and that it could bind to aglycosylated IgG, indicative of a direct protein-protein interaction between CU43 and Fc17. In order to evaluate the formation of a protein complex between CU43 and Fc and its stoichiometry, we incubated catalytically-inactive CU43 (CU43i, (...truncated)