Prof. Marcelo E. Guerin
Structural Glycobiology Lab - The Basque Country - Spain
Therapeutic immunoglobulin G (IgG) antibodies are a prominent and expanding class of drugs used for the treatment of several human disorders including cancer, autoimmunity, and infectious diseases. IgG antibodies are glycoproteins containing a conserved N-linked glycosylation site at residue Asn297 on each of the constant heavy chain 2 (CH2) domains of the fragment crystallizable (Fc) region. The presence and precise chemical structure of this N-linked glycan are critical for IgG function contributing both to Fc γ receptor binding and activation of the complement pathway. IgG antibodies including those produced for clinical use typically exist as mixtures of more than 20 glycoforms, which significantly impacts their efficacies, stabilities and the effector functions. To better control their therapeutic properties, the chemoenzymatic synthesis of homogeneously N-glycosylated antibodies has been developed. Central to this strategy is the use of enzymes with activity on the Asn297 carbohydrate of IgG. The bacteria Streptococcus pyogenes, one of the most common human pathogens, secretes two endoglycosidases, EndoS and EndoS2, which are remarkably specific for IgG. They are multi-domain enzymes that rely on a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) for activity. EndoS has a narrower specificity, recognizing only complex biantennary type N-glycans, while EndoS2 recognizes complex biantennary, hybrid and high-mannose type N-glycans. Together, they can remove the entire repertoire of glycoforms from Asn297, facilitating the bacteria to evade the immune system. Since their discovery, EndoS, EndoS2 and their corresponding glycosynthase mutants have been described in pathways to chemoenzymatically synthesize antibodies with homogenous glycosylation. To understand the molecular basis of N-glycan specificity, we first determined high-resolution X-ray structures of EndoS, both unbound and bound with its specific complex-type glycan (Trastoy et al. Nat. Commun. 9:1874, 2018). Then, we determined X-ray structures of EndoS2 in various forms, including the complexes with complex-type and high-mannose-type glycans, unveiling the structural basis of broad spectrum N-glycan specificity (Klontz and Trastoy et al., ACS Cent Sci. 5:524-538, 2019; Highlighted as F1000Prime article). Why EndoS and EndoS2 are antibody-specific endoglycosidases. We present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. We find that the Fc region is located at the cleft of the “V”-shaped structure of the multi-modular EndoS, between the glycoside hydrolase (GH) and the β-sandwich domains. The EndoS GH domain interacts with the N-glycan and the CH2 domain of the Fc region. Strikingly, the β-sandwich domain of EndoS confers specificity through protein-protein interaction with the Fc region. We show that EndoS- and EndoS2-mediated hydrolysis of the two glycans on Fc or IgG occurs sequentially, concomitant with dissociation of the enzyme:substrate complex after hydrolysis of the first glycan before re-associating to hydrolyze the second glycan (Trastoy et al., doi:10.21203/rs.3.rs-1774503/v1). Finally, we solved crystal structures of EndoBT-3987, a highly specific high mannose (HM) type glycoside hydrolase from Bacteroides thetaiotaomicron, in complex with its glycopeptide substrate and product. Thus, we established the mechanistic basis of substrate recognition and specificity of this HM-hydrolysing enzyme, and an important group of endoglycosidases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown (Trastoy et al., Nat Commun. 11:899, 2020; Highlighted as F1000Prime article). Our studies make important contributions to the understanding on how IgG-specific immunomodulatory endoglycosidases promote immune evasion by major human pathogenic bacteria. The growing understanding of this family of antibody-modifying enzymes will also certainly contribute to modify antibodies in novel ways, so as to unleash the full potential of antibody drugs.
Fecha del seminario: 22/03/2023 12:00
Lugar del seminario: IQFR
