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 of this N-linked glycan is critical for IgG function contributing both to Fc γreceptor binding and activation of the complement pathway. The precise chemical structure of the N-linked glycan modulates the effector functions mediated by the Fc domain. 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.
Streptococcus pyogenes secretes two multidomain antibody-specific endoglycosidases, EndoS and EndoS2, which are central to these pathways. EndoS2 has a broader substrate specificity compared to EndoS, hydrolyzing not only biantennary complex type N-glycans, but also high-mannose, hybrid, and bisecting complex type N-glycans on IgG. Glycosynthase mutants of EndoS2 have also been developed to engineer antibodies with a more diverse set of N-glycans than similar EndoS mutants are capable of creating. In this work, we carried out a detailed study of the structure and function of EndoS and EndoS2, and elucidated the molecular mechanism by which EndoS2 recognizes an expanded repertoire of N-glycans compared to EndoS. Strikingly, this mechanism involves the action not only of the glycoside hydrolase domain, but also that of a carbohydrate binding module, which proved to be essential for IgG recognition and catalysis. These works certainly set the foundation for engineering enzymes to carry out customizable antibody glycosylation reactions for the diagnosis and treatment of human diseases.
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