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

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.

Serum immunoglobulin and the threshold of Fc receptor-mediated immune activation.

Antibodies can mediate immune recruitment or clearance of immune complexes through the interaction of their Fc domain with cellular Fc receptors. Clustering of antibodies is a key step in generating sufficient avidity for efficacious receptor recognition. However, Fc receptors may be saturated with prevailing, endogenous serum immunoglobulin and this raises the threshold by which cellular receptors can be productively engaged. Here, we review the factors controlling serum IgG levels in both healthy and disease states, and discuss how the presence of endogenous IgG is encoded into the functional activation thresholds for low- and high-affinity Fc receptors. We discuss the circumstances where antibody engineering can help overcome these physiological limitations of therapeutic antibodies. Finally, we discuss how the pharmacological control of Fc receptor saturation by endogenous IgG is emerging as a feasible mechanism for the enhancement of antibody therapeutics.

Extensive substrate recognition by the streptococcal antibody-degrading enzymes IdeS and EndoS.

Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a "flipped-out" conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.