H E A L T H I N N O V A T I O N , O V E R C O M I N G D I S E A S E S A N D P A N D E M I C S

S C I E N T I F I C H I G H L I G H T S

3 4 H I G H L I G H T S 2 0 2 3 I

Cryo-EM unearths how a bacterial pathogen evades the human immune system

A combination of X-ray scattering, cryo- electron microscopy experiments and theoretical calculations have revealed the architecture of the complex between two key players of the mitogen- activated protein kinase signalling pathway, p38α, and its activating kinase, MKK6, which triggers inflammation during infection.

Major pathogenic bacteria produce proteins that target components of the immune system, contributing to immune evasion and successful colonisation and invasion of the human host [1]. Streptococcus pyogenes (group A Streptococcus) is a pathogenic bacterium that causes life- threatening conditions and post-infection immune-related diseases, in addition to mild skin and upper respiratory tract infections.

In order to evade the human immune system, S. pyogenes secretes a 108 kDa endoglycosidase, EndoS, that specifically hydrolyses core N-glycans on human immunoglobulin G (IgG) antibodies, dependent on the native IgG fold [2]. This activity contributes to increased survival of S. pyogenes in human blood ex vivo, on account of reduced IgG binding to Fc γ receptors (FcγRs) and impaired complement pathway activation. Injection of EndoS into mice results in the efficient removal of IgG- associated carbohydrate and, accordingly, effectively clears numerous autoimmune conditions in animal models, including rheumatoid arthritis and systemic lupus erythematosus. EndoS releases complex-type (CT) glycans linked to residue Asn297 of the human Fc region CH2 domain, while EndoS2, a similar enzyme from a different S. pyogenes strain, exhibits expanded glycan specificity in that it processes both CT and high- mannose (HM)-type carbohydrates from IgG [3,4]. EndoS and EndoS2 are powerful tools for the chemoenzymatic synthesis of therapeutic IgG monoclonal antibodies with homogenous glycoforms and drug conjugates [5]. EndoS and EndoS2 are the most prominent members of a rare class of carbohydrate-active enzymes that are specific to the protein component of their glycoprotein substrate, not just the glycan. However, no mechanism for protein- specificity of these or any other endoglycosidases have been described to date.

This work defines the mechanism by which EndoS and EndoS2 are IgG-specific (Figure 20). Cryo-electron microscopy (cryo-EM) data was collected at beamline CM01, enabling the determination of the structure of EndoS in complex with the IgG1 Fc fragment. It was found

Fig. 20: Overall structure of EndoS in complex with the Fc region. Schematic representation of Fc N-glycan processing by EndoS.