An Endoglycosidase with Alternative Glycan Specificity Allows Broadened Glycoprotein Remodelling

Abstract

Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reveal that a bacterial endoglycosidase from Streptococcus pyogenes , EndoS, is complementary to other known endoglycosidases (EndoA, EndoH) used for current protein remodeling. It allows processing of complex-type N-linked glycans +/- core fucosylation but does not process oligomannose- or hybrid-type glycans. This biocatalytic activity now addresses previously refractory antibody glycoforms.

Crystal structure ofStreptococcus pyogenesEndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

“…We also generated ab initio models of the protein envelope, all of which exhibited an open shape, and we fit the EndoS crystal structure into the top-ranked model with a correlation coefficient of 0.87. (29), whereas EndoF 3 is specific for both biantennary and triantennary complex oligosaccharides (6); neither have activity on high mannose oligosaccharides. The basic structure of the (β/α) 8 barrel fold and the positions of the active site residues are nearly identical in these two enzymes (SI Appendix, Fig.…”

“…Therapeutically, EndoS is already showing great promise in animal models as a treatment for diverse autoimmune diseases that rely on autoantibodies (11,(16)(17)(18)(19)(20)(21)(22); fine-tuning the specificity and activity of EndoS will be an important aspect of its further development as a protein therapeutic for use in humans. Biotechnologically, EndoS is a unique glycoprotein-modifying enzyme with the capacity to both remove glycans from and, as a glycosynthase variant, attach glycans to antibodies (29,30); expanding the repertoire of homogeneous glycosylated antibodies that can be produced with newly designed EndoS variants will be critical for realizing the full potential of engineered antibodies. Our X-ray crystal structure of EndoS provides a platform for future clinical, therapeutic, and biotechnological progress.…”

“…IgG monoclonal antibodies are used extensively as therapeutics and their activities, as mediated by effector functions, depend on the chemistry of their core glycans. EndoS deglycosylates antibody glycoforms that are refractory to processing by other endoglycosidases (29) and glycosynthase mutants of EndoS efficiently transfer predefined N-glycans to intact IgG (30). Together, these catalytic properties of EndoS enzymes allow for customization of IgG glycoforms that can enhance the therapeutic capacities of monoclonal antibodies.…”

A common glycan structure on immunoglobulin G for enhancement of effector functions

“…During this study, it was discovered that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimal structure that is able to enhance the activities of antibodies against cancer, influenza, and inflammatory diseases. (22) and later applied to antibody glycoengineering (25)(26)(27). The strategy starts with the use of exoglycosidases or endoglycosidases to cleave most of the N-glycans to form a homogeneous glycoform containing a well-defined glycan, followed by extension of the glycan using glycosyltransfer enzymes.…”

Therapeutic potential of deglycosylated antibodies

“…EndoS is an immune evasion factor from Streptococcus pyogenes, which functions by cleaving the glycans from the fragment crystallizable (Fc) domain of IgG molecules (2,3). EndoS is highly unusual in its lack of cross-reactivity to other glycoproteins and has a tight specificity for the biantennary glycans found on serum IgG (4). Hydrolysis of the Fc glycans by EndoS significantly reduces the binding to cellular receptors of the immune system to IgG (5).…”