Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S

Beatriz Trastoy,Alberto Marina,Erik Klontz,Marcelo E Guerin,Jared Orwenyo,Lai-Xi Wang,Eric J Sundberg

doi:10.1038/s41467-018-04300-x

Abstract

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that specifically catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions. It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Here, we report the crystal structure of the full-length EndoS in complex with its oligosaccharide G2 product. The glycoside hydrolase domain contains two well-defined asymmetric grooves that accommodate the complex-type N-linked glycan antennae near the active site. Several loops shape the glycan binding site, thereby governing the strict substrate specificity of EndoS. Comparing the arrangement of these loops within EndoS and related endoglycosidases, reveals distinct-binding site architectures that correlate with the respective glycan specificities, providing a basis for the bioengineering of endoglycosidases to tailor the chemoenzymatic synthesis of monoclonal antibodies.

Highlights

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of immunoglobulin G (IgG) fragment crystallizable (Fc) regions
Endoglycosidase S (EndoS) secreted from Streptococcus pyogenes is a 108 kDa enzyme that catalyzes the hydrolysis of the β-1,4 linkage between the first two Nacetylglucosamine residues of the complex-type N-linked glycan located on N297 of the Fc region of IgG antibodies (Fig. 1)[15,16]
The crystal structure of the full-length catalytically inactive EndoSD233A/E235L in complex with G2 product was solved by molecular replacement methods (EndoSD233A/ E235L-G2 thereafter; Fig. 2; Supplementary Figs. 1 and 2; Supplementary Table 1 and Methods section)[18]

Summary

Introduction

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Endoglycosidase S (EndoS) secreted from Streptococcus pyogenes is a 108 kDa enzyme that catalyzes the hydrolysis of the β-1,4 linkage between the first two Nacetylglucosamine residues of the complex-type N-linked glycan located on N297 of the Fc region of IgG antibodies (Fig. 1)[15,16] This structural modification ablates the effector functions of the host IgG antibodies, markedly contributing to immune evasion by this bacterium[17] because (i) EndoS deglycosylates only IgG glycoforms and no other glycoproteins, and (ii) EndoS glycosynthase variants efficiently transfer predefined complex-type N-linked glycans to intact IgG, this endoglycosidase plays a central role in glycoengineering strategies to develop IgG antibodies with improved therapeutic potential[12,13,14,15,16]. X-ray crystallography, small-angle X-ray scattering (SAXS), site-directed mutagenesis, enzymatic activity, and computational methods are used to define the molecular basis of substrate specificity of EndoS, as well as that of other GH18 endoglycosidase family members

Methods

Results

Conclusion