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

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.

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides

“…Most members able to hydrolyze HM-type N-glycans also hydrolyze Hy-type N-glycans 30 . The N-glycan specificity of the GH18 family members is due to unique interactions between the loops that decorate the β-barrel core of the enzymes and the N-glycan chemical structures 27,51 . EndoBT-3987, EndoF1, and EndoH, which are classified in the first group, show the capacity to hydrolyze HM-type but not CT-type N-glycans 27 .…”

“…In contrast, EndoS and EndoF3 are able to hydrolyze CT-type but not HM-type N-glycans. Binding of EndoS to CT-type N-glycans is predominantly driven by GH domain loops that make contacts with the α (1,3) antenna, a mechanism that is conserved in EndoS2 27,52 . In contrast, our experimental data clearly showed that the HMhydrolyzing enzymes preferentially recognize the α(1,6) antenna 1b of HM-type N-glycans.…”

“…2a-c). The Asn residue is completely exposed to the solvent, accounting for the ability of EndoBT-3987 to process HM-type glycans attached to a broad spectrum of proteins 7,26,27 . The O3 and O6 atoms of the first GlcNAc (+1) residue make hydrogen bonds with the side chains of Y315 and N379, respectively.…”

“…EndoBT-3987 is an endo-β-N-acetylglucosaminidase (ENGase) that specifically catalyzes the hydrolysis of the β1-4 linkage between the first two GlcNAc residues of the HM glycans 7,26,27 but it is not able to hydrolyze complex-type (CT-type) N-glycans 27 . ENGases are endoglycosidases that hydrolyze the chitobiose core of N-linked glycans (EC 3.2.1.96) [28][29][30] .…”

Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods

“…39.6, RMSD = 0.9, %ID = 32). Although the TIM barrel structure is quite similar, loops that link the structure which is known to modulate the selectivity of the enzymes has considerable versatility among ENGase enzymes [36]. The loops surrounding the active site forms a Y-shaped structure which interacts with the "plus" and "minus subsites of the substrate.…”