Hydrolysis of Complex-Type N-Glycans
CD BioGlyco has abundant experience in Antibody Deglycosylation. We expect to become customers' research assistants in the hydrolysis of complex-type N-glycan.
N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases.
EndoS2 glycosynthase mutants from S. pyogenes are key tools to glycoengineer immunotherapeutic IgG monoclonal antibodies. 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. Because of the broad N-glycan specificity of EndoS2, this enzyme can hydrolyze, and as a glycosynthase mutant to generate a more diverse set of N-glycans on antibodies.
CD BioGlyco provides an endoglycosidase that specifically hydrolyzes N-linked glycans in the Fc region of native IgG. The endoglycosidase has excellent specificity and only hydrolyzes glycans from the IgG Fc domain when other glycosylated proteins are present in the system.
The endoglycosidase can be used to rapidly deglycosylate antibodies to reduce sample complexity, or to inactivate Fc-mediated effector functions. However, it has limited activity against high mannose and hybrid glycans. Please click here if you have needs for Hydrolysis of All Fc-Glycans in IgGs.
Our team of experts begins each project with a thorough consultation to understand your specific research goals, the nature of your antibody, and the desired final product. This ensures a customized approach that is perfectly aligned with your project requirements.
Upon receipt of your antibody sample, we perform an initial quality assessment. Our team also assists with or performs sample preparation steps, such as buffer exchange or concentration, to ensure optimal conditions for the deglycosylation reaction.
A proprietary enzymatic cocktail is carefully applied under controlled conditions to achieve selective and efficient hydrolysis of complex N-glycans. Reaction parameters such as time, temperature, and pH are meticulously optimized for each specific antibody to maximize glycan removal while protecting the protein.
Following the enzymatic reaction, the antibody is purified to remove residual enzymes, cleaved glycans, and other reaction byproducts. This crucial step ensures the final product is clean and ready for immediate use in subsequent experiments or applications.
The final deglycosylated antibody is rigorously analyzed using advanced analytical techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS), to confirm complete glycan removal. A comprehensive report detailing the entire process, including raw data and a certificate of analysis, is provided.
Journal: Nature Communications
IF: 14.7
Published: 2018
Results: This study uncovers the structural basis for Endoglycosidase S (EndoS)'s specificity for biantennary complex-type N-linked glycans on IgG Fc regions. The crystal structure of catalytically inactive EndoSD233A/E235L bound to its G2 oligosaccharide product reveals six domains, with the glycoside hydrolase domain featuring two asymmetric grooves that accommodate glycan antennae. Key loops define the binding site, with the N-3HB domain stabilizing the enzyme. Mutational analysis shows critical roles for loops interacting with antenna 2, while those for antenna 1 are dispensable. Structural comparisons with other GH18 endoglycosidases (e.g., EndoF3) highlight distinct loop architectures underpinning glycan specificity—EndoS's unique grooves restrict it to biantennary glycans. These findings enable engineering endoglycosidases for tailored monoclonal antibody synthesis.
Fig.1 Schematic representation of EndoS hydrolytic activity and glycosynthase activity of EndoS mutant. (Trastoy, et al., 2018)
CD BioGlyco constantly updates the technology platform to provide customers with comprehensive and high-quality services for hydrolysis of complex-type N-glycan. We will continue to raise the standard to meet customers' research needs. To learn more, please don't hesitate to contact us to learn how we can help you with your project.
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