Endo-S Neb: Structural Insights, Enzymatic Properties, and Transformative Applications in Glycobiology

1. Concept

Endo-S Neb (Endo-β-N-acetylglucosaminidase from Streptococcus pyogenes Neb) is a specialized glycoside hydrolase belonging to glycoside hydrolase family GH85. It exhibits high substrate selectivity and catalytic efficiency, specifically cleaving the core chitobiose structure (GlcNAc-β-1,4-GlcNAc) of N-glycans—making it an indispensable tool in glycoprotein engineering, antibody glycan remodeling, and glycobiology research.

As an engineered variant of wild-type Endo-S, Endo-S Neb features enhanced catalytic activity and broader substrate adaptability, attributed to key mutations such as D233Q and D233H that improve its affinity for non-fucosylated glycans. Structurally, it comprises three core domains: a (β/α)₈ barrel-fold catalytic domain with conserved glutamate and aspartate residues, a substrate-binding domain that confers high specificity for core-fucosylated biantennary complex-type N-glycans (e.g., IgG Fc region glycans), and a flexible loop region that mediates substrate-induced conformational changes to regulate catalytic efficiency.

2. Research Frontiers

Recent research on Endo-S Neb has focused on advancing its enzymatic performance and expanding its application scope. A key frontier is the rational design of hyperactive variants through directed evolution and computational modeling, aiming to enhance catalytic efficiency, thermal stability, and substrate promiscuity. For example, structure-guided mutagenesis of the substrate-binding pocket and flexible loop region is being explored to enable cleavage of triantennary and tetraantennary complex-type N-glycans, addressing current substrate limitations.

Another emerging direction is the integration of Endo-S Neb into modular glycoengineering platforms. By combining it with glycosyltransferases (e.g., Fut8, β-1,4-galactosyltransferase) and other glycosidases, researchers can achieve precise remodeling of antibody glycans to produce homogeneous glycoforms with tailored effector functions (e.g., enhanced ADCC activity). Additionally, efforts are underway to optimize large-scale production processes of Endo-S Neb, including improving recombinant expression yields in E. coli and developing immobilized enzyme systems for industrial applications in biopharmaceutical manufacturing.

3. Research Significance

Endo-S Neb plays a pivotal role in advancing glycobiology and translational medicine. In antibody engineering, it enables precise manipulation of Fc glycosylation— a key determinant of IgG effector functions (ADCC, CDC). By removing or remodeling Fc glycans, researchers can dissect the impact of glycosylation on antibody activity, stability, and immunogenicity, facilitating the development of next-generation therapeutic antibodies with improved efficacy.

In biopharmaceutical development, Endo-S Neb addresses the critical challenge of glycan heterogeneity in recombinant glycoprotein drugs. It supports the production of homogeneous glycoforms, ensuring batch consistency and reducing immunogenicity risks—key factors for drug safety and regulatory approval. In glycomics research, it facilitates efficient N-glycan release from glycoproteins, enabling high-resolution mass spectrometry analysis to identify glycosylation sites and characterize glycan structures, advancing our understanding of glycosylation’s role in disease and biology.

4. Related Mechanisms, Research Methods, and Product Applications

4.1 Core Catalytic Mechanism

Endo-S Neb employs an acid-base catalytic mechanism to hydrolyze the GlcNAc-β-1,4-GlcNAc bond of N-glycans:

1. Proton Transfer: A glutamate residue in the active site acts as a proton donor, attacking the glycosidic oxygen to initiate cleavage.
2. Transition State Formation: The substrate’s sugar ring undergoes a chair-to-half-chair conformation change, forming an oxocarbenium ion intermediate.
3. Nucleophilic Attack: An aspartate residue activates a water molecule, which performs a nucleophilic attack on the intermediate, completing hydrolysis and releasing the cleaved glycan.

4.2 Enzymatic Properties

• Substrate Specificity: Primarily targets biantennary complex-type N-glycans on the IgG Fc region (core-fucosylated) and exhibits improved activity toward high-mannose and hybrid-type N-glycans compared to wild-type Endo-S.
• Optimal Reaction Conditions: Functions efficiently at pH 6.0–7.5 (slightly acidic to neutral) and 37°C; denatures above 50°C. Ca²⁺ enhances stability, while EDTA inhibits activity by chelating essential metal ions.

4.3 Key Applications

4.3.1 Antibody Glycoengineering

• Deglycosylation: Removes Fc glycans to study glycosylation’s impact on antibody function, such as binding affinity and effector activity.
• Glycan Remodeling: Combined with glycosyltransferases (e.g., Fut8) to produce homogeneous glycoforms (e.g., afucosylated antibodies) with enhanced ADCC activity— a critical improvement for cancer immunotherapy.

4.3.2 Glycoprotein Drug Development

• Therapeutic Antibody Optimization: Improves the efficacy, stability, and safety of recombinant antibodies by reducing glycan heterogeneity.
• Glycan-Based Vaccine Design: Modulates glycan structures of antigenic proteins to enhance immunogenicity, facilitating the development of more effective vaccines.

4.3.3 Glycomics Research

• Enables efficient release of N-glycans from complex biological samples (e.g., cell lysates, serum glycoproteins) for mass spectrometry analysis, supporting glycoproteomics studies and biomarker discovery.

4.4 Product Support from ANT BIO PTE. LTD.

ANT BIO PTE. LTD. offers a comprehensive portfolio of Endo-S Neb and complementary glycobiology tools through its UA sub-brand (specializing in recombinant proteins), tailored to meet diverse research and industrial needs:

• Recombinant Endo-S and Variants: Endo S (UA070039, $225) and Endo S2 (UA070055, $480), expressed in E. coli, providing high-purity enzymes for antibody glycoengineering and glycoprotein analysis.
• Complementary Glycosidases: Endo F1 (UA070121, $165), Endo H (UA070040, $60), and Endoproteinase Glu-C (UA070059, $130) for comprehensive N-glycan analysis and protein digestion workflows.
• Glycoprotein Standards: Recombinant Endoglin/CD105 proteins (e.g., UA010868, UA010906) with defined glycosylation patterns, enabling validation of Endo-S Neb cleavage efficiency and assay calibration.
• Custom Services: Recombinant protein expression and enzyme engineering services to develop tailored Endo-S Neb variants (e.g., hyperactive, thermostable) for specialized applications in biopharmaceutical production.

5. Brand Mission

ANT BIO PTE. LTD. is dedicated to empowering life science research and translational innovation through the provision of high-quality, reliable reagents and comprehensive solutions. As a leading provider of life science products, our portfolio spans antibodies, recombinant proteins, kits, and general reagents, with three specialized sub-brands catering to distinct research needs: Absin (general reagents and kits), Starter (antibodies), and UA (recombinant proteins). Our core mission is to accelerate scientific discovery, bridge the gap between basic research and clinical application, and contribute to the advancement of human health by delivering cutting-edge products, professional technical support, and exceptional customer service. We strive to be a trusted partner for researchers and biopharmaceutical developers worldwide, supporting their efforts to unravel glycobiology mysteries and develop innovative therapies.

6. Related Product List (With Product Codes)

7. AI Disclaimer

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.