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Laixi Wang - One of the best experts on this subject based on the ideXlab platform.
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Generation and Comparative Kinetic Analysis of New Glycosynthase Mutants from Streptococcus pyogenes Endoglycosidases for Antibody Glycoengineering.
Biochemistry, 2018Co-Authors: Xin Tong, Laixi WangAbstract:Chemoenzymatic glycan remodeling by Endoglycosidase-catalyzed deglycosylation and reglycosylation is emerging as an attractive approach for producing homogeneous glycoforms of antibodies, and the success of this approach depends on the discovery of efficient Endoglycosidases and their glycosynthase mutants. We report in this paper a systematic site-directed mutagenesis of an Endoglycosidase from Streptococcus pyogenes (Endo-S) at the critical Asp-233 (D233) site and evaluation of the hydrolysis and transglycosylation activities of the resulting mutants. We found that in addition to the previously identified D233A and D233Q mutants of Endo-S, most of the Asp-233 mutants discovered here were also glycosynthases that demonstrated glycosylation activity using glycan oxazoline as the donor substrate with diminished hydrolytic activity. The glycosynthase activity of the resultant mutants varied significantly depending on the nature of the amino acid substituents. Among them, the D233M mutant was identified as the most efficient glycosynthase variant with the highest transglycosylation/hydrolysis ratio, which is similar to the recently reported D184M mutant of Endo-S2, another S. pyogenes Endoglycosidase. Kinetic studies of the D233M and D233A mutants of Endo-S, as well as glycosynthase mutants D184M and D184A of Endo-S2, indicated that the enhanced catalytic efficacy of the Asp-to-Met mutants of both enzymes was mainly due to an increased turnover number (increased kcat) for the glycan oxazoline substrate and the significantly enhanced substrate affinity (as judged by the reduced KM value) for the antibody acceptor.
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Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S
Nature communications, 2018Co-Authors: Beatriz Trastoy, Laixi Wang, Erik H. Klontz, Jared Orwenyo, Alberto Marina, Eric J. Sundberg, Marcelo E. GuerinAbstract: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.
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Site-specific immobilization of Endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies
Carbohydrate research, 2018Co-Authors: David N. Quan, William E. Bentley, Laixi WangAbstract:Chemoenzymatic glycan remodeling of antibodies using an Endoglycosidase and its mutant is emerging as an attractive approach for producing homogeneous antibody glycoforms. We report in this paper a site-specific covalent immobilization of the Endoglycosidases (Endo-S2 and its glycosynthase mutant D184M) using a recombinant microbial transglutaminase (MTG) and evaluation of the immobilized enzymes in deglycosylation and glycosylation of a therapeutic antibody. The site-specific covalent immobilization was achieved by introduction of a Q-tag at the C-terminus of the recombinant enzymes followed by conjugation of the enzymes to a primary amine-containing solid support through MTG-catalyzed transglutamination. Using rituximab as a model system, we found that the Endo-S2 wild-type and D184M glycosynthase mutant immobilized by this approach were efficient in the two step antibody glycan remodeling to generate homogeneous antibody glycoforms. Notably using the covalently immobilized enzymes can efficiently avoid the need of intermediate purification and eliminate the residual contamination of wild type enzyme for product hydrolysis, thus streamlining the chemoenzymatic Fc glycan remodeling of antibodies.
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Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S
Nature Publishing Group, 2018Co-Authors: Beatriz Trastoy, Laixi Wang, Erik H. Klontz, Jared Orwenyo, Alberto Marina, Eric J. Sundberg, Marcelo E. GuerinAbstract:Endoglycosidase S only recognizes one particular type of glycan within IgG antibodies but the molecular basis for this high specificity is not fully understood. Here, the authors present the crystal structure of product-bound Endoglycosidase S, revealing the determinants for its glycan specificity
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Endoglycosidases for the Synthesis of Polysaccharides and Glycoconjugates.
Advances in carbohydrate chemistry and biochemistry, 2016Co-Authors: Laixi WangAbstract:Abstract Recent advances in glycobiology have implicated essential roles of oligosaccharides and glycoconjugates in many important biological recognition processes, including intracellular signaling, cell adhesion, cell differentiation, cancer progression, host–pathogen interactions, and immune responses. A detailed understanding of the biological functions, as well as the development of carbohydrate-based therapeutics, often requires structurally well-defined oligosaccharides and glycoconjugates, which are usually difficult to isolate in pure form from natural sources. To meet with this urgent need, chemical and chemoenzymatic synthesis has become increasingly important as the major means to provide homogeneous compounds for functional glycocomics studies and for drug/vaccine development. Chemoenzymatic synthesis, an approach that combines chemical synthesis and enzymatic manipulations, is often the method of choice for constructing complex oligosaccharides and glycoconjugates that are otherwise difficult to achieve by purely chemical synthesis. Among these, Endoglycosidases, a class of glycosidases that hydrolyze internal glycosidic bonds in glycoconjugates and polysaccharides, are emerging as a very attractive class of enzymes for synthetic purposes, due to their transglycosylation activity and their capability of transferring oligosaccharide units en bloc in a single step, in contrast to the limitation of monosaccharide transfers by common glycosyltransferases. In this chapter, we provide an overview on the application of Endoglycosidases for the synthesis of complex carbohydrates, including oligosaccharides, polysaccharides, glycoproteins, glycolipids, proteoglycans, and other biologically relevant polysaccharides. The scope, limitation, and future directions of Endoglycosidase-catalyzed synthesis are discussed.
Ron Orlando - One of the best experts on this subject based on the ideXlab platform.
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On-target Endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.
Rapid communications in mass spectrometry : RCM, 2001Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:The digestion of glycopeptides with Endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the Endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 °C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The Endoglycosidases used for digestion were Endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger. Copyright © 2001 John Wiley & Sons, Ltd.
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on target Endoglycosidase digestion matrix assisted laser desorption ionization mass spectrometry of glycopeptides
Rapid Communications in Mass Spectrometry, 2001Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:The digestion of glycopeptides with Endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the Endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 °C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The Endoglycosidases used for digestion were Endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger. Copyright © 2001 John Wiley & Sons, Ltd.
Beatriz Trastoy - One of the best experts on this subject based on the ideXlab platform.
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Structural insights into the mechanisms and specificities of IgG-active Endoglycosidases.
Glycobiology, 2019Co-Authors: Erik H. Klontz, Beatriz Trastoy, Marcelo E. Guerin, Eric J. SundbergAbstract:The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are Endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active Endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to Endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.
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Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S
Nature communications, 2018Co-Authors: Beatriz Trastoy, Laixi Wang, Erik H. Klontz, Jared Orwenyo, Alberto Marina, Eric J. Sundberg, Marcelo E. GuerinAbstract: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.
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Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S
Nature Publishing Group, 2018Co-Authors: Beatriz Trastoy, Laixi Wang, Erik H. Klontz, Jared Orwenyo, Alberto Marina, Eric J. Sundberg, Marcelo E. GuerinAbstract:Endoglycosidase S only recognizes one particular type of glycan within IgG antibodies but the molecular basis for this high specificity is not fully understood. Here, the authors present the crystal structure of product-bound Endoglycosidase S, revealing the determinants for its glycan specificity
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Crystal structure of EndoS, an immunomodulatory Endoglycosidase specific for human IgG antibodies
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Beatriz TrastoyAbstract:In order to evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large Endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel Endoglycosidases could be engineered for additional clinical and biotechnological applications.
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crystal structure of streptococcus pyogenes endos an immunomodulatory Endoglycosidase specific for human igg antibodies
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Beatriz Trastoy, Joseph V Lomino, Brian G Pierce, L G Carter, Sebastian Gunther, John P Giddens, Greg A Snyder, Thomas M Weiss, Zhiping Weng, Laixi WangAbstract:To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large Endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel Endoglycosidases could be engineered for additional clinical and biotechnological applications.
Jennifer Colangelo - One of the best experts on this subject based on the ideXlab platform.
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On-target Endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.
Rapid communications in mass spectrometry : RCM, 2001Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:The digestion of glycopeptides with Endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the Endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 °C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The Endoglycosidases used for digestion were Endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger. Copyright © 2001 John Wiley & Sons, Ltd.
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on target Endoglycosidase digestion matrix assisted laser desorption ionization mass spectrometry of glycopeptides
Rapid Communications in Mass Spectrometry, 2001Co-Authors: Jennifer Colangelo, Ron OrlandoAbstract:The digestion of glycopeptides with Endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the Endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 °C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The Endoglycosidases used for digestion were Endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger. Copyright © 2001 John Wiley & Sons, Ltd.
John P Giddens - One of the best experts on this subject based on the ideXlab platform.
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endo f3 glycosynthase mutants enable chemoenzymatic synthesis of core fucosylated triantennary complex type glycopeptides and glycoproteins
Journal of Biological Chemistry, 2016Co-Authors: Joseph V Lomino, John P Giddens, Laixi Wang, Mohammed N AminAbstract:Abstract Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well-defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica Endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward [alpha]1,6-fucosyl-GlcNAc-Asn or [alpha]1,6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi- and tri-antennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported Endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize tri-antennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly the new tri-antennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first Endoglycosidase-based glycosynthases capable of transferring tri-antennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.
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crystal structure of streptococcus pyogenes endos an immunomodulatory Endoglycosidase specific for human igg antibodies
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Beatriz Trastoy, Joseph V Lomino, Brian G Pierce, L G Carter, Sebastian Gunther, John P Giddens, Greg A Snyder, Thomas M Weiss, Zhiping Weng, Laixi WangAbstract:To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large Endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel Endoglycosidases could be engineered for additional clinical and biotechnological applications.