Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

Wang, Lai-Xi

doi:10.1016/j.carres.2008.03.025

Cited by 124 publications

(116 citation statements)

References 86 publications

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“…Furthermore, as NGTs are encoded on the genome of relevant pathogens, it will be interesting to study how the immune system of the hosts reacts to this type of N-glycosylation. Finally, NGTs represent a promising tool that complements established platforms for production of glycoconjugates and for site-specific modification of proteins (35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Schwarz

Fan

Schubert

et al. 2011

Journal of Biological Chemistry

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N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life. This process involves the transfer of a preassembled oligosaccharide from a lipid donor to asparagine side chains of polypeptides and is catalyzed by the membrane-bound oligosaccharyltransferase (OST). We characterized an alternative bacterial pathway wherein a cytoplasmic N-glycosyltransferase uses nucleotide-activated monosaccharides as donors to modify asparagine residues of peptides and proteins. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the NX(S/T) consensus sequence. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. The identification of an enzyme that integrates some of the features of OST in a cytoplasmic pathway defines a novel class of N-linked protein glycosylation found in pathogenic bacteria.N-Linked glycosylation is characterized by an N-glycosidic linkage between the side chain amide of an asparagine residue of proteins and an oligosaccharide. This type of glycosylation occurs in both prokaryotes and eukaryotes and requires the assembly of an oligosaccharide on a polyisoprenoid lipid by sequential addition of monosaccharides, catalyzed by cytosolic glycosyltransferase. The resulting lipid-linked oligosaccharide is translocated to the luminal side of the endoplasmic reticulum membrane or the plasma membrane of prokaryotes, where it may be further elongated. The glycan is then transferred to the ␦-amino group of asparagine residues within the consensus sequence NX(S/T) of polypeptides. This reaction is catalyzed by oligosaccharyltransferase (OST), 4 a membrane-bound enzyme that can be composed of several different subunits (1). As oligosaccharide transfer takes place in the endoplasmic reticulum or in the periplasm, N-glycosylation affects proteins trafficking along the secretory pathway.N-Glycosylation exhibits important physiological functions. In the early secretory pathway of eukaryotes, N-glycans present on newly synthesized proteins orchestrate the folding of glycoproteins and act as a signal for directing misfolded polypeptides to degradation (2). After being processed in the Golgi organelle, N-linked glycans are relevant, among other processes, for the modulation of the immune system and for the control of immune cell homeostasis and inflammation (3, 4). The importance of this protein modification is supported by its incidence: more than half of all eukaryotic proteins are glycosylated (5).About 8 years ago, a study uncovered that the extracellular HMW1A adhesin of the Gram-negative bacterium Haemophilus influenzae is modified with hexose monosaccharides on asparagine residues (6). The pioneering work of St Geme and co-workers (7, 8) subsequently showed that the modified asparagine residues are found within the same consensus sequence recognized by OST (i.e. NX(S/T)) and that the enzyme responsible for this modification is the HMW1C p...

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Section: Discussionmentioning

confidence: 99%

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Schwarz

Fan

Schubert

et al. 2011

Journal of Biological Chemistry

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“…Bacterial biosynthesis of cellulosefor so-called reverse hydrolysis or hydrolasecatalyzed transglycosylation in low water concentration reactions (Wang, 2008). Xyloglucan endotransglycosylases (EC 2.4.1.207) catalyze both the cleavage and religation of polysaccharides and are enzymes related to the production of cellulose in plants (Steele et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

et al. 2009

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In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 1C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

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“…Endoglycosidases, including EndoS, have also been used extensively for in vitro glycan remodeling to modulate the properties of glycoproteins (24). Natural glycoproteins exist as mixtures of glycoforms, of which only one or a few typically exhibit maximal activity.…”

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confidence: 99%

Crystal structure ofStreptococcus pyogenesEndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

Trastoy

Lomino

Pierce

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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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Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

Cited by 124 publications

References 86 publications

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

Crystal structure ofStreptococcus pyogenesEndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

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