Large‐Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered <i>Escherichia coli</i>

Antoine, T.; Priem, Bernard; Heyraud, A.; Greffe, Lionel; Gilbert, Michel; Wakarchuk, Warren W.; Lam, Joseph S.; Samain, E.

doi:10.1002/cbic.200200540

Cited by 81 publications

(55 citation statements)

References 31 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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“…One limitation is that bacterial strains will have to be engineered to produce the activated sugar donors not available in E. coli under standard growth conditions, for example, GDPfucose and CMP-sialic acid. However, strains producing these activated sugars have been constructed (34). A main focus of future work should be the thorough analysis and optimization of metabolic pathways and the expression and control of activity of glycosyltransferases originating from various species, in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Exploiting Bacterial Glycosylation Machineries for the Synthesis of a Lewis Antigen-containing Glycoprotein

Hug

Zheng

Reiz

et al. 2011

Journal of Biological Chemistry

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Background: Bacterial glycosylation systems appear to be ideal for glycoengineering purposes. Results: A Lewis x containing glycoprotein was synthesized using bacterial enzymes from four different species using in vivo and in vitro steps. Conclusion: Glycosylation systems in bacteria open new avenues for the production of engineered glycoproteins. Significance: Bacterial glycoengineering is a promising technology for the production of therapeutically valuable glycoproteins with expected high precision and low cost.

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“…It is not surprising that LgtD could act as a galactosyltransferase because it has already been shown that this enzyme can use UDP-Gal with a low efficiency with Gb3 as the acceptor [27]. In addition, there are several reports of GalNAc-transferases that have a side galactosyltransferase activity [28] and the blood groups A and B glycosyltransferases are known to differ only by a few amino acids [29]. What is quite remarkable in Gb5 production by the MR15 strain is the fact that LgtD specifically adds a galactosyl residue onto Gb4 but does not galactosylate Gb3 to produce the globotetraose analog Galb-3Gala-4Galb-4Glc.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of globopentaose using a novel β1,3‐galactosyltransferase activity of the Haemophilus influenzae β1,3‐N‐acetylgalactosaminyltransferase LgtD

Randriantsoa¹,

Drouillard²,

Breton³

et al. 2007

FEBS Letters

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We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding b1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87,[197][198][199][200][201][202][203]. Here, we found that LgtD has an additional b1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galb-3GalNAcb-3Gala-4Galb-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both b1,3-GalT and b1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.

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Large‐Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

Cited by 81 publications

References 31 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

Exploiting Bacterial Glycosylation Machineries for the Synthesis of a Lewis Antigen-containing Glycoprotein

Synthesis of globopentaose using a novel β1,3‐galactosyltransferase activity of the Haemophilus influenzae β1,3‐N‐acetylgalactosaminyltransferase LgtD

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