Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

Yavuz, Elif; Maffioli, Carola; Ilg, Karin; Aebi, Markus; Priem, Bernard

doi:10.1007/s10719-010-9322-1

Cited by 16 publications

(6 citation statements)

References 36 publications

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“…In contrast, display of carbohydrate antigens on OMVs requires the coordinated expression of multiple heterologous glycosyltransferases for directing the synthesis of desired glycans onto bacterial lipid carriers that subsequently localize to the outer membrane and become constituents of released OMVs. Despite the challenges, several groups including ours have used glycoengineering as a tool to remodel the bacterial outer membrane with mammalian glycotopes of interest including ganglioside GM3 (Ilg et al, 2010), Lewis Y (Le Y ) antigen (Yavuz et al, 2011), and trimannosyl core N- glycan (Valderrama-Rincon et al, 2012). Presumably, expression of these different cell surface glycans in a hypervesiculating host strain would yield uniquely glycosylated OMVs, although this remains to be shown.…”

Section: Discussionmentioning

confidence: 99%

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

Valentine

Chen

Perregaux

et al. 2016

Cell Chemical Biology

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Summary The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

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Section: Discussionmentioning

confidence: 99%

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

Valentine

Chen

Perregaux

et al. 2016

Cell Chemical Biology

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“…Compared to the smaller glycans constructed above, these structures could provide greater specificity in a variety of applications including the targeting and inhibition of galectins, siglecs, and lectins on human and pathogenic cells 44,46,57,58 as well as the adjuvanting of vaccines by installing Lewis-X glycan structures that bind DC-SIGN receptors on dendritic cells 62 . While some combinations of these GTs have been used to create free oligosaccharides or glycolipids 37–40,63–65 , the products resulting from interactions between their specificities have not been systematically studied in the context of a protein substrate. We used GlycoPRIME to test all pairwise combinations of these five GTs, expressing each of them in separate CFPS reactions and then mixing two of those crude lysates in equal volumes with CFPS reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 2 µM NmLgtB.…”

Section: Resultsmentioning

confidence: 99%

“…By elaborating this glucose residue, we generated a diverse library of therapeutically relevant glycosylation motifs from the bottom-up in vitro. Of the 23 unique glycosylation motifs for which biosynthetic pathways were discovered in this work, several have been synthesized as free 37–40,63,64 or lipid-linked 37,38 oligosaccharides or by remodeling existing glycoproteins 6,30,42 ; however, to our knowledge, only glucose 16,22,28 , dextran 16 , lactose 28 , LacNAc 65 , and polysialyllactose 28 have been previously produced as glycoprotein conjugates in bacterial systems. The 18 synthetic glycosylation pathways leading to novel glycan motifs on proteins discovered in this work represent the largest addition made by any single bacterial glycoengineering study to date.…”

Section: Discussionmentioning

confidence: 99%

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

et al. 2019

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Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

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“…Poly‐LacNAc can be modified by a combination of sialyl and fucosyl residues resulting in Lewis and sialyl‐Lewis X antigens. Lewis X trisaccharide can be synthesized in metabolically engineered E. coli to produce soluble oligosaccharides or modified LOS surfaces (Dumon et al ., ; ; Yavuz et al ., ). One major limitation of this technique is the appearance of unwanted glycoforms like fucose linked to Glc.…”

Section: Discussionmentioning

confidence: 97%

Glycoengineering of host mimicking type‐2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces

Mally

Fontana

LeibundGut‐Landmann

et al. 2012

Molecular Microbiology

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Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galb1-4(Fuca1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Galb1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The polyLacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.

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Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

Cited by 16 publications

References 36 publications

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

Glycoengineering of host mimicking type‐2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces

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