Metabolic labeling of fucosylated glycoproteins in Bacteroidales species

Besanceney-Webler, Christen; Jiang, Hao; Wang, Wei; Baughn, Anthony D.; Wu, Peng

doi:10.1016/j.bmcl.2011.05.038

Cited by 53 publications

(61 citation statements)

References 20 publications

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“…Indeed, reports of metabolic labeling of glycoproteins with azide-modified pseudaminic acid in C. jejuni (54) and with alkyne-bearing fucosylated sugars in Bacteroidales sp. (99) suggest that this glycoproteomic approach should transfer readily to these organisms. The ability to use a general metabolic precursor rather than a specific glycan-binding reagent will facilitate the detection and discovery of glycoproteins in a broad range of bacteria.…”

Section: Discussionmentioning

confidence: 99%

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Champasa

Longwell

Eldridge

et al. 2013

Molecular & Cellular Proteomics

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Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.

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Section: Discussionmentioning

confidence: 99%

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Champasa

Longwell

Eldridge

et al. 2013

Molecular & Cellular Proteomics

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“…Indeed, unnatural sugars have been incorporated into Haemophilus ducreyi [26] , Campylobacter jejuni [27] , Escherichia coli [28] , and Bacteroidales sp. [29] , suggesting that targeting strategies against these pathogens are possibilities.…”

Section: Discussionmentioning

confidence: 99%

Recruiting the Host's Immune System to Target Helicobacter pylori's Surface Glycans

2013

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Due to the increased prevalence of bacterial strains that are resistant to existing antibiotics, there is an urgent need for new antibacterial strategies. Bacterial glycans are an attractive target for new treatments, as they are frequently linked to pathogenesis and contain distinctive structures that are absent in humans. We set out to develop a novel targeting strategy based on surface glycans present on the gastric pathogen Helicobacter pylori (Hp). In this study, metabolic labeling of bacterial glycans with an azide-containing sugar allowed selective delivery of immune stimulants to azide-covered Hp. We established that Hp’s surface glycans are labeled by treatment with the metabolic substrate peracetylated N-azidoacetylglucosamine (Ac4GlcNAz). By contrast, mammalian cells treated with Ac4GlcNAz exhibit no incorporation of the chemical label within extracellular glycans. We further demonstrated that the Staudinger ligation between azides and phosphines proceeds under acidic conditions with only a small loss of efficiency. We then targeted azide-covered Hp with phosphines conjugated to the immune stimulant 2,4-dinitrophenyl (DNP), a compound capable of directing a host immune response against these cells. Finally, we report that immune effector cells catalyze selective damage in vitro to DNP-covered Hp in the presence of anti-DNP antibodies. The technology reported herein represents a novel strategy to target Hp based on its glycans.

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“…Pioneered by Reutter and Bertozzi, MOE offers a method for glycan detection. Labeling and detecting sialylated 8,11 , fucosylated 8,12–14 , and mucin-type O-linked glycans 8,15 in live cells and model organisms has become routine practice using this approach. Harnessing the glycan metabolic machinery to incorporate unnatural monosaccharide substrates to enable downstream imaging is a powerful application of this approach, however, two complications can be encountered (Figure 1).…”

Section: Complex Glycosylation: Diversity and Challengesmentioning

confidence: 99%

Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling

et al. 2017

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The study of cellular glycosylation presents many challenges due, in large part, to the non-template driven nature of glycan biosynthesis and their structural complexity. Chemoenzymatic glycan labeling (CEGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CEGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CEGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CEGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.

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Metabolic labeling of fucosylated glycoproteins in Bacteroidales species

Cited by 53 publications

References 20 publications

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Recruiting the Host's Immune System to Target Helicobacter pylori's Surface Glycans

Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling

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