Differentiation between Structurally Homologous Shiga 1 and Shiga 2 Toxins by Using Synthetic Glycoconjugates

Kale, Ramesh R.; McGannon, Colleen M.; Fuller-Schaefer, Cynthia; Hatch, Duane M.; Flagler, Michael J.; Gamage, Shantini D.; Weiss, Alison A.; Iyer, Suri S.

doi:10.1002/ange.200703680

Cited by 23 publications

(33 citation statements)

References 25 publications

(28 reference statements)

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“…3, inserts). As reported previously (43,44), Stx1 bound to Pk but Stx2a and Stx2c did not. Stx2b and Stx2d did not bind to Pk either (Fig.…”

Section: Resultssupporting

confidence: 86%

“…2). In previous studies, while Stx1 bound to an analogue displaying the native Pk-trisaccharide, Stx2 did not bind (43,44). Instead, Stx2 bound to a synthetic variant, NAc-Pk (GalNAc␣1-4Gal␤1-4Glc), which did not engage Stx1.…”

Section: Resultsmentioning

confidence: 90%

“…Shiga toxin-containing supernatants were produced as previously described (43,46). Toxin concentrations were determined by quantitative Western blot assays with mouse monoclonal antibody 11E10 to the Stx2 A subunit and a rabbit polyclonal antibody to Stx1.…”

Section: Methodsmentioning

confidence: 99%

“…2). Pk-trisaccharide and NAc-Pktrisaccharide analogs of Gb3 can differentiate between Stx1 and Stx2a (43). Di-and trisaccharides with and without an N-acetyl group on the terminal or penultimate galactose unit allow for understanding of the contributions of individual glycans in the trisaccharide.…”

mentioning

confidence: 99%

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Binding of Pk-Trisaccharide Analogs of Globotriaosylceramide to Shiga Toxin Variants

2013

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The two major forms of Shiga toxin, Stx1 and Stx2, use the glycolipid globotriaosylceramide (Gb3) as their cellular receptor. Stx1 primarily recognizes the Pk-trisaccharide portion and has three Pk binding sites per B monomer. The Stx2a subtype requires glycolipid residues in addition to Pk. We synthesized analogs of Pk to examine the binding preferences of Stx1 and Stx2 subtypes a to d. Furthermore, to determine how many binding sites must be engaged, the Pk analogues were conjugated to biotinylated mono-and biantennary platforms, allowing for the display of two to four Pk analogues per streptavidin molecule. Stx binding to Pk analogues immobilized on streptavidin-coated plates was assessed by enzyme-linked immunosorbent assay (ELISA). Stx1, but not the Stx2 subtypes, bound to native Pk. Stx2a and Stx2c bound to the Pk analog with a terminal GalNAc (NAc-Pk), while Stx1, Stx2b, and Stx2d did not bind to this analog. Interestingly, the purified Stx2d B subunit bound to NAc-Pk, suggesting that the A subunit of Stx2d interferes with binding. Disaccharide analogs (Gal␣1-4Gal, GalNAc␣1-4Gal, and Gal␣1-4GalNAc) did not support the binding of any of the Stx forms, indicating that the trisaccharide is necessary for binding. Studies with monoantennary and biantennary analogs and mixtures suggest that Stx1, Stx2a, and Stx2c need to engage at least three Pk analogues for effective binding. To our knowledge, this is the first study examining the minimum number of Pk analogs required for effective binding and the first report documenting the role of the A subunit in influencing Stx2 binding.

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“…3, inserts). As reported previously (43,44), Stx1 bound to Pk but Stx2a and Stx2c did not. Stx2b and Stx2d did not bind to Pk either (Fig.…”

Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Binding of Pk-Trisaccharide Analogs of Globotriaosylceramide to Shiga Toxin Variants

2013

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“…Epidemiological evidence suggests that E. coli strains expressing different subtypes of Stx2 display differences in virulence, including the likelihood of progression to HUS (15,27,36,50). Since different subtypes also display differences in receptor preference (9,14,21,26,27,43), we wanted to determine if any of the amino acid polymorphisms map to the Gb3 binding sites in the Stx2 B subunit. The structure of Stx2a bound to Gb3 has not been determined; however, the structure of a highly homologous form, Stx2e (GT3), with bound Gb3 has been determined (31).…”

Section: Resultsmentioning

confidence: 99%

Shiga Toxin Subtypes Display Dramatic Differences in Potency

et al. 2011

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Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

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Molecular Recognition Elements for Toxin and Pathogen Detection

2011

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Rapid, sensitive, and selective detection of infectious agents in food, water, the environment, and patient samples is critical for appropriate countermeasures, particularly in the event of a suspected outbreak. The biosensors should be inexpensive, portable, and easy to use, require minimal sample preparation and reagents, and should be able to detect emerging variants for wide acceptance. The vast number of biological threats, each with its own unique characteristics, as well as the stringent requirements of a biosensor, dictates a multidisciplinary approach to overcome the scientific and technological hurdles necessary to develop a "real-time" biosensor. While advances in several areas are required, one of the most critical components of a biosensor is the recognition molecule. Ultimately, the success of a biosensor is highly dependent on the specificity, sensitivity, reproducibility, and broad applicability of the recognition element. In this chapter, we review the state-of-the-art advancements in the molecular recognition of toxins and pathogens. We discuss different recognition molecules in detail with particular emphasis on the use of emerging recognition molecules that include antimicrobial peptides, carbohydrates, molecularly imprinted polymers, aptamers, and phage for detection of infectious agents. As this chapter indicates, there are advantages and limitations to each molecule. However, significant advances in the area of molecular recognition for toxins and pathogens are being made. As the field evolves, continued improvements in molecular recognition and other components of the biosensor such as transducer Chemosensors: Principles, Strategies, and Applications, First Edition. Edited by Binghe Wang and Eric V. Anslyn.

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Differentiation between Structurally Homologous Shiga 1 and Shiga 2 Toxins by Using Synthetic Glycoconjugates

Cited by 23 publications

References 25 publications

Binding of Pk-Trisaccharide Analogs of Globotriaosylceramide to Shiga Toxin Variants

Binding of Pk-Trisaccharide Analogs of Globotriaosylceramide to Shiga Toxin Variants

Shiga Toxin Subtypes Display Dramatic Differences in Potency

Molecular Recognition Elements for Toxin and Pathogen Detection

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