Factors Affecting Protein–Glycan Specificity: Effect of Spacers and Incubation Time

Lewallen, Daniel M.; Siler, David A.; Iyer, Suri S.

doi:10.1002/cbic.200900211

Cited by 36 publications

(43 citation statements)

References 14 publications

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“…Additionally, the linker used to attach the glycans to the array surface matrix can influence toxin binding [24], [41], [42]. Glycan 120 and glycan 119 share the identical glycan trisaccharide, but are attached with different linkers.…”

Section: Resultsmentioning

confidence: 99%

“…There are several explanations for the different apparent K d values obtained in different studies. ELISA has been shown to be more sensitive than SPR [16], [43], possibly because the longer incubation periods in the static ELISA allows the toxin to achieve optimal interacting conformation compared to the dynamic flow conditions of SPR [42]. Additionally, we incubated the plates at 4°C, while the SPR studies were done at room temperature.…”

Section: Resultsmentioning

confidence: 99%

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Shiga Toxin Binding to Glycolipids and Glycans

et al. 2012

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BackgroundImmunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable.MethodologyWe used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells.ResultsBy ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells.ConclusionsStx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED50) of protein synthesis was about 10−11 M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Shiga Toxin Binding to Glycolipids and Glycans

et al. 2012

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“…We and others have observed this phenomenon before, binding of glycans to their respective analytes are highly dependent on glycan density and presentation. 14,25 Exposure to H3N2 strain A/Aichi/2/1968 resulted in similar results (Figure 3C); however, all compounds bound to H1N1 A/Solomon Islands/3/2006, indicating that this virus strain is more accommodating in its binding preferences. (Figure 3B).…”

Section: Resultsmentioning

confidence: 62%

Glycan based Detection and Drug Susceptibility of Influenza Virus

et al. 2014

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We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (101 plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains.

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“…Syntheses of the scaffolds have been reported previously by our group. [18, 19],[20] Synthesis of the glycoconjugates containing Gal-α(1,4)-Gal and Gal-α(1,4)-GalNHAc oligosaccharide have been described below.…”

Section: Resultsmentioning

confidence: 99%

Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus

Yosief

Weiss

Iyer³

2013

ChemBioChem

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Biotinylated mono and bi- antennary di/trisaccharides were synthesized to evaluate their ability to capture Escherichia coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di/trisaccharides contain Galα1-4Gal, GalNHAcα1-4Gal, Galα1-4GalNHAc, Galα1-4Galβ1-4Glc and GalNHAcα1-4Galβ1-4Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin coated magnetic beads, incubated with different strains of live E. coli. Capturing ability was assessed using a luciferase assay which detects bacterial ATP. The trisaccharides containing Galα1-4Galβ1-4Glc and the disaccharides containing Galα1-4Gal as the epitopes exhibited strong capturing ability for uropathogenic E.coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E.coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharide containing GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the non-reducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.

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Factors Affecting Protein–Glycan Specificity: Effect of Spacers and Incubation Time

Cited by 36 publications

References 14 publications

Shiga Toxin Binding to Glycolipids and Glycans

Shiga Toxin Binding to Glycolipids and Glycans

Glycan based Detection and Drug Susceptibility of Influenza Virus

Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus

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