GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900MHz NMR analysis

Pukin, Aliaksei V.; Weijers, C.A.G.M.; Lagen, Barend van; Wechselberger, Rainer; Sun, Bin; Gilbert, Michel; Karwaski, Marie-France; Florack, D.E.A.; Jacobs, Bart C.; Tio‐Gillen, Anne P.; Belkum, Alex van; Endtz, Hubert P.; Visser, Gerben M.; Zuilhof, Han

doi:10.1016/j.carres.2008.01.007

Cited by 36 publications

(35 citation statements)

References 72 publications

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“…[10] IC 50 values were found to be four to five orders of magnitude lower than those for monovalent GM1 derivative GM1-1. [24] In the present study we found that these compounds are also active against the B-pentamer of E. coli heat-labile toxin (LTBh) which has identical GM1 binding sites to CTB. [25] Although there are subtle differences in the binding affinities of CTB and LTB isoforms for a range of ligands, [26] CTB and LTB are often used interchangeably when testing inhibitors.…”

Section: Resultsmentioning

confidence: 94%

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The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

et al. 2009

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Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregates.

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Section: Resultsmentioning

confidence: 94%

“…[32,[41][42][43] Experimental Section Sample preparation: Compounds GM1-1, GM1-2 and GM1-4 were prepared as reported previously. [10,24] Buffers and other reagents were bought from Sigma or Fisher unless stated otherwise.…”

Section: Resultsmentioning

confidence: 99%

The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

et al. 2009

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“…[13] The GM1os ligand was prepared by first synthesizing Bocprotected aminooxy alkyne 3 (Scheme 1) according to a reported procedure. [14] GM1 azide 2 was prepared using a chemo-enzymatic approach [15] before ligation to aminooxy alkyne 3 employing a copper-catalyzed alkyne azide cycloaddition (CuAAC) at room temperature with stirring for 48 hours. Attempts were made to use microwave-assisted CuAAC for the synthesis, [16] but proved unsuccessful in this case.…”

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confidence: 99%

A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

et al. 2014

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Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC 50 ) of 104 pm for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.

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“…CMP-Neu5Gc was synthesized from Neu5Gc and CTP using the recombinant CMP-Neu5Ac synthetase (construct NSY-05) from Neisseria meningitidis (40). 3GSLN was synthesized from CMP-Neu5Gc and N-acetyllactosamine using the recombinant Cst-I ␣-2,3-sialyltransferase (construct CST-06) from Campylobacter jejuni (41). The synthesized 3GSLN was purified by size exclusion chromatography on a Superdex peptide column (10 mm by 30 cm; GE Healthcare) using 20 mM NH 4 CO 3 as the eluent.…”

Section: Methodsmentioning

confidence: 99%

Crystallographic and Glycan Microarray Analysis of Human Polyomavirus 9 VP1 Identifies N -Glycolyl Neuraminic Acid as a Receptor Candidate

Khan

Liu

Neu

et al. 2014

J Virol

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Human polyomavirus 9 (HPyV9) is a closely related homologue of simian B-lymphotropic polyomavirus (LPyV). In order to define the architecture and receptor binding properties of HPyV9, we solved high-resolution crystal structures of its major capsid protein, VP1, in complex with three putative oligosaccharide receptors identified by glycan microarray screening. Comparison of the properties of HPyV9 VP1 with the known structure and glycan-binding properties of LPyV VP1 revealed that both viruses engage short sialylated oligosaccharides, but small yet important differences in specificity were detected. Surprisingly, HPyV9 VP1 preferentially binds sialyllactosamine compounds terminating in 5-N-glycolyl neuraminic acid (Neu5Gc) over those terminating in 5-N-acetyl neuraminic acid (Neu5Ac), whereas LPyV does not exhibit such a preference. The structural analysis demonstrated that HPyV9 makes specific contacts, via hydrogen bonds, with the extra hydroxyl group present in Neu5Gc. An equivalent hydrogen bond cannot be formed by LPyV VP1. IMPORTANCEThe most common sialic acid in humans is 5-N-acetyl neuraminic acid (Neu5Ac), but various modifications give rise to more than 50 different sialic acid variants that decorate the cell surface. Unlike most mammals, humans cannot synthesize the sialic acid variant 5-N-glycolyl neuraminic acid (Neu5Gc) due to a gene defect. Humans can, however, still acquire this compound from dietary sources. The role of Neu5Gc in receptor engagement and in defining viral tropism is only beginning to emerge, and structural analyses defining the differences in specificity for Neu5Ac and Neu5Gc are still rare. Using glycan microarray screening and high-resolution protein crystallography, we have examined the receptor specificity of a recently discovered human polyomavirus, HPyV9, and compared it to that of the closely related simian polyomavirus LPyV. Our study highlights critical differences in the specificities of both viruses, contributing to an enhanced understanding of the principles that underlie pathogen selectivity for modified sialic acids.

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GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900MHz NMR analysis

Cited by 36 publications

References 72 publications

The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

Crystallographic and Glycan Microarray Analysis of Human Polyomavirus 9 VP1 Identifies N -Glycolyl Neuraminic Acid as a Receptor Candidate

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