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Identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands.

Coombs PJ, Harrison R, Pemberton S, Quintero-Martinez A, Parry S, Haslam SM, Dell A, Taylor ME, Drickamer K - J. Mol. Biol. (2009)

Bottom Line: First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity.Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans.Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London SW7 2AZ, UK.

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Determination of the affinity of DC-SIGN for glycopeptide and glycoprotein ligands. (a) and (b) Binding of monomeric CRD to immobilized SBA and glycopeptide derived from SBA. Data were fit to simple first-order binding curves with a linear increase in nonspecific background binding to derive dissociation constants. (c) Binding of the glycopeptide from SBA to immobilized CRD from DC-SIGN. KD was derived from fitting as in the previous experiments. (d) Binding competition assays in which immobilized CRD from DC-SIGN was probed with 125I-labeled mannose-BSA in the presence of competing ligands. Data were fit to simple first-order competition curves to derive KI values.
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fig2: Determination of the affinity of DC-SIGN for glycopeptide and glycoprotein ligands. (a) and (b) Binding of monomeric CRD to immobilized SBA and glycopeptide derived from SBA. Data were fit to simple first-order binding curves with a linear increase in nonspecific background binding to derive dissociation constants. (c) Binding of the glycopeptide from SBA to immobilized CRD from DC-SIGN. KD was derived from fitting as in the previous experiments. (d) Binding competition assays in which immobilized CRD from DC-SIGN was probed with 125I-labeled mannose-BSA in the presence of competing ligands. Data were fit to simple first-order competition curves to derive KI values.

Mentions: In initial studies, DC-SIGN was used to demonstrate the importance of some potential factors in enhancing glycoprotein binding to a CRD and to validate appropriate assay formats. The availability of a highly soluble, monomeric CRD from DC-SIGN and the fact that high-mannose oligosaccharides bind to this receptor with affinities that fall within the range that can be determined using surface plasmon resonance provided an opportunity to measure the interaction directly. Making use of the fact that each subunit of soybean agglutinin (SBA) bears a single, uniform Man9GlcNAc2 oligosaccharide,20 N-hydroxysuccinimide chemistry was used to prepare surfaces coated with the intact glycoprotein and with the glycopeptide isolated from it, in order to assess the effect of glycan presentation on a protein backbone (Fig. 1d). Effects of valency were eliminated by analyzing the interaction with monomeric CRDs. Because of the rapid rates of association and dissociation, interactions were measured under steady-state conditions and analyzed by fitting the data to a simple saturation binding model (Fig. 2a and b). The results suggest that presentation of the oligosaccharide on a glycoprotein results in a roughly 15-fold enhancement of the binding affinity. However, the data for the glycopeptide binding experiment fall mostly below the apparent KD obtained from fitting the curve, because of limits on the protein concentration that could be achieved. A more definitive result was obtained by immobilizing the CRD, because higher concentrations of glycopeptide could be achieved in the fluid phase (Fig. 2c). The measured KD of 76 μM suggests that the enhancement resulting from presentation of the Man9GlcNAc2 oligosaccharide on a protein scaffold is closer to 20-fold.


Identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands.

Coombs PJ, Harrison R, Pemberton S, Quintero-Martinez A, Parry S, Haslam SM, Dell A, Taylor ME, Drickamer K - J. Mol. Biol. (2009)

Determination of the affinity of DC-SIGN for glycopeptide and glycoprotein ligands. (a) and (b) Binding of monomeric CRD to immobilized SBA and glycopeptide derived from SBA. Data were fit to simple first-order binding curves with a linear increase in nonspecific background binding to derive dissociation constants. (c) Binding of the glycopeptide from SBA to immobilized CRD from DC-SIGN. KD was derived from fitting as in the previous experiments. (d) Binding competition assays in which immobilized CRD from DC-SIGN was probed with 125I-labeled mannose-BSA in the presence of competing ligands. Data were fit to simple first-order competition curves to derive KI values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2824085&req=5

fig2: Determination of the affinity of DC-SIGN for glycopeptide and glycoprotein ligands. (a) and (b) Binding of monomeric CRD to immobilized SBA and glycopeptide derived from SBA. Data were fit to simple first-order binding curves with a linear increase in nonspecific background binding to derive dissociation constants. (c) Binding of the glycopeptide from SBA to immobilized CRD from DC-SIGN. KD was derived from fitting as in the previous experiments. (d) Binding competition assays in which immobilized CRD from DC-SIGN was probed with 125I-labeled mannose-BSA in the presence of competing ligands. Data were fit to simple first-order competition curves to derive KI values.
Mentions: In initial studies, DC-SIGN was used to demonstrate the importance of some potential factors in enhancing glycoprotein binding to a CRD and to validate appropriate assay formats. The availability of a highly soluble, monomeric CRD from DC-SIGN and the fact that high-mannose oligosaccharides bind to this receptor with affinities that fall within the range that can be determined using surface plasmon resonance provided an opportunity to measure the interaction directly. Making use of the fact that each subunit of soybean agglutinin (SBA) bears a single, uniform Man9GlcNAc2 oligosaccharide,20 N-hydroxysuccinimide chemistry was used to prepare surfaces coated with the intact glycoprotein and with the glycopeptide isolated from it, in order to assess the effect of glycan presentation on a protein backbone (Fig. 1d). Effects of valency were eliminated by analyzing the interaction with monomeric CRDs. Because of the rapid rates of association and dissociation, interactions were measured under steady-state conditions and analyzed by fitting the data to a simple saturation binding model (Fig. 2a and b). The results suggest that presentation of the oligosaccharide on a glycoprotein results in a roughly 15-fold enhancement of the binding affinity. However, the data for the glycopeptide binding experiment fall mostly below the apparent KD obtained from fitting the curve, because of limits on the protein concentration that could be achieved. A more definitive result was obtained by immobilizing the CRD, because higher concentrations of glycopeptide could be achieved in the fluid phase (Fig. 2c). The measured KD of 76 μM suggests that the enhancement resulting from presentation of the Man9GlcNAc2 oligosaccharide on a protein scaffold is closer to 20-fold.

Bottom Line: First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity.Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans.Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London SW7 2AZ, UK.

Show MeSH
Related in: MedlinePlus