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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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Binding competition assays comparing binding of a tri-antennary glycopeptide to MGL. Binding curves for monomeric CRD are shown in blue and curves for trimeric extracellular domain are shown in red. The KI values obtained from these experiments are summarized in Table 2.
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fig3: Binding competition assays comparing binding of a tri-antennary glycopeptide to MGL. Binding curves for monomeric CRD are shown in blue and curves for trimeric extracellular domain are shown in red. The KI values obtained from these experiments are summarized in Table 2.

Mentions: The effect of glycan branching on interaction with MGL was investigated by probing both monomeric CRDs and trimeric extracellular domains of MGL with a desialylated tri-antennary glycopeptide, isolated from fetuin, in the competition binding assay (Fig. 3). The relative affinity of the CRD for the glycopeptide compared to galactose is 21.5 (Table 1), which is 7.2-fold higher than the expected threefold enhancement that would be expected based solely on the presence of three terminal galactose residues. This roughly sevenfold enhanced affinity for branched glycans is somewhat like that observed for DC-SIGN (illustrated in Fig. 1b). Binding to the trimeric extracellular domain was enhanced a further 1.6-fold (Table 1), suggesting that binding of multiple terminal sugars by multiple CRDs in the extracellular domain (illustrated in Fig. 1f) makes only a small contribution to the overall affinity of the intact receptor for a branched glycan. The effect of branching on binding to MGL is consistent with previous results from testing this protein against a glycan array, because several of the ligands giving the highest signals for MGL are branched structures terminating in galactose,30 as well as quantitative binding assays with linear and branched sugar structures.31,32 However, the present results demonstrate that, as in the case of DC-SIGN, this enhancement results primarily from multiple interactions within a CRD and that only a limited further enhancement results from simultaneous interactions with multiple CRDs in a receptor oligomer.


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)

Binding competition assays comparing binding of a tri-antennary glycopeptide to MGL. Binding curves for monomeric CRD are shown in blue and curves for trimeric extracellular domain are shown in red. The KI values obtained from these experiments are summarized in Table 2.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Binding competition assays comparing binding of a tri-antennary glycopeptide to MGL. Binding curves for monomeric CRD are shown in blue and curves for trimeric extracellular domain are shown in red. The KI values obtained from these experiments are summarized in Table 2.
Mentions: The effect of glycan branching on interaction with MGL was investigated by probing both monomeric CRDs and trimeric extracellular domains of MGL with a desialylated tri-antennary glycopeptide, isolated from fetuin, in the competition binding assay (Fig. 3). The relative affinity of the CRD for the glycopeptide compared to galactose is 21.5 (Table 1), which is 7.2-fold higher than the expected threefold enhancement that would be expected based solely on the presence of three terminal galactose residues. This roughly sevenfold enhanced affinity for branched glycans is somewhat like that observed for DC-SIGN (illustrated in Fig. 1b). Binding to the trimeric extracellular domain was enhanced a further 1.6-fold (Table 1), suggesting that binding of multiple terminal sugars by multiple CRDs in the extracellular domain (illustrated in Fig. 1f) makes only a small contribution to the overall affinity of the intact receptor for a branched glycan. The effect of branching on binding to MGL is consistent with previous results from testing this protein against a glycan array, because several of the ligands giving the highest signals for MGL are branched structures terminating in galactose,30 as well as quantitative binding assays with linear and branched sugar structures.31,32 However, the present results demonstrate that, as in the case of DC-SIGN, this enhancement results primarily from multiple interactions within a CRD and that only a limited further enhancement results from simultaneous interactions with multiple CRDs in a receptor oligomer.

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