Identification of Novel Contributions to High-affinity Glycoprotein–Receptor Interactions using Engineered Ligands

Abstract

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. 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. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.

Specificity analysis of the C-type lectin from rattlesnake venom, and its selectivity towards Gal- or GalNAc-terminated glycoproteins

“…Its reaction with asialotransferrin, which has two biantennary N-linked glycans, was only an order of magnitude greater than with the isolated glycopeptides, an increase similar to that previously found for glycoproteins vs their glycopeptides [5]. It also reacted minimally with C. jejuni glycoproteins that had only a few GalNAc-terminated glycans, further confirming the importance of the multivalency of the ligand.…”

“…These glycopeptides had similar affinities to the monosaccharides, indicating that bi-or tri-antennary structures do not form multiple attachments to individual RSVL molecules. The asialotransferrin was bound an order of magnitude more strongly than its glycopeptide, an effect that has been previously reported with other C-type lectins [5]. The two anomeric forms of p-nitrophenyl GalNAc had similar association constants, indicating that the lack of β-GalNAc compounds in the array results (Table 2) may be due to the scarcity of them in the ligand set rather than lower affinities for RSVL.…”

“…Their specificities arise from several causes, such as additional subsites near the main calcium one, steric exclusion, and the geometric arrangement of the carbohydrate recognition domains (CRDs) caused by ancillary domains [4]. They also show a greater affinity for glycoproteins than free glycans [5].…”

The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces

“…It is possible that GbpA binding to mucin depends on the glycan-protein linkage, as is not uncommon for other glycan binding proteins [35], or the binding avidity of GbpA for mucin that cannot be simulated on the glycan binding array [36].…”

Organization of the extracellular portion of the macrophage galactose receptor: A trimeric cluster of simple binding sites for N-acetylgalactosamine

“…Beyond the recognition of individual glycans through these mechanisms, higher order specificity is often achieved by placing multiple CRDs in groups, either as a string of CRDs in a single polypeptide or as a cluster of CRDs in an oligomer of receptor polypeptides (Dam and Brewer 2008; Coombs et al 2010). From the organization of the relatively few receptors that have been examined in molecular detail, two general paradigms for oligomer organization have emerged.…”