Polyvalent binding to carbohydrates immobilized on an insoluble resin

Abstract

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate-protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate-protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound inf luence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate-protein interactions should be studied.Interactions between carbohydrates on the surface of one cell type and proteins on the surface of another cell type play critical roles in a wide variety of biochemical recognition processes (1, 2). However, the details of these interactions are poorly understood. Typically, receptor-ligand binding events are studied by making derivatives of the ligand and directly quantitating the affinity. Applying this approach to cell surface carbohydrates has been problematic because carbohydrates are notoriously difficult to synthesize; it is usually not feasible to make more than a small number of derivatives, and even that can take years (3). Moreover, it is difficult to measure the binding affinities by using direct methods because individual carbohydrates bind weakly (K d Ϸ 10 Ϫ3 M) to their protein receptors. Therefore, the relative affinities of carbohydrates are obtained from the concentrations of ligand required to inhibit some event or process-e.g., cell agglutination-that is caused by interactions between the protein receptor and carbohydrates presented on the cell surface. These inhibition assays have shown that many carbohydrate-binding proteins can bind a variety of different structures with similar affinities (4). The broad specificity makes it hard to evaluate which structural features are critical for recognition.Despite the low affinity and broad specificity of individual carbohydrate-protein interactions, carbohydrates function as very specific signals in a wide variety of cell-cell recognition events. Proteins involved...

Nonstatistical binding of a protein to clustered carbohydrates

“…Initial experiments addressed whether the mixed SAMs were an appropriate model system to investigate specific interactions between surface-bound carbohydrates and proteins. Previous studies on TentaGel beads had shown that BP lectin recognizes ligands 1a and 2a in the presence of other structurally similar ligands, suggesting a high degree of specificity in the polyvalent interactions (6,7). Mixed SAMs presenting 1c (Fig.…”

“…Carbohydrate-protein binding events usually involve several simultaneous contacts between carbohydrates that are clustered on cell surfaces and protein receptors that contain multiple carbohydrate-binding sites. Numerous studies on model systems have demonstrated that polyvalent displays of carbohydrates can lead to remarkably high binding avidities (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Because avidity and specificity are not necessarily correlated (6,7), however, polyvalency does not completely resolve the paradox of how marginally selective carbohydrate-binding interactions can produce highly specific responses.…”

“…Simplified model systems are usually used to study polyvalent carbohydrate-protein interactions (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). We recently demonstrated that carbohydrates presented on TentaGel beads exhibit polyvalent binding and may therefore be useful as model systems to investigate the interactions of carbohydrates on surfaces (6, 7).…”

The Mannose/N-Acetylgalactosamine-4-SO4Receptor Displays Greater Specificity for Multivalent than Monovalent Ligands

“…Investigators have previously observed greater specificity on the part of lectins for polyvalent ligands as compared with their monovalent counterparts (15)(16)(17)(18)(19)(20)(21)(22). The specificity for multivalent ligands has been attributed to a requirement for unique spacing patterns (15)(16)(17)(18), secondary protein-protein and protein-carbohydrate interactions (19,20), and amplification in the polyvalent state of small differences in the affinities between monovalent forms (21). One prediction of a model in which small differences in affinity of monovalent ligands are amplified in the polyvalent state is that the affinities obtained for Mu11 binding to immobilized S4GGnM-BSA and S3GGnM-BSA would differ but that the B max obtained at saturation would be the same.…”

Cationic Antimicrobial Peptides

“…Furthermore, the reactive centers are located at the terminus of the glycol spacers. These properties provide the opportunities for synthesizing combinatorial libraries using organic solvents followed by bioassays in aqueous media [147,148]. These resins have various size distributions ranging from TentaGel M NH 2 (diameter  10 m) to TentaGel MB NH 2 (diameter ~ 300 m) ( Table 9).…”