Laura E. Strong

Synthetic multivalent ligands in the exploration of cell-surface interactions

Processes such as cell-cell recognition and the initiation of signal transduction often depend on the formation of multiple receptor-ligand complexes at the cell surface. Synthetic multivalent ligands are unique probes of these complex cell-surface-binding events. Multivalent ligands can be used as inhibitors of receptor-ligand interactions or as activators of signal transduction pathways. Emerging from these complementary applications is insight into how cells exploit multivalent interactions to bind with increased avidity and specificity and how cell-surface receptor organization influences signaling and the cellular responses that result.

Synthesis of end-labeled multivalent ligands for exploring cell-surface-receptor–ligand interactions

BACKGROUND Ring-opening metathesis polymerization (ROMP) is a powerful synthetic method for generating unique materials. The functional group tolerance of ruthenium ROMP initiators allows the synthesis of a wide range of biologically active polymers. We generated multivalent ligands that inhibit cell surface L-selectin, a protein that mediates lymphocyte homing and leukocyte recruitment in inflammation. We hypothesized that these ligands function through specific, multivalent binding to L-selection. To examine this and to develop a general method for synthesizing multivalent materials with end-labels, we investigated functionalized enol ethers as capping agents in ruthenium-initiated ROMP. RESULTS We synthesized a bifunctional molecule that introduces a unique end group by terminating ruthenium-initiated ROMP reactions. This agent contains an enol ether at one end and a masked carboxylic acid at the other. We conjugated a fluorescein derivative to an end-capped neoglycopolymer that had previously been shown to inhibit L-selection function. We used fluorescence microscopy to visualize neoglycopolymer binding to cells displaying L-selectin. Our results suggest that the neoglycopolymers bind specifically to cell surface L-selectin through multivalent interactions. CONCLUSIONS Ruthenium-initiated ROMP can be used to generate biologically active, multivalent ligands terminated with a latent functional group. The functionalized polymers can be labeled with a variety of molecular tags, including fluorescent molecules, biotin, lipids or antibodies. The ability to conjugate reporter groups to ROMP polymers using this strategy has broad applications in the material and biological sciences.

Neoglycopolymer inhibitors of the selectins

Abstract The selectin class of proteins plays an important role in the inflammatory response. These proteins, which bind saccharide ligands, facilitate the recruitment of leukocytes to the inflamed endothelium. The ring-opening metathesis polymerization (ROMP) has been used to generate synthetic multidentate ligands, which display multiple copies of sulfated saccharide residues. By altering the structure of the appended saccharide residues, multivalent ligands that selectively target one member of the selectin family, P-selectin, were created. The biological activities of materials prepared from the same monomer unit varied, depending on the method of polymer preparation. This result suggests that polymers containing more repeat elements exhibit higher selectin inhibitory activities.

Tuning chemotactic responses with synthetic multivalent ligands

BACKGROUND Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses. Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency. How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined. The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function. Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response. RESULTS To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization. We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis. The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency. Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane. CONCLUSIONS Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria. This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both. Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses. Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses.

Cell Aggregation by Scaffolded Receptor Clusters

The aggregation of cells by lectins or antibodies is important for biotechnological and therapeutic applications. One strategy to augment the avidity and aggregating properties of these mediators is to maximize the number of their ligand binding sites. The valency of lectins and antibodies, however, is limited by their quaternary structures. To overcome this limitation, we explored the use of polymers generated by ring-opening metathesis polymerization (ROMP) as scaffolds to noncovalently assemble multiple copies of a lectin, the tetravalent protein concanavalin A (Con A). We demonstrate that complexes between Con A and multivalent scaffolds aggregate cells of a T cell leukemia line (Jurkat) more effectively than Con A alone. We anticipate that synthetic scaffolds will offer a new means of facilitating processes that rely on cell aggregation, such as pathogen clearance and immune recognition.

Chapter 29. Principles for multivalent ligand design

Publisher Summary This chapter focuses on principles for multivalent ligand design. Multivalent interactions control a wide variety of cellular processes including cell surface recognition events. Examples of specific cell–cell binding events can be found in diverse processes, such as inflammation, tumor metastasis, and fertilization. An understanding of the mechanistic principles that underlie multivalent binding events facilitates the generation of new classes of therapeutic agents and biomaterials. Synthetic multivalent ligands can be used to illuminate and exploit biological processes that benefit from multipoint contacts. The use of antibodies can provide information about the involvement of multivalent interactions in a particular process. Understanding the structural requirements for multivalent ligand activity requires a wider variety of multivalent ligands whose structure, including size and valency, can be controlled and tailored. The chapter focuses on the principles for designing synthetic multivalent ligands and the interplay between ligand structure and biological activity. An overview of size and three dimensional shape is presented and flexibility of the Scaffold is discussed. Incorporation of ancillary functional groups is elaborated and design of multivalent scaffolds is also analyzed.

Visualization of Single Multivalent Receptor–Ligand Complexes by Transmission Electron Microscopy

Multivalent ligands have the capacity to interact simultaneously with multiple receptors. The binding of a divalent ligand, for example, can bring two receptors together. Many natural and synthetic ligands are not divalent, however, but rather contain many possible receptor binding sites. The complexation of such multivalent ligands with multiple receptors may be important for their biological activities. Unfortunately, whether multiple receptors bind to a multi-valent ligand and how many associate in a complex often can only be inferred. The difficulties associated with investigating such molecular details are exacerbated by the lack of tools available to characterize these binding events. Previous efforts to study multivalent receptor ± ligand complexes have used light scattering, [6] fluorescence resonance energy transfer, [7±9] capillary electrophoresis, [10] or analytical ultracentrifugation. Transmission electron micros-copy (TEM) methods are complementary to these indirect measurements as they allow direct visualization of complexes. Another advantage of TEM experiments is that since single molecules or complexes can be viewed the amount of material needed is typically less than that required for other techniques. Prior applications of electron micros-copy to examine multiple receptors interacting with a ligand have focused on large complexes that can be imaged directly. Many important receptor ± ligand complexes, however, are too small or of insufficient density to be directly imaged in this way. Here we report a strategy that extends the range of receptor ± ligand complexes that can be imaged by TEM. We reasoned that a method that increased the contrast of receptors would allow visualization of receptors bound to a given ligand and facilitate the characterization of individual small receptor ± ligand complexes. Our solution employs colloidal gold particles as labels to monitor the receptor position in the presence of a ligand. Because of its density, colloidal gold allows high-contrast imaging by electron microscopy. This approach is routinely taken in immunohis-tochemical applications, in which cellular proteins are located by colloidal gold particles attached to antibodies. Proteins other than antibodies can be attached to colloidal gold, and streptavidin-conjugated gold particles of different sizes are readily available. Such particles are used commonly because CUO molecule and the Ar atoms of the model cages. We are examining these interactions carefully, especially in light of the recent report of the new stable argon molecule HArF. The potential energy surfaces (PES) along the bending coordinate are quite complicated for both the

Glycoprotein-inspired materials promote the proteolytic release of cell surface l-Selectin

The proteolytic release, or shedding, of a cell surface protein can serve a regulatory role; the process liberates a soluble form of the protein into circulation while downregulating its cell surface concentration. The characteristics that render a protein susceptible to proteolytic cleavage are not known. We hypothesized that the clustering of a protein at the cell surface might target it for proteolysis. To test this hypothesis, we synthesized molecules that display multiple copies of sulfated galactose residues, termed neoglycopolymers, that are designed to mimic natural ligands for the cell adhesion protein L-selectin. We found that treatment of human neutrophils with the neoglycopolymers resulted in a dose-dependent loss of L-selectin from the cell surface, while monovalent compounds and unsulfated neoglycopolymers had no effect. Because L-selectin is an important mediator in the inflammatory response, such compounds could lead to novel antiinflammatory drugs. Moreover, molecules that control receptor shedding can be used to alter cellular responsiveness to specific ligands or to promote responses at distal sites; consequently, these results have broad implications for regulating the location and presentation of important biomolecules.

Designed potent multivalent chemoattractants for Escherichia coli

Bacterial chemotactic responses are initiated when certain small molecules (i.e., carbohydrates, amino acids) interact with bacterial chemoreceptors. Although bacterial chemotaxis has been the subject of intense investigations, few have explored the influence of attractant structure on signal generation and chemotaxis. Previously, we found that polymers bearing multiple copies of galactose interact with the chemoreceptor Trg via the periplasmic binding protein glucose/galactose binding protein (GGBP). These synthetic multivalent ligands were potent agonists of Escherichia coli chemotaxis. Here, we report on the development of a second generation of multivalent attractants that possess increased chemotactic activities. Strikingly, the new ligands can alter bacterial behavior at concentrations 10-fold lower than those required with the original displays; thus, they are some of the most potent synthetic chemoattractants known. The potency depends on the number of galactose moieties attached to the oligomer backbone and the length of the linker tethering these carbohydrates. Our investigations reveal the plasticity of GGBP; it can bind and mediate responses to several carbohydrates and carbohydrate derivatives. These attributes of GGBP may underlie the ability of bacteria to sense a variety of ligands with relatively few receptors. Our results provide insight into the design and development of compounds that can modulate bacterial chemotaxis and pathogenicity.

Conserved Amplification of Chemotactic Responses through Chemoreceptor Interactions

Many bacteria concentrate their chemoreceptors at the cell poles. Chemoreceptor location is important in Escherichia coli, since chemosensory responses are sensitive to receptor proximity. It is not known, however, whether chemotaxis in other bacteria is similarly regulated. To investigate the importance of receptor-receptor interactions in other bacterial species, we synthesized saccharide-bearing multivalent ligands that are designed to cluster relevant chemoreceptors. As has been shown with E. coli, we demonstrate that the behaviors of Bacillus subtilis, Spirochaete aurantia, and Vibrio furnissii are sensitive to the valence of the chemoattractant. Moreover, in B. subtilis, chemotactic responses to serine were increased by pretreatment with saccharide-bearing multivalent ligands. This result indicates that, as in E. coli, signaling information is transferred among chemoreceptors in B. subtilis. These results suggest that interreceptor communication may be a general mechanism for modulating chemotactic responses in bacteria.