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Christopher W Cairo

Publications and source records attributed to Christopher W Cairo.

10 recordsLinked to original sources
PubMed2006

Mechanisms of Cellular Avidity Regulation in CD2-CD58-Mediated T Cell Adhesion.

The CD2 receptor on T lymphocytes is essential for T cell adhesion and stimulation by antigen presenting cells (APCs). Blockade of CD2 function is immunosuppressive in both model systems and humans, indicating the importance of CD2 for the cellular immune response. Although the affinity of the molecular interaction between CD2 and its counter-receptor, CD58, is relatively low when measured in solution, this interaction mediates tight adhesion within the 2D cell-cell interface. To understand the mechanisms responsible for regulating the avidity of the CD2-CD58 interaction, we measured the number, affinity, and lateral mobility of CD2 molecules on resting and activated T cells. Cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and the 2D affinity of CD2 for CD58 increased by 2.5-fold. The combination of T cell activation and CD2 ligation to CD58 decreased the laterally mobile fraction of the ligated CD2. Together, these changes would substantially enhance CD2 avidity and strengthen T cell-APC adhesion. The change in CD2 mobile fraction suggests that the cell uses cytoskeletal regulators to immobilize the receptor selectively at the site of contact with surfaces expressing CD58. Our observations are consistent with a model in which T cell activation initially induces increased CD2 2D affinity, cell surface receptor expression, and lateral mobility, allowing the CD2 molecules to diffuse to sites of contact with CD58-bearing APCs. Subsequently, T cell activation causes the CD58-bound CD2 to be recognized and immobilized at sites of cell-cell contact, thereby strengthening T cell-APC adhesion.

Antibody Affinity↗
PubMed2006

Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules.

We formulate a general analysis to determine the two-dimensional dissociation constant (2D Kd), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.

CD2 Antigens↗
PubMed2006

Cytoskeletal regulation couples LFA-1 conformational changes to receptor lateral mobility and clustering.

The alpha(L)beta(2) integrin (leukocyte function-associated antigen-1 [LFA-1]) is regulated to engage and maintain T cell adhesion. Conformational changes in the receptor are associated with changes in receptor-ligand affinity and are necessary for firm adhesion. Less well understood is the relationship between receptor conformation and the regulation of its lateral mobility. We have used fluorescence photobleaching recovery and single-particle tracking to measure the lateral mobility of specific conformations of LFA-1. These measurements show that different receptor conformations have distinct diffusion profiles and that these profiles vary according to the activation state of the cell. Notably, a high-affinity conformation of LFA-1 is mobile on resting cells but immobile on phorbol-12-myristate-13-acetate-activated cells. This activation-induced immobilization is prevented by a calpain inhibitor and by an allosteric LFA-1 inhibitor. Our results suggest that current models of LFA-1 regulation are incomplete and that LFA-1 confinement by cytoskeletal attachment regulates cell adhesion both negatively and positively.

Animals↗
PubMed2002

Influencing receptor-ligand binding mechanisms with multivalent ligand architecture.

Multivalent ligands can function as inhibitors or effectors of biological processes. Potent inhibitory activity can arise from the high functional affinities of multivalent ligand-receptor interactions. Effector functions, however, are influenced not only by apparent affinities but also by alternate factors, including the ability of a ligand to cluster receptors. Little is known about the molecular features of a multivalent ligand that determine whether it will function as an inhibitor or effector. We envisioned that, by altering multivalent ligand architecture, ligands with preferences for different binding mechanisms would be generated. To this end, a series of 28 ligands possessing structural diversity was synthesized. This series provides the means to explore the effects of ligand architecture on the inhibition and clustering of a model protein, the lectin concanavalin A (Con A). The structural parameters that were varied include scaffold shape, size, valency, and density of binding elements. We found that ligands with certain architectures are effective inhibitors, but others mediate receptor clustering. Specifically, high molecular weight, polydisperse polyvalent ligands are effective inhibitors of Con A binding, whereas linear oligomeric ligands generated by the ring-opening metathesis polymerization have structural properties that favor clustering. The shape of a multivalent ligand also influences specific aspects of receptor clustering. These include the rate at which the receptor is clustered, the number of receptors in the clusters, and the average interreceptor distance. Our results indicate that the architecture of a multivalent ligand is a key parameter in determining its activity as an inhibitor or effector. Diversity-oriented syntheses of multivalent ligands coupled with effective assays that can be used to compare the contributions of different binding parameters may afford ligands that function by specific mechanisms.

Chemical Precipitation↗
PubMed2002

Affinity-based inhibition of beta-amyloid toxicity.

Strategies for interfering with protein aggregation are important for elucidating and controlling the pathologies of amyloid diseases. We have previously identified compounds that block the cellular toxicity of the beta-amyloid peptide, but the relationship between their ability to inhibit toxicity and their affinity for A beta is unknown. To elucidate this relationship, we have developed an assay capable of measuring the affinities of small molecules for beta-amyloid peptide. Our approach employs immobilized beta-amyloid peptide at low density to minimize the problems that arise from variability in the beta-amyloid aggregation state. We found that low-molecular weight (MW of 700-1700) ligands for beta-amyloid can be identified readily by using surface plasmon resonance. The best of these bound effectively (K(d) approximately 40 microM) to beta-amyloid. The affinities measured for peptides in the SPR assay correspond to results from A beta cell toxicity assays. The most potent ligands for immobilized beta-amyloid are the most potent inhibitors of the neuronal cell toxicity of beta-amyloid. Compounds with dissociation constants above approximately 100 microM did not show significant activity in the cell toxicity assays. Our data support the hypothesis that ligands exhibiting greater affinity for the beta-amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity.

Amino Acid Sequence↗
PubMed2002

Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy.

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.

Amines↗
PubMed2002

Control of multivalent interactions by binding epitope density.

Receptor clustering by multivalent ligands can activate signaling pathways. In principle, multivalent ligand features can control clustering and the downstream signals that result, but the influence of ligand structure on these processes is incompletely understood. Using a series of synthetic polymers that vary systematically, we studied the influence of multivalent ligand binding epitope density on the clustering of a model receptor, concanavalin A (Con A). We analyze three aspects of receptor clustering: the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Our experiments reveal that the density of binding sites on a multivalent ligand strongly influences each of these parameters. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, however, are the most efficient on a binding epitope basis. Our results provide insight into the design of ligands for controlling receptor-receptor interactions and can be used to illuminate mechanisms by which natural multivalent displays function.

Concanavalin A↗
PubMed2002

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.

Cell Aggregation↗