Kinetics of Protein-Nucleic Acid Interactions: Use of Salt Effects to Probe Mechanisms of Interactio

Abstract

Fc receptors (FcRs) are immunoglobulin-binding structures that enable antibodies to perform a variety of functions by forming connections between specific recognition and effector cells. Besides eliciting cytotoxicity, inducing secretion of mediators and endocytosis of opsonized particles, FcRs are involved in the regulation of antibody production, both as integral membrane proteins and as soluble molecules released from the cell surface. Most FcRs belong to the same family of proteins as their ligands (immunoglobulin superfamily). This review contains recent data obtained by use of monoclonal antibodies and cloning studies on FcRs and FcR-like molecules. The importance of fine specificity of receptor binding site(s) — that of the conformation of FcRs and their ligands in triggering signaling mechanisms — is analyzed. The regulatory function of membrane-bound and -released FcRs; the correlation between cell cycle, FcR expression, and release; as well as the possible mechanisms of these phenomena are discussed.     

The two binding-site models of human IgG binding Fc gamma receptors

Fc receptors (FcR) are immunoglobulin-binding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and with their ligands. Recent data on the structure of IgG binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity Fc gamma RI possess one active binding site specific for contact residues that is located at the N-proximal end of the CH2 domain and is able to mediate both binding and signal transfer. The low-affinity Fc gamma RIII has two active binding sites: the CH3 domain-specific site, which mediates only binding; and the CH2 domain-specific site, which is responsible for binding and signaling. Similarly, the low-affinity Fc gamma RII on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc gamma RII on B cells correlates with the cell cycle.     

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.     

Antibody against extracellular vaccinia virus (EV) protects mice through complement and Fc receptors

Protein-based subunit smallpox vaccines have shown their potential as effective alternatives to live virus vaccines in animal model challenge studies. We vaccinated mice with combinations of three different vaccinia virus (VACV) proteins (A33, B5, L1) and examined how the combined antibody responses to these proteins cooperate to effectively neutralize the extracellular virus (EV) infectious form of VACV. Antibodies against these targets were generated in the presence or absence of CpG adjuvant so that Th1-biased antibody responses could be compared to Th2-biased responses to the proteins with aluminum hydroxide alone, specifically with interest in looking at the ability of anti-B5 and anti-A33 polyclonal antibodies (pAb) to utilize complement-mediated neutralization in vitro. We found that neutralization of EV by anti-A33 or anti-B5 pAb can be enhanced in the presence of complement if Th1-biased antibody (IgG2a) is generated. Mechanistic differences found for complement-mediated neutralization showed that anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

Fc receptors and immunity to parasites

Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.     

Human antibody-Fc receptor interactions illuminated by crystal structures

Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies.     

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.     

Phage presentation

There has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins. Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non-proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies. The display of proteins or their functional domains provides a system for the analysis of structure-function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties. The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by-pass hybridoma technology in the generation of monoclonal antibodies.     

Glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration

Cell surface complex carbohydrates have emerged as key recognition molecules, mediating physiological interactions between cells. Typically, glycans on one cell surface are engaged by complementary carbohydrate binding proteins (lectins) on an apposing cell, initiating appropriate cellular responses. Although many cell surface lectins have been identified in vertebrates, only a few of their endogenous carbohydrate ligands have been established. Each major class of cell surface glycans-glycoproteins, glycolipids, and proteoglycans-has been implicated as physiologically relevant lectin ligands. The current minireview focuses on findings that implicate glycosphingolipids as especially important molecules in cell-cell recognition in two different systems: the recognition of human leukocytes by E-selectin on the vascular endothelium during inflammation and the recognition of nerve cell axons by myelin-associated glycoprotein in myelin-axon stabilization and the regulation of axon regeneration.     

NMR study on the major mite allergen Der f 2: its refined tertiary structure, epitopes for monoclonal antibodies and characteristics shared by ML protein group members

Group 2 major mite allergens Der f 2 and Der p 2 are classified into the recently identified group of MD-2-related lipid-recognition (ML) proteins, but the ligands and biological functions of these allergens are unknown. We have obtained a high-quality NMR structure for Der f 2, and found that it is more similar to the crystal structure of NPC2, a distant homologue, than to that of Der p 2, in terms of the separation and angle between the two major beta-sheets. This made us propose that ML proteins undergo clamshell-like motions that change the sizes of ligand-binding spaces inside their immunoglobulin-fold beta-sandwich to accommodate lipid molecules. This type of motion in lipopolysaccaride recognition of MD-2 is suggested to be likely as well by structural models. We also report the applicability of NMR differential exchange broadening experiments for complexes of intact monoclonal antibodies and antigens; using this technique, we have detected the conformational epitopes for monoclonal antibodies 15E11 and 13A4 as two separate surface patches.     

A family of highly diverse human and mouse genes structurally links leukocyte FcR,gp42 and PECAM-1

A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.     

Supramolecularity creates nonstandard protein ligands

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.     

Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension

DM-GRASP is an immunoglobulin superfamily cell adhesion molecule that is expressed in both the developing nervous and immune system. Specific populations of neurons respond to DM-GRASP substrates appears to require homophilic interactions between DM-GRASP molecules. We were interested in determining whether DM-GRASP interacts heterophilically with other ligands as well. We have found that eleven proteins from embryonic chick brain membranes consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion molecules were not detected in the eluate. The DM- GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreased neurite extension on laminin, suggesting that the antibody is not toxic or generally inhibiting motility. We present two possible models for the DM-GRASP-NgCAM association and a hypothesis for neural cell adhesion function that features the dimerization of cell adhesion molecules.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

Immunohistochemical Validation and Expression Profiling of NKG2D Ligands in a Wide Spectrum of Human Epithelial Neoplasms

The MHC class I-chain-related proteins (MICs) and the UL16-binding proteins (ULBPs) are inducible stress response molecules that work as activators of a specific receptor, NKG2D, which is expressed on effector cells, such as NK cells and subsets of T cells. In this study, we sought to explore the biological significance of NKG2D ligands in human neoplasms by comprehensively examining the immunohistochemical expression profile of NKG2D ligands in a variety of human epithelial neoplasms. Following careful validation of the immunohistochemical specificity and availability of anti-human ULBP antibodies for formalin-fixed paraffin-embedded (FFPE) materials, the expression of NKG2D ligands was analyzed in FFPE tissue microarrays comprising 22 types of epithelial neoplastic tissue with their non-neoplastic counterpart from various organs. Hierarchical cluster analysis demonstrated a positive relationship among ULBP2/6, ULBP3, ULBP1, and ULBP5, whose expression patterns were similar across all of the neoplastic tissues examined. In contrast, MICA/B, as well as ULBP4, did not appear to be related to any other ligand. These expression profiles of NKG2D ligands in human neoplasms based on well-validated specific antibodies, followed by hierarchical cluster analysis, should help to clarify some functional aspects of these molecules in cancer biology, and also provide a path to the development of novel tumor-type-specific treatment strategies.     

Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.