Henry Metzger

The Receptor with High Affinity for IgE

The cDNAs for each of the three types of polypeptide that form the high affinity IgE receptor have been cloned and sequenced. Analysis of the predicted amino acid sequence and other data suggests that the four-chained structure (alpha beta gamma 2) contains seven transmembrane segments. The alpha chain resembles the immunoglobulin-binding chain found in other Fc receptors, but the beta and gamma chain sequences do not resemble other known proteins. (The one exception: the transmembrane segment of the gamma chains, which is homologous to the corresponding segment of the zeta chain of the CD3 complex found on T lymphocytes). Efficient expression of IgE binding by the rat receptor in COS cells was observed only when the coding sequences for each of the three chains were co-transfected. So far, only the cDNA for the human alpha chain has been successfully cloned. We attempted to express this chain by co-transfecting its cDNA with those for the rat beta and gamma chains. Surprisingly, co-transfection with the cDNA for the gamma chain was sufficient, although when the beta and gamma chains were both co-transfected, expression of alpha beta gamma oligomers was evident. Approaches being used to define by genetic manipulation the functional role of various parts of the receptor are discussed.

Structure and Function of γ Macroglobulins

Publisher Summary This chapter provides a critical evaluation of the various procedures employed to extract antigens and to characterize them chemically and physically. The various biological activities of the histocompatibility antigens and their use in assay systems for antigen and antibody detection and quantitation are also described in the chapter. One of the most active areas of immunologic research involves the participation and interactions of various cell types in the immune response. The recognition of the requirement for cell interaction in the antibody response can make the problem complicated related to the interpretation of much experimental data. Excellent yields of purified rat γM macroglobulin are reported by Fisher and Canning using sucrose density gradients in the BIV zonal centrifuge. This method allows for large batch processing and may be particularly useful for the isolation of γM from small animal sources. A very wide range of sedimentation constants for each of the components is reported in studies of large numbers of macroglobulinemic sera (as opposed to isolated macroglobulins).

Effect of Antigen Binding on the Properties of Antibody

Publisher Summary This chapter describes the currently available data on the structure of intact immunoglobulins and their degradation products, the characteristics of antigen-antibody complexes, and the requiredments for antigen-induced antibody function in complement fixation and stimulation of immunoglobulin-bearing cells. The structural data indicate that immunoglobulins are organized into domains consisting of strongly interacting homology regions. The domains appear to interact relatively little. Similarly, the evidence is good that the Fab and Fc regions behave quite independently of each other. What lack of flexibility exists in the molecule is more likely secondary to steric inhibition of movement rather than strong interactions at domains of bonding. Thus, from structural considerations alone, it seems unlikely that antigens reacting with the combining site at the NH 2 -terminal end of the molecule could induce changes in the more distal regions.

Investigation of Early Events in FcεRI-Mediated Signaling Using a Detailed Mathematical Model

Aggregation of FcεRI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the β and γ subunits of FcεRI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving FcεRI, Lyn, Syk, and a bivalent ligand that aggregates FcεRI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the β- and γ-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the β ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the β ITAM will act as an inhibitor.

The Effect of Antigen on Antibodies: Recent Studies

In 1972–1973 I reviewed what was known about the effect of antigen on the properties of antibodies (Metzger, 1974). Several possibilities which had been suggested by various investigators were considered. According to the allosteric model, antigen by virtue of its chemical structure induces conformational changes distal to the combining site. These changes would in turn lead to changes in the molecule (receptor) which was activated by the antigen-antibody complex. According to the distortive model, the topological distribution of antigenic determinants leads to a distortion of a semirigid antibody molecule, resulting in effects similar to those postulated for the allosteric model. Finally a simple associative model of antigen action was considered. Here polymerization of the antibody by a multideterminant antigen would be the critical and perhaps sufficient role played by antigen. I concluded that “... the weight of evidence speaks against both the allosteric and distortive models for antigen action. We are left with the associative model. I know of no data that are inconsistent with this model ... and there is much evidence in favor of it.”

Studies with a monoclonal antibody to the β subunit of the receptor with high affinity for immunoglobulin E

The receptor with high affinity for IgE consists of a tetrameric complex of polypeptides, one of which (alpha), contains the binding site for IgE. The function of the other chains--a single beta and two disulfide-linked gamma chains--is unknown. We report the cloning of a murine hybridoma that secretes an IgG1 antibody which specifically reacts with the beta subunit. Studies with this monoclonal antibody show that the subunit stoichiometry of the receptor is unaffected by the presence or absence of bound IgE. We also found that under certain conditions where the alpha beta gamma 2 complex dissociates, beta remains attached to the dimer of gamma chains, indicating that these chains contact each other in the native receptor. In rat basophilic leukemia cells--a neoplastic line of mucosal-type mast cells--all of the beta subunits expressed by the cells appeared to be associated with the high affinity receptor. However, in at least one cell line which has no high affinity receptors--a putative rat lymphoma line--beta or beta-like polypeptides were also expressed.

Further characterization of the β-component of the receptor for immunoglobulin E

Abstract A 30,000 mol. wt component (β) is associated in a 1:1 ratio with the 50,000 mol. wt glycoprotein (a) which binds immunoglobulin E (IgE) on mast cells and related tumor cells. We show that α and β are associated in membrane preparations. This is consistent with previous results which showed labeling of β with the hydrophobic probe 5-iodonaphthyl-1-azide (INA). The β-polypeptide is susceptible to proteolytic cleavage during preparation of the membranes and when this occurs a 20,000 fragment can be labeled with INA and remains associated with α. No incorporation of carbohydrate precursors into β was observed. Since β is also not modified when cells are surface-labeled, it may not be exposed on the cell surface. Rigorous washing of IgE-receptor complexes with non-ionic detergent results in dissociation of β from the α-IgE complex. The latter will then not reassociate with β when exposed to crude detergent extracts of the tumor cells.

Association of the receptor for immunoglobulin E with an endogenous polypeptide on rat basophilic leukemia cells.

AbstractChemical crosslinking studies have been carried out to investigate the structural organization of the receptor for immunoglobulin E (IgE) on rat basophilic leukemia cells both before and after solubilization of the receptor with non-ionic detergent. A previously unidentified polypeptide with an apparent molecular weight of 30,000 -35,000 was found to crosslink to the IgE-binding polypeptide in a |:| stoichiometry when either whole cells or soluble cell extract were crosslinked at 4°C with dimethyl suberimidate or its cleavable analog, dimethyl 3,3′-dithiobis-propionimidate (DTBP). This polypeptide can be biosynthetically labeled with a mixture of 3H-amino acids but is not surface radioiodinated by the lactoperoxidase method. A biosynthetically labeled polypeptide of the same size copurifies with the uncrosslinked IgE-binding polypeptide to a variable extent, and this component is probably identical to that which crosslinks to the receptor.Bound IgE is crosslinked to the IgE-binding polypeptide by ...

Distribution and valency of receptor for IgE on rodent mast cells and related tumour cells

MONOMERIC IgE binds tightly to the surface of mast cells and basophils without, by itself, causing any known perturbation. Interaction of the IgE with an appropriate antigen (or experimentally with anti-IgE) causes the cells to degranulate. Little is known about the initial molecular events in IgE-mediated triggering, but there is considerable evidence that crosslinking of the IgE molecules is a critical step (see refs 1 and 2 for discussion). The available data suggest that only limited bridging rather than an aggregation-induced extensive redistribution of the surface IgE3,4 is required; indeed if the bridging is extensive enough to induce such gross redistribution within the time that degranulation would ordinarily be expected, the latter is inhibited3,5. Since bridging of the IgE, and therefore of the receptor to which it is bound, seems to be a critical signal, it is important to know if the cellular binding sites for IgE are integrated into larger functional units; the receptor molecules themselves might be multivalent, or individual receptors might be connected to each other by some other component (Fig. 1). The experiments described here were directed towards investigating this point. We saturated cells with a mixture of two distinguishable types of rat IgE and determined whether by redistributing one of them with specific antibody, the other would comigrate.

Kinetic proofreading models for cell signaling predict ways to escape kinetic proofreading

In the context of cell signaling, kinetic proofreading was introduced to explain how cells can discriminate among ligands based on a kinetic parameter, the ligand-receptor dissociation rate constant. In the kinetic proofreading model of cell signaling, responses occur only when a bound receptor undergoes a complete series of modifications. If the ligand dissociates prematurely, the receptor returns to its basal state and signaling is frustrated. We extend the model to deal with systems where aggregation of receptors is essential to signal transduction, and present a version of the model for systems where signaling depends on an extrinsic kinase. We also investigate the kinetics of signaling molecules, “messengers,” that are generated by aggregated receptors but do not remain associated with the receptor complex. We show that the extended model predicts modes of signaling that exhibit kinetic discrimination for some range of parameters but for other parameter values show little or no discrimination and thus escape kinetic proofreading. We compare model predictions with experimental data.