Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcεRIα.

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (FcεRI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human Cε3 domains comprise the binding sites for FcεRIα and crystal structure determination has shown that amino acids in four sites contribute to the high affinity of the interaction. The role of homologous residues within canine IgE-Fc, i.e. amino acids located at Cε2-Cε3 interface (residues 332-337), loop BC (residues 362-365), loop DE (residues 393-396), and loop FG (residues 424-427) in canine Cε3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of ß-hexosaminidase from RBL cells transfected with canine or human FcεRIα and (ii) the affinity of the mutants for the soluble extracellular domain of the α-chain expressed in Pichia pastoris were determined by Surface Plasmon Resonance (SPR). Kinetic analysis supports the observed effects of IgE mutations on stimulus secretion coupling. Potential applications of this study, leading to the generation of an IgE variant with a disabled FcεRIα binding site, are discussed.

The role of Cε2, Cε3, and Cε4 domains in human and canine IgE and their contribution to FcεRIα interaction.

The Cε2 and Cε4 domains are considered as scaffolds, allowing Cε3 domains to assume an appropriate orientation to interact with FcεRI (Wurzburg and Jardetzky, 2002; Hunter et al., 2008). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of Cε2 and Cε4 domains to bind to and induce target cell degranulation via FcεRIα. Our results indicate that for (1) Cε3 domains in IgE of canine and human origin are the only necessary region for binding to FcεRIα. (2) The interaction of canine IgE with human sFcεRIα is significantly enhanced by contributions from both Cε2 and Cε4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine Cε2 and Cε4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the Cε3 domain to facilitate stable interaction with canine FcRIα. Kinetic constants for the binding of this chimera to the soluble extracellular domain of the receptor indicate an approximate 120-fold decrease in the affinity for canine sFcεRIα (ka=5.30 × 10(2)M(-1)s(-1)) and a 330-fold increase in the dissociation from canine sFcεRIα (KD=6.9 × 10(-6)M(-1)), compared to the wild type IgE kinetic constants (Ka=6.30 × 10(4)M(-1)s(-1); KD=2.1 × 10(-8)M(-1)). Although canine IgE does engage human FcεRIα, canine Cε2 and Cε4 do not contribute to the high-affinity of interaction with human FcεRIα. Upon replacement of human Cε2 and Cε4 domain by the canine homologues, human IgE Cε3 only retains a low affinity for the human receptor, which shows that Cε2 and Cε4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor.

Fc receptor-γ subunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells.

The high-affinity IgE receptor (FcɛRI) is formed by the IgE-binding α subunit, ß subunit and γ subunits homodimer. All three subunits are required for proper expression of the receptor on the plasma membrane of mast cells and basophils. However, the exact molecular mechanism of inter-subunit interactions required for correct expression and function of the FcɛRI complex remains to be identified. A recent study suggested that polar aspartate at position 194 within the transmembrane domain of the α subunit could interact by hydrogen bonding with polar threonine at position 22 in the transmembrane domains of the γ subunits. To verify this, we used previously isolated rat basophilic leukemia (RBL)-2H3 variant cells deficient in the expression of the FcɛRI-γ subunit (FcR-γ), and transfected them with DNA vectors coding for FcR-γ of the wild-type or mutants in which T22 was substituted for nonpolar alanine (T22A mutant) or polar serine (T22S mutant). Analysis of the transfectants showed that both T22A and T22S mutants were capable to restore surface expression of the FcɛRI similar to wild-type FcR-γ. Furthermore, cells transfected with wild-type, T22A or T22S FcR-γ showed comparably enhanced FcɛRI-mediated degranulation. Our data indicate that substitution of FcR-γ T22 with non-polar amino acid does not interfere with surface expression of the FcɛRI and its signaling capacity.

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A-B-loop mimetics of the Cε3 domain of human IgE.

The disruption of the human immunolobulin E-high affinity receptor I (IgE-FcεRI) protein-protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A-B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC(50) ∼660 µM) is displayed by the peptide containing a 2,2'-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A-B loop epitope in IgE.

A proteomics approach to the study of the molecular consequence of IgE-mediated cell signalling in RBL-2H3.1 cells and 2D reference map preparation for the RBL-2H3.1 cell line.

A highly reproducible 2D (two-dimensional) map for the proteome and a pattern of protein phosphorylation of high secretory variant of RBL-2H3 cells (RBL-2H3.1) (a model cell in allergy studies) in resting and treated cells with IgE or IgE+Ag are presented. Major molecular changes were seen in the proteome of 3 h-activated cells with IgE+Ag, especially for proteins of ∼17 kDa compared with the control. We have identified 13 proteins on 11 corresponding spots as up-regulated proteins in response to IgE+Ag activation. Also, protein identification on 55 spots with MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) and ESI-MS (electrospray ionization mass spectrometry) resulted in a reliable 2D reference map and an opportunity for the subsequent use of a 1 min-activated cell map for a phosphoproteomics study.

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction.

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.

Proteomic identification and characterization of secreted N-glycosylated NPC2 following cross-linking of the high-affinity receptor for IgE on mast cells.

Allergen-mediated cross-linking of the high-affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL-2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol-binding glycoprotein, NPC2/RE1 (Niemann-Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE-mediated stimulus and co-localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD-2-related lipid-recognition) protein family (155 members), which includes the Toll-like receptor co-factors, MD-1 and MD-2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.

Characterization of an early signaling defect following Fc epsilonRI activation in the canine mastocytoma cell line, C2.

A comparison of IgE recognition by cognate receptors expressed on the C2 canine mastocytoma cell line with analogous events in a rat basophilic leukemia cell line transfected with the alpha-chain subunit of the canine high-affinity IgE receptor using flow cytometry show that canine IgE recognizes the alpha-chain of its cognate receptor on both cell lines. Our study confirms the expression of functional IgE receptors in both cell lines, but receptor-mediated signaling in the C2 line only supports the early stages of downstream signaling as shown by the phosphorylation of the gamma-chain and the failure to effect the phosphorylation of Syk. In contrast RBL-2H3 cells respond to sensitization with IgE and challenge with cognate antigen with tyrosine phosphorylation of the gamma-subunits of the receptor complex followed by downstream phosphorylation of Syk and Ca(2+) mobilization, culminating in beta-hexosaminidase release. We propose that the identification of the precise signaling defect in C2 cells will yield useful information regarding the pathway leading to mast cell exocytosis and facilitate the restoration of the complete signaling cascade through complementation of the missing/defective signal transducer since signaling events downstream of Ca(2+) mobilization are intact as demonstrated by beta-hexosaminidase release following non-immunologic stimulation with the calcium ionophore, A23187.

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha.

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.

The importance of Lys-352 of human immunoglobulin E in FcepsilonRII/CD23 recognition.

The interaction of immunoglobulin E (IgE) with its low affinity receptor (FcepsilonRII/CD23) plays a central role in the initiation and regulation of type I hypersensitivity responses. We have previously identified the importance of amino acid residues in the A-B loop of the Cepsilon3 domain of human IgE and implicated a region close to the glycosylation site at asparagine 371 as contributing to IgE-CD23 interaction. These residues were now targeted by site-directed mutagenesis. The IgE-CD23 interaction was assessed by semiquantitative flow cytometry. Replacement of the entire Cepsilon3 A-B loop (residues 341-356) with the homologous rat IgE sequence resulted in complete loss of human CD23 recognition, as did replacement of residues 346-353, indicating that class-specific effector residue(s) are contained within these eight amino acids. Lysine 352 within the A-B loop was identified as contributing directly to human CD23 interaction. Mutation to the rodent homologue glycine or glutamate resulted in a significant reduction in binding compared with native IgE, whereas conservative substitution with arginine effected a small, but statistically significant, enhancement of CD23 binding. Mutation of the Cepsilon3 glycosylation site at asparagine 371 to threonine or glutamine did not significantly affect CD23 recognition. Our results yield new insights into the structural basis of the hIgE-CD23 interaction and hold promise for the rational design of drugs that can manipulate IgE-mediated regulation of the allergic response.

Solid-phase synthesis of an A-B loop mimetic of the Cepsilon3 domain of human IgE: macrocyclization by Sonogashira coupling.

The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B loop of the Cepsilon3 domain of human immunoglobulin E has been carried out. The key macrocyclization step to form the 65-membered ring is achieved in approximately 15% yield via an "on-resin" Sonogashira coupling reaction which concomitantly installs a diphenylacetylene amino acid conformational constraint within the loop.