[Eukaryotic expression, purification of human IgECepsilon2-4 protein and its target fishing].

Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography. The interaction between high-affinity IgE receptor (FcepsilonR I) of KU812 cell surface and IgECepsilon2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECepsilon2-4 were captured from human serum by SPR technique. Results The recombinant plasmid containing the signal peptide III showed the highest expression (6.2 mg/L). Highly purified recombinant protein IgECepsilon2-4 was obtained by affinity purification. Immunofluorescence cytochemistry showed that the recombinant protein IgECepsilon2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECepsilon2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECepsilon2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECepsilon2-4 was likely to interact with 39 kinds of proteins in human serum.

Histone H3k9 and H3k27 Acetylation Regulates IL-4/STAT6-Mediated Igε Transcription in B Lymphocytes.

IL-4 activates STAT6 and causes the subsequent up-regulation of Ig heavy chain germline Igε via chromatin remodeling involved in B lymphocytes development. STAT6 acts as a molecular switch to regulate the higher-order chromatin remodeling via dynamically orchestrating co-activators (CBP/Tudor-SN) and co-repressors (HDAC1/PSF). Here, we demonstrated that STAT6/Tudor-SN/PSF form a complex, balancing the acetylation and deacetylation states to co-regulate IL-4/STAT6 gene transcription. In addition, we confirmed that IL-4 treatment increased the HATs activity in Ramos cells. As "active" markers, the expression of H3K9ac and H3K27ac increased after treatment with IL-4. However, transcriptional repressors such as H3K9me3 and H3K27me3 decreased in response to IL-4 stimulation. Moreover, IL-4 treatment enhanced H3 acetylation at the Igε promoter regions. Our results revealed that the Igε gene transcription is regulated by histone modifications in the IL-4/STAT6 pathway. The study will provide novel insights into the pathogenesis of allergic diseases.

TRIF signaling is essential for TLR4-driven IgE class switching.

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.

Control of the STAT6-BCL6 antagonism by SWAP-70 determines IgE production.

Asthma and allergies are major health concerns in which Ig isotype E plays a pivotal role. Ag-bound IgE drives mast cells and basophils into exocytosis, thereby promoting allergic and potentially anaphylactic reactions. The importance of tightly regulated IgE production is underscored by severe immunological conditions in humans with elevated IgE levels. Cytokines direct IgH class-switching to a particular isotype by initiation of germline transcription (GLT) from isotype-specific intronic (I) promoters. The switch to IgE depends on IL-4, which stimulates GLT of the Iε promoter, but is specifically and strongly impaired in Swap-70(-/-) mice. Although early events in IL-4 signal transduction (i.e., activation of the JAK/STAT6 pathway) do not require SWAP-70, SWAP-70 deficiency results in impaired Iε GLT. The affinity of STAT6 to chromatin is reduced in absence of SWAP-70. Chromatin immunoprecipitation revealed that SWAP-70 binds to Iε and is required for association of STAT6 with Iε. BCL6, known to antagonize STAT6 particularly at Iε, is increased on Iε in absence of SWAP-70. Other promoters bound by BCL6 and STAT6 were found unaffected. We conclude that SWAP-70 controls IgE production through regulation of the antagonistic STAT6 and BCL6 occupancy of Iε. The identification of this mechanism opens new avenues to inhibit allergic reactions triggered by IgE.

Unique epitopes on C epsilon mX in IgE-B cell receptors are potentially applicable for targeting IgE-committed B cells.

Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgE(+) B cells, the membrane-bound epsilon-chain (mepsilon) exists predominantly in the long isoform, mepsilon(L), containing an extra 52 aa CepsilonmX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of mepsilon, mepsilon(S), exists in minor proportions. CepsilonmX thus provides an attractive site for immunologic targeting of mIgE(+) B cells. In this study, we show that nine newly prepared CepsilonmX-specific mAbs, as well as the previously reported a20, bound to mIgE.Fc(L)-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.Fc(L)-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CepsilonmX. Expression of Igalpha and Igbeta on the mIgE.Fc(L)-CHO cells reduces the binding of a20 to CepsilonmX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.Fc(L)-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated Ab-dependent cellular cytotoxicity toward mIgE.Fc(L)-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 and an immunogen using the peptide segments recognized by these mAbs are potentially useful for targeting mIgE(+) B cells to control IgE production.

Flavones suppress type I IL-4 receptor signaling by down-regulating the expression of common gamma chain.

Immunoglobulin E (IgE) production is induced by interleukin (IL)-4 signaling mediated by type I IL-4 receptor (IL-4R) in B cells. We found that flavones inhibited IL-4-induced epsilon germline transcription which is essential for IgE class switching, and the phosphorylation of signal transducer and activator of transcription 6, janus kinase 3, and IL-4Ralpha, whereas IL-4 signaling mediated through type II IL-4R was unaffected by flavones. Furthermore, flavones reduced the expression of common gamma chain, a characteristic constituent subunit of type I IL-4R, suggesting that flavones suppress type I IL-4R signaling.

Catalytic folding of the Cepsilon3 domain by its high affinity receptor.

The interaction of immunoglobulin E (IgE) with its cellular receptor FcepsilonRIalpha is a central regulator of allergy. Structural studies have identified the third domain (Cepsilon3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cepsilon3 domain is a "molten globule" that becomes structured upon binding of the FcepsilonRIalpha ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcepsilonRIalpha in the folding of the monomeric Cepsilon3 domain of IgE. Soluble FcepsilonRIalpha is shown to display characteristic properties of a catalyst for the folding of Cepsilon3, with the rate of Cepsilon3 folding being dependent on the concentration of the receptor.

Modification of Cepsilon mRNA expression by EBV-encoded latent membrane protein 1.

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cepsilon mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cepsilon mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cepsilon mRNA levels were decreased in the splenocytes derived from the immunized rats. Cepsilon mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.

Dissecting the molecular mechanisms of TCR zeta chain downregulation and T cell signaling abnormalities in human systemic lupus erythematosus.

Abnormal expression of key signaling molecules and defective function of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Probing on altered expression of genes that may predispose to SLE revealed that the expression of TCR zeta chain is defective in the majority of SLE patients. Current research has been directed towards understanding the molecular basis of TCR zeta chain deficiency and dissecting the T cell signalling abnormalities in SLE T cells. Latest developments suggest that interplay of abnormal transcriptional factor expression, aberrant mRNA processing/editing, unbiquitination, proteolysis, and the effects of oxidative stress as well as changes in chromatin structure invariably contribute to TCR zeta chain deficiency in SLE T cells. On the other hand, multiple factors, including altered receptor structure, modulation of membrane clustering, lipid-raft distribution of signaling molecules, and defective signal silencing mechanisms, play a key role in delivering the increased TCR/CD3-mediated intracellular calcium response in SLE T cells.

CD40-mediated tumor necrosis factor receptor-associated factor 3 signaling upregulates IL-4-induced germline Cepsilon transcription in a human B cell line.

Induction of germline C epsilon transcription in B cells by IL-4, which is a critical initiating step for IgE class switching, is enhanced by CD40 engagement. Although signaling by CD40 is initiated by the binding of tumor necrosis factor receptor-associated factor (TRAF) family members to its cytoplasmic domain, whether those TRAF family proteins mediate enhancement of germline Cepsilon transcription is not evident. We report here that CD40-induced TRAF3-dependent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) is involved in the upregulation of IL-4-driven germline C epsilon transcription in a human Burkitt's lymphoma B cell line, DG75. Among the six known TRAF proteins, TRAF2, 3, 5, and 6 associated with CD40 in an unstimulated state, and the levels of these four proteins were unaffected by anti-CD40 stimulation. Antisense oligodeoxynucleotide (ODN) for TRAF3 inhibited CD40-induced activation of MEK1-ERK pathway by decreasing expression of TRAF3 protein, but antisense ODNs for TRAF2, 5, and 6 were ineffective. Furthermore, CD40-mediated enhancement of IL-4-driven germline C epsilon transcription was inhibited by antisense ODN for TRAF3 and by a MEK1 inhibitor, PD98059. These results suggest that in DG75 cells, TRAF3-induced MEK1 activation may be involved in CD40-mediated upregulation of IL-4-driven germline C epsilon transcription.

Myb proteins repress human Ig epsilon germline transcription by inhibiting STAT6-dependent promoter activation.

Cytokine-dependent induction of correctly spliced germline (GL) transcripts is required to target the appropriate switch region for class switch recombination. GL transcription is linked to the cell cycle and the number of cell divisions through mechanisms that have not been defined. The human proximal epsilon GL promoter contains an IL-4 responsive element (IL-4RE) that binds STAT6 and is sufficient to confer IL-4 inducibility to a heterologous promoter in transient transfection studies. We show herein that the IL-4RE contains a novel Myb binding motif that overlaps the 3' end of the STAT6 palindrome. EMSA analysis showed binding to the IL-4RE of endogenous Myb proteins expressed in BL-2 B cells and Jurkat T cells. However, double occupancy of a probe spanning both STAT6 and Myb binding motifs could not be detected. Thus, binding of either factor may prevent protein/DNA interactions at the other site, raising the possibility that Myb binding may interfere with STAT6-dependent activation of the IL-4RE. Indeed, cotransfection of A-Myb or c-Myb expression vectors in HEK293 and BL-2 cells suppressed STAT6-dependent transcription from a reporter construct containing four copies of the IL-4RE cloned upstream of a minimal thymidine kinase promoter. Most importantly, overexpression of A-Myb was sufficient to suppress IL-4-induced endogenous epsilon GL transcription in BL-2 cells. Our results indicate that Myb proteins, which are known to act as cell cycle sensors, may play an important mechanistic role in the in vivo regulation of epsilon GL transcription in human B cells.

Differential regulation of mouse germline Ig gamma 1 and epsilon promoters by IL-4 and CD40.

Before Ig class switching, RNA transcription through the specific S regions undergoing recombination is induced by cytokines and other activators that induce and direct switching. The resulting germline (GL) transcripts are essential for switch recombination. To understand the differential regulation of mouse IgG1 and IgE, we compared the promoters for GL gamma1 and epsilon transcripts. We addressed the question of why the promoter that regulates GL epsilon transcription is more responsive to IL-4 than the gamma1 promoter and also why GL epsilon transcription is more dependent on IL-4 than is gamma1 transcription. We found that the IL-4-responsive region of the GL epsilon promoter is more inducible than that of the gamma1 promoter, although each promoter contains a binding site for the IL-4-inducible transcription factor Stat6, located immediately adjacent to a binding site for a basic region leucine zipper (bZip) family protein. However, the arrangement and sequences of the sites differ between the epsilon and gamma1 promoters. The GL epsilon promoter binds Stat6 with a 10-fold higher affinity than does the gamma1 promoter. Furthermore, the bZip elements of the two promoters bind different transcription factors, as the GL epsilon promoter binds and is activated by AP-1, whereas the gamma1 promoter binds and is activated by activating transcription factor 2. C/EBPbeta and C/EBPgamma also bind the gamma1 bZip element, although they inhibit rather than activate transcription. However, inhibition of promoter activity by C/EBPbeta does not require the bZip element and may instead occur via inhibiting the activity of NF-kappaB.

Activation of the mouse Ig germline epsilon promoter by IL-4 is dependent on AP-1 transcription factors.

Induction of germline (GL) epsilon transcripts, an essential step preceding Ig isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g., CD40 ligand or LPS. We demonstrate that AP-1 (Fos and Jun), induced transiently by CD40 ligand or LPS, binds a DNA element in the mouse GL epsilon promoter. AP-1 synergizes with Stat6 to activate both the intact GL epsilon promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL epsilon promoter. By contrast, C/EBP beta, which trans-activates the human GL epsilon promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not trans-activate the human GL epsilon promoter. Thus, induction of GL epsilon transcripts in mice and humans may be regulated differently. In addition, although mouse GL epsilon transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h, and the promoter appears to be active for at least 2 days after B cell activation. Altogether, these data suggest that induction of AP-1 activity, although transient, is required for activation of the mouse GL epsilon promoter by IL-4-induced Stat6.

The novel human IgE epsilon heavy chain, epsilon tailpiece, is present in plasma as part of a covalent complex.

Several splice variants of the secreted human epsilon heavy chain have previously been identified by reverse transcription-PCR. The heavy chain of one isoform, IgE tailpiece, differs from the originally identified IgE, IgE classic, by the replacement of the 2 carboxy-terminal amino acids by 8 novel amino acids including a carboxy-terminal cysteine residue. Recombinant human epsilon tailpiece and epsilon classic heavy chains were expressed and secreted as H2L2 monomers in Sp2/0 murine myeloma cells. We have investigated the in vitro function and in vivo occurrence of epsilon tailpiece heavy chains using receptor binding assays, granule release assays, flow cytometry, half-life studies, immunoprecipitation, SDS-PAGE, two-dimensional SDS-PAGE, and Western blotting. IgE tailpiece and IgE classic exhibited similar in vivo half-lives in BALB/c mice, bound the human high- and low-affinity IgE receptors with similar affinities and triggered equivalent levels of high affinity IgE receptor induced degranulation. In humans, IgE classic is present as a 190 kD circulating protein in vivo. In contrast, we found that in plasma epsilon tailpiece was primarily present as part of covalent complexes of approximately 300 and 338 kD. Dissociation of the complexes revealed that two species of epsilon tailpiece heavy chains were present therein and surprisingly, these in vivo derived epsilon tailpiece heavy chains were approximately 5 and 10 kD smaller than the recombinant expressed epsilon tailpiece or epsilon classic heavy chains. These results show that epsilon tailpiece is present in novel covalent complexes in humans.

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry.

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.

IL-4 induces the intracellular expression of the alpha chain of the high-affinity receptor for IgE in in vitro-generated dendritic cells.

BACKGROUND: Recent findings have shown that the surface expression of the high-affinity receptor for IgE (FcepsilonRI) on human CD1a(+) Langerhans cells (LC) and related dendritic cells (DC) in the skin, despite a constant intracellular expression of its alpha chain (FcepsilonRIalpha), is highly up-regulated in atopic dermatitis. Moreover, this surface expression correlates with the IgE serum level, strongly suggesting yet-to-be-defined common signals in the regulation of FcepsilonRI display on LC/DC and IgE synthesis. OBJECTIVES: In this study we examined the influence of different cytokines on the expression of FcepsilonRI on in vitro-generated CD1a(+) LC/DC. METHODS: CD34(+) precursor cells were isolated from cord blood with use of high-gradient magnetic cell sorting, cultured with GM-CSF, TNF-alpha, IL-4, or IFN-gamma, and surface and cytoplasmic staining for flow cytometry were performed. RESULTS: IL-4 strongly enhanced the generation of CD1a(+) LC/DC and also up-regulated the expression of the skin-homing structures E-cadherin and cutaneous lymphocyte antigen. In contrast, IFN-gamma was found to suppress the E-cadherin expression and to be a strong antagonist of IL-4 by inhibiting the production of CD1a(+) cells. Most important, IL-4 induced the cytoplasmic expression of FcepsilonRIalpha in CD1a(+) LC/DC but not its surface expression. This up-regulation was antagonized by IFN-gamma. CONCLUSION: IL-4 is not only a key cytokine in the regulation of IgE but also induces the expression of its receptor binding chain as well as up-regulation of skin homing molecules on LC/DC. Expression of these structures during generation of LC/DC reflects the in vivo situation encountered in LC.

Synergistic activation of the germline epsilon promoter mediated by Stat6 and C/EBP beta.

Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the epsilon germline transcript is induced by IL-4 and requires the integrity of a composite IL-4 response element. The element is bound by the IL-4-inducible transcription factor Stat6 and one or more members of the CAAT/enhancer-binding protein (C/EBP) family, a constitutively expressed class of transcription factors. Here, we show that Stat6 and C/EBP beta cooperate to synergistically activate transcription from the epsilon element. The effect was most pronounced in lymphoid cells, and the activation domains of both proteins were required to achieve this synergy. Although other members of the C/EBP family are able to bind the element, very little cooperativity was seen with C/EBP alpha and none with C/EBP gamma. In fact, C/EBP gamma was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBP beta bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is slowed when C/EBP beta binds at the neighboring site. These data suggest a mechanism whereby C/EBP beta stabilizes Stat6 binding at this element, thereby increasing the likelihood that both of their activation domains will interact, possibly with other factors, to activate transcription in an IL-4-dependent manner.

Interaction of the low-affinity receptor CD23/Fc epsilonRII lectin domain with the Fc epsilon3-4 fragment of human immunoglobulin E.

CD23/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD23 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD23, corresponding to the soluble lectin domain of CD23, and Fc epsilon3-4, a dimer of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD23 and Fc epsilon3-4 indicate that IgE has two binding sites for CD23, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (delta H(degree)To = -2.1 +/- 3.3 kcal mol(-1)) and heat capacity (delta Cp(degree) = -320 +/- 320 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (delta H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (delta Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD23, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD23 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the 2:1 complex between CD23 and IgE is consistent with the thermodynamic data presented.

Altered patterns of tyrosine phosphorylation and Syk activation for sterically restricted cyclic dimers of IgE-Fc epsilonRI.

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis-[[epsilon-(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystin e ((DCT)2-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilonRI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilonRI and two (DCT)2-cys (Posner et al. (1995) J. Immunol. 155, 3601-3609). These cyclic dimers do not trigger Ca2+ or degranulation responses under a variety of conditions. However, we find that the linearly cross-linked IgE-Fc epsilonRI formed at higher concentrations of (DCT)2-cys do trigger degranulation in the presence of cytochalasin D, an inhibitor of actin polymerization. We further investigated stimulation by (DCT)2-cys of the earliest known events in the functional response, i.e., tyrosine phosphorylation of the beta and gamma subunits of Fc epsilonRI. At the higher (DCT)2-cys concentrations corresponding to linear dimers and maximal degranulation, tyrosine phosphorylation of both beta and gamma are observed. At lower (DCT)2-cys concentrations where cross-linking is maximal and cyclic dimers are overwhelmingly dominant, only gamma tyrosine phosphorylation is observed. Cytochalasin D does not affect these phosphorylation patterns, but instead appears to enhance coupling to downstream signaling events. Phosphorylation of Syk occurs at the higher (DCT)2-cys concentrations in parallel with beta phosphorylation but does not occur in its absence at the lower (DCT)2-cys concentrations. These results suggest that cyclic dimers of IgE-Fc epsilonRI are sterically restricted such that they stimulate tyrosine phosphorylation of gamma but not beta, and this is not sufficient for Syk binding and/or activation.