[Screening and characterization of aptamers of Cepsilon3-Cepsilon4 protein].

In order to obtain nucleotides aptamers bind to IgE, 80 bp nucleotides single-stranded DNA library containing 40 random nucleotides was designed and synthesized. Oligonucleotides that bind to human Cepsilon3-Cepsilon4 protein were isolated from ssDNA pools by the systematic evolution of ligands by exponential enrichment (SELEX) method using nitrocellulose filters as screening medium. Through the optimization of critical PCR and asymmetric PCR parameters including annealing temperature, cycles, and molar ratios of target protein and ssDNA etc, a suitable screening system was established. The aptamers of Cepsilon3-Cepsilon4 protein with high affinity and high specificity were identified by ELISA with biotin-streptavidin-horseradish peroxidase system, and its primary sequence and second structure were analyzed by DNAMAN package and DNA folding sever after being cloned and sequenced. Moreover, target protein was bound to one aptamer and another aptamer modified with biotion together forming a sandwich-like complex, which was captured in microwell to detect IgE concentration using the optimal combination in the sandwich method named enzyme-linked aptamers sorption assay (ELASA). The method could be used for the quantitative detection of human IgE, and whose sensitivity reached to 120 ng x mL(-1).

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.

The involvement of immunoglobulin E isotype switch in scleroderma skin tissue.

BACKGROUND: The involvement of mast cell, which is activated by immunoglobulin E (IgE), has been reported in the formation of systemic sclerosis (SSc) abnormality. IgE is generated with isotype switch. During isotype switch, switch circles resulting from direct mu to epsilon, or from sequential mu to gamma via epsilon switching will be created. OBJECTIVE: We studied whether switching occurs in SSc. METHODS: We used nested polymerase chain reaction to analyze the S fragments from switch circles. Fifty-two patients with SSc, and 62 healthy women were studied. RESULTS: Neither of 62 normal skin tissues showed direct switch, nor sequential switch. Neither of seven normal whole blood cells showed direct switch, nor sequential switch. In 52SSc skin tissues, three (5.8%) showed direct switch, and two (3.8%) showed sequential switch. As a result, five (9.6%) of SSc skin tissue showed immunogobulin E class switch. These results were confirmed by DNA sequencing. CONCLUSION: These results demonstrated that isotype switch to the epsilon locus achieved by direct and/or sequential switch are involved in SSc skin.

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.

Monoclonal antibodies against the C(epsilon)mX domain of human membrane-bound IgE and their potential use for targeting IgE-expressing B cells.

BACKGROUND: IgE mediates immediate-type hypersensitivity reactions responsible for various allergic symptoms. It is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on their surface. The epsilon-chain of human mIgE contains a membrane-anchoring peptide and an extra 52-amino-acid (a.a.)-long domain (referred to as C(epsilon)mX) between the membrane anchor and the CH4 domain. OBJECTIVE: The study was designed to evaluate the effects of C(epsilon)mX-specific monoclonal antibodies (mAbs) to target IgE-expressing B cells and decrease IgE production. METHODS: A C(epsilon)mX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting C(epsilon)mX-specific mAbs were obtained. RESULTS: Characterization of the mAbs using ELISA, immunoprecipitation, and immunoblotting methods showed that they could bind to both native and denatured forms of C(epsilon)mX. The mAbs exhibited mutual inhibition of binding to mIgE. Epitope mapping using synthetic peptides revealed that all five mAbs recognize the same epitope, RADWPGPP, located near the C-terminus of C(epsilon)mX. Binding of one of the mAbs to mIgE on SKO-007 cells induced the cross-linking of mIgE molecules on the cell surface, resulting in their patching and capping. In vitro functional analysis revealed that mAbs are able to cause complement-mediated cytotoxicity on transfectants expressing the Fc portion of mIgE. CONCLUSION: We have prepared several human mIgE-specific mAbs. The potential of the mAbs on targeting mIgE+ B cells was demonstrated by CDC analysis.

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat (Felis domesticus) Ig epsilon constant region.

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.

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.

Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.

BACKGROUND: Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform. OBJECTIVES: We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific. STUDY DESIGN: Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas. RESULTS: Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE. CONCLUSION: Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.

Characterization of two dog IgE-specific antibodies elicited by different recombinant fragments of the epsilon chain in hens.

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.

Automated hydrodynamic modelling of a complex between a human IgE fragment (Fc epsilon 3-4) and the IgE high affinity receptor Fc epsilon RI alpha-chain.

The binding of IgE to its high affinity receptor Fc epsilon RI plays an important role in the allergic response. The interaction between soluble Fc epsilon RI alpha-chain (sFc epsilon RI alpha) and Fc epsilon 3-4, a fragment of IgE consisting of the C epsilon 3 and C epsilon 4 heavy chain constant domains, has been studied using analytical ultracentrifugation (Keown et al. this volume). Here we describe the development of a simple automated hydrodynamic modelling technique and its application to this interaction. This procedure utilises sphere models of the two molecules and performs an automated systematic translational search of sFc epsilon RI alpha relative to Fc epsilon 3-4. The result of this is the generation of 40,359 individual models of how the receptor can be placed relative to Fc epsilon 3-4. These are then assessed for consistency by comparing the sedimentation coefficients generated for the models to the experimentally determined sedimentation coefficients, and are displayed graphically to show allowed and disallowed complexes. From this analysis, it is clear that the complex between sFc epsilon RI alpha and Fc epsilon 3-4 is compact, with the most elongated models being excluded. In addition, sFc epsilon RI alpha appears not to interact with the C-terminal end of Fc epsilon 3-4, and probably binds either to the sides or face, observations which are consistent with other experimental data on the Fc epsilon RI alpha/IgE interaction. Automated hydrodynamic modelling also has the potential to be used for other interactions, providing a simple way of looking at a large number of models, and making rigorous studies of interacting components more feasible.

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.

Cross-reactivity and molecular mass of the epsilon chains of the IgE antibodies in dogs, humans, rats, and mice.

We report the cross-reactivities and comparative molecular masses of the IgE epsilon chains in humans, rats, mice, and dogs. Monoclonal human, rat, and mouse IgE, and our purified polyclonal dog IgE were used in the study. IgE of the 4 species, separated by SDS-PAGE, were analyzed by immunoblotting with polyclonal and monoclonal antihuman IgE, polyclonal and monoclonal antimouse IgE, monoclonal antirat IgE, and polyclonal antidog IgE antibodies. The polyclonal antihuman and polyclonal antimouse IgE cross-reacted with the IgE of the other 3 species, while their monoclonal forms cross-reacted with dog IgE only. Polyclonal antidog IgE cross-reacted with human and mouse IgE, while the monoclonal antirat IgE did not cross-react with any other species. "Reverse' passive cutaneous anaphylaxis in ragweed-sensitized dogs revealed that polyclonal antihuman and polyclonal antimouse IgE were able to elicit positive skin responses, and monoclonal antihuman, antirat, and antimouse IgE antibodies were not. The molecular masses of the epsilon chains were 77 kDa for mice, 75 kDa for rats and dogs, and 70 kDa for humans.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Identification of the high affinity receptor binding region in human immunoglobulin E.

We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.

Characterization of the human immunoglobulin epsilon mRNAs and their polyadenylation sites.

Several IgE heavy (H) chain transcripts are produced by alternative splicing between constant region (CH3 and CH4) and membrane (M1 and M2) exons and by differential cleavage-polyadenylation at poly(A) sites downstream of the CH4 and M2 exons. We have now characterized the poly(A) signal of the epsilon transcripts that contain membrane exon sequences (epsilon CH4-M1'-M2, epsilon CH4-M1-M2, epsilon CH4-M2' and epsilon CH4-M2") and have determined the complete sequence of the M2 exon and 1.4 kb of downstream genomic DNA. The membrane locus poly(A) site was identified by RACE-PCR analysis of epsilon transcripts obtained from IgE-producing myeloma cells and normal peripheral blood lymphocytes (PBL). All membrane exon transcripts were found to be polyadenylated following a CA dinucleotide located 1046 nt from the beginning of the M2 exon. An AGTAAA hexamer, located 13 nt upstream from the site of cleavage and polyadenylation, was the only poly(A) signal sequence present in the 1.4 kb of genomic DNA downstream of the M2 exon. A (G+T)-rich region, which is also conserved in most poly(A) signals, was present 50 nt downstream of the AGTAAA hexamer. Northern blot analysis confirmed that this poly(A) site is used by the membrane exon epsilon mRNAs expressed by the U266 myeloma. The four membrane exon transcripts were detected in different relative amounts in PBL and IgE-producing myeloma cells, which could reflect different epsilon mRNA splicing patterns during B-cell differentiation.

Partial sequence of the equine immunoglobulin epsilon heavy chain cDNA.

In order to isolate a part of the immunoglobulin E (IgE) heavy chain cDNA of the horse, primers have been designed based upon well conserved sequences in humans, sheep and rats. The PCR resulted in a 500 bp fragment which hybridised with a human IgE constant region probe. The fragment was cloned and sequenced and its derived protein sequence compared with the corresponding sequences in humans, sheep and mice. Most amino acids common to these three species are also shared by the horse.