Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.     

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

The binding of IgE to murine Fc epsilon RII is calcium-dependent but not inhibited by carbohydrate

Despite extensive study, little is known about the functions of the moderate affinity IgE receptors (Fc epsilon RII) on B cells. Recent cDNA and genomic cloning studies have demonstrated that, in contrast to other FcR, Fc epsilon RII is not a member of the Ig gene superfamily. Moreover, it uniquely expresses a region that is highly homologous with a membrane-associated, calcium-dependent binding lectin, the asialoglycoprotein receptor. We now report that the interaction between IgE and the Fc epsilon RII of murine B cells and macrophages requires calcium. Furthermore, as might be expected of asialoglycoprotein lectins, this binding was pH-dependent and resulted in ligand internalization. However, although 125I-Fc epsilon RII bound in a calcium-dependent manner to monosaccharide-agarose beads, high concentrations of mono- and disaccharides did not inhibit the interaction between either 125I-IgE and intact B cells or 125I-Fc epsilon RII (from surface-labeled B cells) and IgE-Sepharose. These results suggest that although murine Fc epsilon RII is a lectin, it is not strictly dependent upon IgE oligosaccharides for its binding to IgE.     

Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.     

Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin.

IgE-binding protein (epsilon BP) was originally identified by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain. The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains. The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage. In this study, human epsilon BP was first expressed in Escherichia coli, and the carboxyl-terminal domain (epsilon BP-C) was then generated by collagenase digestion of epsilon BP. By equilibrium dialysis, the association constants of epsilon BP and epsilon BP-C for lactose were found to be similar (6.0 +/- 0.70) x 10(4) M-1 and (4.7 +/- 0.27) x 10(4) M-1, respectively. Both polypeptides contain only one lactose-binding site/molecule. By an assay involving binding of 125I-labeled epsilon BP or epsilon BP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that epsilon BP-C retains the saccharide specificity of epsilon BP. Importantly, although unlabeled epsilon BP-C inhibited the binding of the radiolabeled epsilon BP to IgE, unlabeled epsilon BP caused increased binding to IgE, suggesting self-association among epsilon BP molecules. Oligomeric structures resulting from self-association of epsilon BP were confirmed by chemical cross-linking studies. Furthermore, epsilon BP possesses hemagglutination activity on rabbit erythrocytes, whereas epsilon BP-C lacks such activity. Based on these results, we propose a structural model for multivalency of epsilon BP: dimerization or oligomerization of epsilon BP occurs through intermolecular interaction involving the amino-terminal domain.     

Heterogeneous IgE glycoforms characterized by differential recognition of an endogenous lectin (IgE-binding protein)

IgE is highly glycosylated, but the function of the oligosaccharide side chains is largely unknown. The previous discovery of an animal lectin, IgE-binding protein (epsilon BP), affords an opportunity to study potential carbohydrate-dependent effector functions of IgE. epsilon BP is a beta-galactoside-specific lectin with binding affinity for IgE and is now known to be equivalent to carbohydrate-binding protein 35 and the Mac-2 Ag; thus, it may have multiple functions in addition to IgE binding. We have previously shown that rat r epsilon BP recognizes sialidase-treated human myeloma IgE to a much greater extent than the untreated IgE. In contrast, human epsilon BP binds essentially equivalently to a monoclonal murine IgE with or without sialidase pretreatment. To validate a possible role for epsilon BP in the IgE system, we investigated the pattern of recognition of epsilon BP for various polyclonal human IgE samples. We show that polyclonal IgE derived from four individuals with hyper-IgE syndrome or atopic dermatitis recognizes epsilon BP and that there is individual variation in the proportion of IgE recognized by epsilon BP, ranging from greater than 60% for one sample to almost undetectable levels in another. We conclude that epsilon BP does indeed recognize polyclonal IgE and that this recognition is modulated by sialylation of IgE oligosaccharides. Furthermore, there exist different IgE glycoforms, varying in the degree of sialylation, and these are distributed in a distinct manner in different individuals.     

Activated macrophages and antibodies against the plant lectin, GSI-B4, recognize the same tumor-associated structure (TAS)

Activated macrophages that were stabilized with either formalin or glutaraldehyde absorbed two polypeptides (Mr 100,000 and 60,000) from detergent extracts of all of the tumor cell lines tested, but not from detergent extracts of normal human peripheral blood lymphocytes. A major polypeptide (Mr 95,000) was retained from spent culture media of tumor cell lines. Polypeptides with molecular sizes of 100,000 and 60,000 daltons were also adsorbed by activated macrophages from detergent extracts of chicken embryo cell membranes, suggesting an oncofetal nature for these proteins. The 100,000 dalton polypeptide, but not the 60,000 dalton component, was found to be available to lactoperoxidase-catalyzed cell surface iodination. Polypeptides with identical molecular sizes could be adsorbed to immobilized galactopyranoside, indicating that they are vertebrate lectins. Activated macrophages and affinity adsorbents prepared by the covalent coupling of galactopyranoside to agarose also bind the plant lectin isolectin B4 prepared from the seeds of Griffonia simplicifolia. On the basis of these findings, we put forth the hypothesis that macromolecules of the same specificity, that is affinity to galactopyranosyl residues, must show homologies in their binding sites. We have predicted therefore that antisera prepared against this plant lectin should cross-react with galactopyranosyl-binding vertebrate lectins present on the surface of tumor cells. In this communication, we also report the generation of hybridomas that produce antibodies reactive with both the plant and vertebrate lectins. Inhibition experiments that make use of various mono- and disaccharides suggest that the specificities of these antibodies are for determinants intimately associated with the galactosyl binding site on the lectin molecule. Two of the antibodies were found to have moderate selectivity for tumor cells when tested in an immunohistochemical procedure that made use of fresh-frozen or paraffin-embedded sections of human biopsy material. These two antibodies on immunoblots of tumor cell membrane extracts reacted with a polypeptide with an apparent molecular size of 100,000 daltons.     

Galectin-9 Enhances Cytokine Secretion,but Suppresses Survival and Degranulation,in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.     

Lectin-binding ability of immunoglobulin E and its partipication in triggering of mast cells

Eleven purified lectins were compared in their IgE-precipitating, mast-cell-degranulating and histamine-releasing activity and in eliciting skin allergic reactions in atopic or normal people. A significantly positive relationship could be proved between their IgE-precipitating activity and degranulation of IgE-sensitized rat mast cells. A similar and often significant relation was observed in skin reactions and histamine released from human leukocytes. Soybean lectin reacted with concanavalin A by a precipitation line in coalescence with the IgE showing the possibility of a common structural component. Correlation tests between different lectins show that all but three elicit the cell activation by similar ways, but different from IgE antiserum. Soybean lectin, Triticum vulgare and Vicia cracca reacted in other ways and correlated better with histamine release caused by the IgE antiserum. A certain degree of the heterogeneity in the IgE sugar component together with the complexity of the lectin structure may be responsible for a wide mode of activation on cell membranes. The significance of this non-immunological binding for sensitization, natural desensitization and cell regulation by physiological histamine release was discussed.     

Molecular structure of human lymphocyte receptor for immunoglobulin E

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.     

Death of a dogma or enforcing the artificial: monomeric IgE binding may initiate mast cell response by inducing its receptor aggregation

Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, Fc epsilon RI, on mast cells induces important responses. These observations contradict the notion that it is the aggregation of this receptor that is essential for initiating mast cell response. In the present study, we suggest that the most probable causes for the reported observations are the experimental protocol used combined with the high expression levels of the Fc epsilon RI by mast cells. Specifically, we suggest using the published data and physicochemical calculations that the exceptionally high number of cell surface Fc epsilon RI-bound monoclonal IgE yields, in the two-dimensions of the cells' membranes, a situation where even a low affinity of these mIgE for epitopes on their own structure or on another cell surface component may lead to their aggregation. Hence, we hypothesize that the reported response to mIgE binding is a result of such an Fc epsilon RI-IgE induced aggregation.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Establishment and characterization of hybrid rat mast cells

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.     

Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.