Relationship among IgE-binding factors with various molecular weights

The rat-mouse T cell hybridoma 23B6 forms IgE-binding factors on incubation with IgE. The hybridoma cells incubated with IgE contained intracellular IgE-binding factors of 60K, 30K, 14K, and 10K daltons, and secreted the 60K, 30K, and 14K IgE-binding factors. Both intracellular and extracellular IgE-binding factors of 60K and 14K daltons selectively suppressed the IgE response, whereas the 30K and 10K factors failed to do so. Reduction and alkylation of the extracellular 60K IgE-binding factors yielded fragments of 30K, 14K, and 10K daltons with IgE-binding activity, and the same treatment of the 30K "inactive" IgE-binding factor yielded the 14K and 10K fragments. Among the fragments obtained by reduction and alkylation, only the 14K IgE-binding factors selectively suppressed the IgE response. Monoclonal antibody OX 3, which recognizes an Ia determinant, bound the 60K and 30K IgE-binding factor, and the 30K fragment obtained by reduction and alkylation. None of the 14K and 10K IgE-binding factors and fragments of comparable size obtained by reduction and alkylation contained the antigenic determinant. The results indicate that 30K, 14K, and 10K IgE-binding factors are derived from the 60K precursor molecules. Transfection of COS 7 monkey kidney cells with a single cloned cDNA, 8.3, which encodes rodent IgE-binding factor, resulted in the formation of IgE-binding factors of 60K and 11K daltons. Both species of the factors had affinity for lentil lectin and selectively potentiated the IgE response, but only the 60K IgE-binding factor bound to the monoclonal anti-Ia antibody. Reduction and alkylation of the 60K IgE-binding factors from the cDNA clone yielded an 11K fragment having the same properties as the 11K IgE-binding factor, with respect to the affinity for lentil lectin, and the biologic activities. Because the cDNA clone was constructed from mRNA of 23B6 cells, it appears that the 60K IgE-binding factor molecule from 23B6 cells is composed of a single peptide chain, and that the naturally formed 30K, 14K, and 10K IgE-binding factors are formed by post-translational modification of the 60K peptide.     

Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9.

Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, is a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis. Approximately 40-65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T-cell response against this yeast is found in atopic dermatitis patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli. The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the searched database. The proteins encoded by the complete cDNA sequences were expressed in E. coli as recombinant proteins. Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M. furfur extract could specifically inhibit this IgE binding.     

IgE-binding factors from mouse T lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon

Incubation of normal mouse spleen cells with homologous IgE resulted in the formation of soluble factors that inhibited rosette formation of mouse Fc epsilon R+ cells with IgE-coated ox erythrocytes. The soluble factors could be absorbed with mouse or rat IgE coupled to Sepharose and recovered from the beads by acid elution. However, the factors had no affinity for either human IgE or mouse IgG. The IgE-binding factors were derived from T cells. Production of the factors required Lyt1+ T cells and Fc gamma R+ cells, which suggests that the factors are derived from Fc gamma R+ Lyt 1+ T cells. The molecular size of IgE-binding factors was approximately 15,000 daltons. When IgE-binding factors were formed by BALB/c spleen cells, nearly one-half of the factors had affinity for lentil lectin, and the remaining half of the factors failed to bind to the lectin. The proportion of the two species of IgE-binding factors differed depending on mouse strains. The majority of the factors formed by B6D2F1 spleen cells had affinity for lentil lectin, but those formed by SJL spleen cells failed to bind to the lectin. The IgE-binding factors were also induced by incubation of normal spleen cells with polyinosinic-polycytidylic acid (pI:pC). The nucleotide stimulated splenic adherent cells to form "inducers" of IgE-binding factors, which in turn induced normal lymphocytes to form IgE-binding factors. The inducers of IgE-binding factors were inactivated (or neutralized) by antibodies specific for mouse Type I interferon. It was also found that purified mouse beta interferon could induce the formation of IgE-binding factors. IgE-binding factors induced by pI:pC consisted of two different molecules: one had a m.w. of 15,000 daltons, and another had a m.w. of between 40,000 and 60,000 daltons.     

Patient-tailored cloning of allergens by phage display: Peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10¹¹ cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( -proline)-Sepharose and yielded 30 mg l⁻¹ culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.     

Modulation of the biologic activities of IgE-binding factor. II. Physicochemical properties and cell sources of glycosylation-enhancing factor

T lymphocytes of rats treated with Bordetella pertussis vaccine (BP) formed a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis, and provided the latter factors with the biologic activity to potentiate the IgE response. The present experiments demonstrated that pertussigen (leukocytosis-promoting factor) from BP induced normal rat spleen cells to form the glycosylation-enhancing factor. The same factor was obtained by incubation of normal spleen cells with 5 micrograms/ml, but not 2 micrograms/ml, concanavalin A. When normal rat mesenteric lymph node cells were incubated with the glycosylation-enhancing factor together with IgE, IgE-binding factors formed by the cells selectively potentiated the IgE response. The IgE-binding factors formed by the same cells upon incubation with IgE alone neither enhanced nor suppressed the IgE response. The glycosylation-enhancing factor changed the nature of IgE-binding factors formed by the rat-mouse T cell hybridoma, 23A4. IgE-binding factors induced by IgE alone lacked affinity for lentil lectin, whereas those induced by IgE in the presence of the glycosylation-enhancing factor had affinity for the lectin. The cell source of the glycosylation-enhancing factor appeared to be W 3/25+ Fc gamma R+ T cells. The glycosylation-enhancing factor was protein in nature and had a m.w. of about 25,000. The factor had affinity for acid-treated Sepharose and could be recovered from the beads by elution with lactose. The factor was different from interleukin 2 with respect to both its affinity for galactose and its isoelectric point.     

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.     

Cloning allergens via phage display

Although an impressive list of allergenic structures has been elucidated during the last decade by classical cloning methods, the size of the repertoire of molecular structures able to elicit allergic reactions is still unknown. Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based high-throughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by Escherichia coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures.     

Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E

The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites.     

Presence of an antigen-specific T cell subset that forms IgE-suppressive factor and IgG-suppressive factor on antigenic stimulation

B6D2F1 mice were given three i.v. injections of ovalbumin (OA), and antigen-specific T cell clones were established from their spleen cells. One of the FcR+ T cell clones formed IgE-binding factors on incubation with OA-pulsed syngeneic macrophages. Neither soluble antigen nor macrophages alone induced factor formation. T cell hybridomas were constructed by fusion of the antigen-specific T cell clone with BW 5147 cells. Among 11 T cell hybridomas established, six clones produced IgE-binding factors on incubation with OA-pulsed BDF1 macrophages. Mouse IgE also induced the same hybridoma to form IgE-binding factors. The majority of IgE-binding factors formed by two T hybridomas and by those produced by the parent T cell clone had affinity for peanut agglutinin but for neither lentil lectin nor Con A. These hybridomas and the original T cell clone spontaneously released glycosylation-inhibiting factor, which inhibits the assembly of N-linked oligosaccharide(s) on IgE-binding factors. On antigenic stimulation, the T cell hybridomas produced both IgE-binding factors and IgG-binding factors. The IgE-binding factors consisted of three species with m.w. of 60,000, 30,000, and 15,000. Both the 60K and 15K IgE-binding factors selectively suppressed the IgE response of DNP-OA-primed rat mesenteric lymph node cells, whereas IgG-binding factors selectively suppressed the IgG response. The results indicate that antigen-primed FcR+ T cells produced IgE-suppressive factors and IgG-suppressive factors on antigenic stimulation. However, the T cell hybridomas were not committed to suppressive activity. When the hybridomas were stimulated by antigen in the presence of glycosylation-enhancing factor (GEF), the 60K, 30K, and 15K IgE-binding factors formed by the cells selectively potentiated the IgE response. IgG-binding factors formed by the cells in the presence of GEF failed to suppress the IgG response. It appears that antigen-specific FcR+ T cells regulate the antibody response through the formation of Ig-binding factors, but that the function of the cells could be switched from suppression to enhancement, depending on the environment of the cells.     

Modulation of the biologic activities of IgE-binding factors. III. Switching of a T cell hybrid clone from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor

Incubation of rat-mouse T cell hybridoma cells, 23B6, with rat immunoglobulin E (IgE) results in the formation of the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor, which has neither potentiating activity nor suppressive activity on the IgE response. Another T cell hybridoma, 23A4 cells, produces the 30,000-dalton "inactive" IgE-binding factor upon incubation with IgE. Both the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor lacked affinity for lentil lectin but bound to peanut agglutinin. When the 23B6 cells were incubated with IgE in the presence of lysolecithin, the majority of the 15,000-dalton IgE-binding factor formed by the cells gained affinity for lentil lectin, and this factor selectively potentiated the IgE response. The glycosylation-enhancing factor, which was formed by stimulation of normal spleen cells with lymphocytosis-promoting factor (LPF or pertussigen), also switched 23B6 cells from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor. It was also found that the 30,000-dalton "inactive" IgE-binding factor, formed by both 23B6 and 23A4 cells, gained the ability to potentiate the IgE response, when the cells were cultured with IgE in the presence of glycosylation-enhancing factor. The results indicate that IgE-potentiating factor and IgE-suppressive factor share common precursors, and that biologic activities of IgE-binding factors are decided by their carbohydrate moieties. Incubation of the two hybridoma cells with lysolecithin or glycosylation-enhancing factor results in an increase in the proportion of FC epsilon R+ cells, suggesting that the assembly of N-linked oligosaccharide to precursor molecules is intrinsic for the expression of FC epsilon R.     

Specific IgE determination to epitope peptides of omega-5 gliadin and high molecular weight glutenin subunit is a useful tool for diagnosis of wheat-dependent exercise-induced anaphylaxis

Wheat omega-5 gliadin and a high m.w. glutenin subunit (HMW-glutenin) have been reported as major allergens in wheat-dependent exercise-induced anaphylaxis. A simultaneous detection of specific IgE to epitope sequences of both proteins is considered to be a reliable method for diagnosis of wheat-dependent exercise-induced anaphylaxis. However, the IgE-binding epitope of HMW-glutenin remains unknown. The aim of this study was to determine the IgE-binding epitopes of HMW-glutenin to establish a useful system of identifying patients with wheat-dependent exercise-induced anaphylaxis. For determination of IgE-binding epitopes of HMW-glutenin overlapping peptides were synthesized and reactivities of IgE Abs in the sera of patients to those peptides were analyzed. Three IgE-binding epitopes, QQPGQ, QQPGQGQQ, and QQSGQGQ, were identified within primary sequence of HMW-glutenin. Epitope peptides, which include IgE-binding sequences of omega-5 gliadin and a HMW-glutenin, were synthesized and peptide-specific IgE Abs were measured by CAP-System fluorescent enzyme immunoassay. Twenty-nine of 30 patients with wheat-dependent exercise-induced anaphylaxis had specific IgE Abs to these epitope peptides. None of the 25 sera from healthy subjects reacted to both epitope peptides. Twenty-five patients with atopic dermatitis who had specific IgE to wheat and/or gluten had very low or nonexistent levels of epitope peptide-specific IgE Abs. These results indicated that measurement of IgE levels specific to epitope peptides of omega-5 gliadin and HMW-glutenin is useful as an in vitro diagnostic method for the assessment of patients with wheat-dependent exercise-induced anaphylaxis.     

Nucleotide sequence of the intracisternal A-particle genome inserted 5'' to the interleukin-3 gene of the leukemia cell line WEHI-3B.

The nucleotide sequence of the intracisternal A-particle genome IAP-IL3 is presented. This IAP element was found to have inserted upstream of the promoter of the interleukin-3 gene of the leukemia cell line WEHI-3B. IAP-IL3 is 5095 bp in length, with identical long terminal repeats (LTRs) of 337 bp. The LTRs show many of the conserved sequence elements identified in other retroviruses. Comparison with other available sequences of IAP genomes indicates that IAP-IL3 is a deleted type I element. It carries a deletion covering the 3' end of the putative IAP gag gene and extending into the 5' end of the putative IAP pol gene. IAP-IL3 has extensive sequence homology with an IgE-binding factor cDNA and evidence is presented indicating that it was derived from a member of the mouse IAP sequence family. Comparison between the pol region of IAP-IL3 and other retroviruses suggests that IAP-IL3 is most closely related to type B and type D retroviruses.     

Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase.

Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS). Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMala s 9) from this yeast. By combining high throughput screening and phage surface display techniques, 27 complete and partial IgE-binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialis allergens by RACE-PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones. The coding sequences of one of the clones showed similarity to the heat shock protein (HSP) family and the other to manganese superoxide dismutase (MnSOD), and both had a high degree of homology to human counterparts. The coding sequences were expressed in Escherichia coli as six-histidine tagged recombinant proteins and generated products with molecular masses of 86.1 kDa for HSP and 22.4 kDa for MnSOD. Their IgE-binding frequencies were shown to be 69% and 75%, respectively, to 28 sera from AEDS patients with serum IgE to M. sympodialis extract, indicating that HSP and MnSOD are major M. sympodialis allergens. In inhibition immunoblotting, M. sympodialis extract could inhibit the binding of serum IgE from AEDS patients to rHSP and rMnSOD in a concentration-dependent manner. The high frequency of sera from AEDS patients, showing IgE binding to both HSP and MnSOD, indicates that these allergens, designated Mala s 10 and Mala s 11, could play a role in AEDS.     

A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect α-amylase: cDNA cloning and chromosomal location of the gene

A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of -amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric -amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosome 2, 3, 4, 6 and 7).     

Two-dimensional electrophoresis of Malassezia allergens for atopic dermatitis and isolation of Mal f 4 homologs with mitochondrial malate dehydrogenase.

The yeast Malassezia furfur is a natural inhabitant of the human skin microflora that induces an allergic reaction in atopic dermatitis. To identify allergens of M. furfur, we separated a crude preparation of M. furfur antigens as discrete spots by 2-D PAGE and detected IgE-binding proteins using sera of atopic dermatitis patients. We identified the known allergens, Mal f 2 and Mal f 3, and determined N-terminal amino acid sequences of six new IgE-binding proteins including Mal f 4. The cDNA and genomic DNA encoding Mal f 4 were cloned and sequenced. The gene was mitochondrial malate dehydrogenase and encoded Mal f 4 composed of 315 amino acids and a signal sequence of 27 amino acids. We purified Mal f 4, which had a molecular mass of 35 kDa from a membrane fraction of a lysate of cultured cells. Thirty of 36 M. furfur-allergic atopic dermatitis patients (83.3%) had elevated serum levels of IgE to purified Mal f 4, indicating that Mal f 4 is a major allergen. There was a significant correlation of the Phadebas RAST unit values of Mal f 4 and the crude antigen, but not between Mal f 4 and the known allergen Mal f 2.     

Molecular cloning and the allergenic characterization of tropomyosin from Tyrophagus putrescentiae

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.     

Comparison of mRNA and protein levels of four members of the protein 4.1 family: the type II brain 4.1/4.1B/KIAA0987 is the most predominant member of the protein 4.1 family in rat brain

The human gene for the fourth member of the protein 4.1 family, KIAA0987, was recently identified by comprehensive cDNA analysis. To further characterize the corresponding gene and its product in rats, we cloned and sequenced rat KIAA0987 cDNA. RNA blot analyses revealed that the rat KIAA0987 gene was abundantly expressed only in the brain, kidney, and testis. Although we have previously reported that the third member of the protein 4.1 family, the KIAA0338 gene product, is predominantly expressed in rat brain, and thus was named brain 4.1, quantitative RNA blot analyses indicated that KIAA0987 should be called something other than brain 4.1 because the level of KIAA0987 mRNA was found to be of the same order as that of KIAA0338 mRNA. Our quantitative immunoblot analysis showed that the most predominant member of the protein 4.1 family at the protein level was the product of the KIAA0987 gene, not that of the KIAA0338 gene. Taking these results together, we consider it reasonable to name the KIAA0338 and KIAA0987 gene products 'type I brain 4.1' and 'type II brain 4.1,' respectively, because these two products were found to be more prominently produced in rat brain than the other two members of the protein 4.1 family, erythroid 4.1 and 4.1G.     

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.     

The gene for the human mast cell high-affinity IgE receptor alpha chain: chromosomal localization to Iq21-q23 and RFLP analysis.

We have used a cDNA probe for the human IgE receptor alpha chain to determine the chromosomal location for the human gene. A combination of Southern blot analysis of panels of somatic-cell hybrid DNAs and chromosomal in situ hybridization has localized the gene to the long arm of chromosome 1 at q21-q23. With this cDNA probe, we have also identified an RsaI RFLP which is inherited in a Mendelian fashion. This polymorphic marker on chromosome 1 should be valuable for studies directed at the identification both of diseases involving the IgE receptor alpha chain and of genetic factors affecting the allergic response.     

Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments. All of these fusion polypeptides bound IgE, and the C-terminal 94-residue fragment appeared to bind more IgE. Using synthetic peptides we identified S219-N233 (S(219)GFAPEFLKEAFGVN(233)) as the dominant IgE-binding epitope. Alanine scanning of this epitope indicated that six amino acids (E224, F225, L226, F230, G231, and V232) contributed most to IgE binding. Among these amino acids, only G231 of soybean G2a is not conserved in soybean G1a (S234) and peanut Ara h 3 (Q256). Synthetic peptides corresponding to the equivalent regions in G1a and Ara h 3 bound IgE in the order Ara h 3>/=soybean G2a>soybean G1a. This sequence represents a new IgE-binding epitope that occurs in a highly conserved region present in legume glycinins. Such IgE-binding sites could provide a molecular explanation for the IgE cross-reactivity observed between soybean and peanut proteins.