Toshihiko Jyo

Cloning and characterization of a new allergen, Mag 3, from the house dust mite, Dermatophagoides farinae: Cross-reactivity with high-molecular-weight allergen

A new immunoreactive clone whose sequence is not homologous to that of any of the previously identified mite allergens was isolated by successive immunoscreening of a Dermatophagoides farinae cDNA library with rabbit antisera to an extract of the house dust mite and IgE in pooled sera from patients allergic to mites. Rabbit antibodies specific for the recombinant protein recognized a 177 kD protein in a mite body extract. This immunoreactive protein was located in the circumferential tissues of esophagus, gut and the other internal organs in mites. The reaction of human IgE to the purified natural antigen was inhibited competitively to 30% by the recombinant antigen. In terms of the frequency and the intensity of response to specific IgE in sera from asthmatic patients, the natural protein was similar to Der f2, while the recombinant protein was slightly less allergenic by these criteria. We conclude that the natural protein from the house dust mite, D. farinae, is an important allergen.

Cloning and Characterization of cDNA Coding for a New Allergen from the House Dust Mite, Dermatophagoides farinae

A cDNA clone encoding a new allergen from the Dermatophagoides farinae cDNA lambda gt11 library was isolated and sequenced. There was no amino acid sequence homology with other known allergens. The gene product, beta-galactosidase fusion protein, of the truncated cDNA on blot reacted with IgE in 13 of 43 sera from patients allergic to mites. The affinity-purified fusion protein had a potent ability to release histamine from washed blood cells of the mite-allergic patients. Human specific IgE eluted from the fusion protein band on blots recognized a 39-kD component on blots of a mite body extract.

An epitope residing in carbohydrate chains of a sea squirt antigen termed Gi-rep

O-Glycosidically linked oligosaccharides (Gp-1 beta-b) were liberated from a large size glycopeptide (GP) fraction (Gp-1) in a Pronase digest of a sea squirt antigen termed Gi-rep by the treatment with 0.1 N NaOH per 1 mol/L of NaBH4. Gp-1 beta-b as well as Gp-1 and the intact antigen was capable of inducing a remarkable erythema in the skin of patients with asthma with sea squirt allergy at its concentration of 10 micrograms/ml. N-Glycoside GP fractions (Gp-1 beta-a and Gp-2 beta) having the allergenic activity were also prepared as alkali-resistant GPs from Gp-1 and the other relatively small size GP fraction (Gp-2) of the Pronase digest, respectively, after the alkali treatment. Chemically, the three allergenically active preparations were rich in galactosamine, glucosamine, and fucose in common, although the N-linked carbohydrates were much larger in size than O-linked carbohydrates. Accordingly, it has been expected that the epitope of the sea squirt antigen corresponds to certain oligosaccharide units residing in both of the O- and N-linked carbohydrate chains of the antigen. This suggestion was consistent with the observation that the allergenic activity of Gi-rep was significantly unstable to the periodate oxidation but substantially stable not only to acid, alkali, and heat treatments but also to the enzymatic proteolysis with Pronase E.

Therapeutic effect and titers of the specific IgE and IgG antibodies in patients with sea squirt Allergy (Hoya asthma) under a long-term hyposensitization with three sea squirt antigens

Hyposensitization therapy with each of three sea squirt antigens, Gi-rep (molecular weight [MW] 106,000), Ei-M (MW 22,800), and DIIIa (MW 9980), have succeeded in 72%, 90%, and 36% of patients with sea squirt allergy, respectively, within the first year, and the effect has been maintained during subsequent 4-year maintenance therapy. All available sera from some of the hyposensitized patients were also examined for IgE and IgG titers against the most effective therapeutic antigen, Ei-M, and it was revealed that the Ei-M-specific IgG titer increased rapidly in the successfully hyposensitized patients, regardless of the therapeutic antigen used, except for a few patients. Furthermore, the high specific IgG titer, as well as the therapeutic effect, was maintained during the subsequent maintenance therapy. No such increase in the specific IgG titer was detected in all unsuccessfully hyposensitized patients. In contrast, the Ei-M-specific IgE titer was practically unchanged in all allergic patients, independent of the therapy. Therefore, the effect of the hyposensitization therapy was closely dependent on the induction of the specific IgG capable of competing with the specific IgE for a certain asthma-inducing antigen, like DIIIa. The apparently low therapeutic efficiency of DIIIa was attributed to its relatively low immunogenicity to induce the specific IgG.

Structure of IgE epitopes on a new 39-kD allergen molecule from the house dust mite, Dermatophagoides farinae

Two IgE epitope sequences comprising Ser56-Pro-Val-Thr-Lys-Arg-Ala-Ser-Leu-Lys-Ile-Asp-Ser-Lys-Lys70 and Asp104-Val-Glu-Leu-Ser-Leu-Arg-Ser-Ser-Asp-Ile-Ala115 were identified by deletion analysis of the cDNA encoding a new 39-kD protein of mite allergen. A synthetic dodecapeptide corresponding to the latter epitope sequence functioned as a monovalent and mite-specific hapten. Replacement of each of the 12 amino acid residues with Gly, using site-directed mutagenesis, indicated that Arg110 may play a central role in IgE binding. However, the 8 allergic sera tested exhibited a wide variation in their amino acid residues required for reactivity to IgE in allergic sera.

B Cell Mitogenic Activity of Sea Squirt Antigen

The activity of sea squirt antigen, one of the allergy-inducing substances for humans, on murine and human lymphocytes was studied in vitro. Sea squirt antigen stimulated normal mouse spleen cells to proliferate, as detected by [3H]-TdR incorporation, in a dose-dependent manner. The responder cells are B cells because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement and passing through a nylon wool column, but not with anti-Thy-1 antibody and complement. Spleen cells of C3H/HeJ mice, which are lipopolysaccharide low responders, were also stimulated as well as spleen cells of C3H/HeN mice, suggesting that this response is not due to lipopolysaccharide in the antigen fraction. Sea squirt antigen stimulated not only proliferative response of B cells, but also polyclonal immunoglobulin production. Furthermore, sea squirt antigen also stimulated human lymphocytes to proliferate and to produce immunoglobulin. All these results suggest that sea squirt antigen has mitogenic activity on B cells, and this ability is concerned with the induction of allergic reaction.

Purification of two types of sea-squirt antigens.

Under monitoring the antigenic activity by radioimmunoassay (RIA), one of the major sea-squirt antigens was isolated by gel chromatography after treatment of the extract from internal organs of sea-squirt with QAE-Sephadex. This antigen was fairly homogenous as judged from its elution profile in chromatography on a column of Ultrogel AcA 44 with respect not only of the antigenic activity but also to UV absorption. Furthermore, the antigen inhibited the binding of the 125I-labeled tracer antigen to rabbit anti-sea-squirt antibody in a similar mode to the case of Ei-2, one of the sea-squirt antigens previously prepared, but its specific activity was as high as 2.33 times that of Ei-2. Accordingly this isolated antigen was corresponded to the major antigenic substance in Ei-2 and referred to as Ei-M. In addition, the other antigen of Gi-x was also isolated by chromatography on a column of Sepharose 6B as the major antigenic substance in Gi-2, the other one of the previous antigens. Gi-x was apparently different from E-M in the mode of inhibition of the binding of the tracer antigen to the antibody, although its antigenic activity against the asthmatic patients in vivo was as high as that of Ei-M. Thus, it was considered that sea-squirt contained at least two types of antigens of Ei-M and GI-X.

Identification of T-cell epitope sequences on an important mite antigen

Background T‐cell epitopes on Der 1 and Der 2 groups, the major mite allergens, have been intensively analysed, while those on the other important allergens remain to be elucidated. We have cloned four cDNAs coding for important mite allergens on the basis of frequency and capacity of IgE binding. Stimulatory action of glutathione S‐transferase‐fused Mag1 on lymphocytes from mite‐allergic patients was relatively high among them.

T-cell epitope analysis of Mag 3, an important allergen from the house dust mite, Dermatophagoides farinae.

Here we describe the detection of T-cell epitope region on the house dust mite allergen Mag 3, which has been shown to trigger T-cell proliferation in mite-allergic asthmatic patients. We first examined murine T-cell epitope using T-cell fraction prepared from recombinant Mag 3 (r-Mag 3)-primed H-2k mice. Initial proliferation assay with truncated r-Mag 3 indicated that N-terminal 113 amino acid region was required for triggering T-cell activation. Subsequent epitope scanning with synthetic overlapping peptides revealed that T-cell reactive region was assigned within amino acid range 56-75. We also explored human T-cell determinant using specific T-cells from mite-allergic patients. Intriguingly, we found that amino acid range 56-85, a portion partially overlapping with that identified in r-Mag 3-primed mice, was exclusively recognized by T-cells from different patients. Further investigation of unique T-cell epitope region found in this study would provide insight into the development of animal therapeutic model and/or peptide vaccine for asthma.