ABSTRACT
Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100µg/dose) or rTAT-Che a 3 (100µg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-ß secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-ß- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.
Subject(s)
Allergens/administration & dosage , Antigens, Plant/administration & dosage , Calcium-Binding Proteins/administration & dosage , Gene Products, tat/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Rhinitis, Allergic, Seasonal/therapy , Sublingual Immunotherapy , Animals , Antigens, Plant/genetics , Calcium-Binding Proteins/genetics , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Female , Forkhead Transcription Factors/genetics , Gene Products, tat/genetics , Mice , Mice, Inbred BALB C , Rhinitis, Allergic, Seasonal/immunology , Spleen/cytology , Spleen/immunology , T-Box Domain Proteins/genetics , T-Lymphocytes/immunologyABSTRACT
Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Escherichia coli/metabolism , Magnoliopsida/chemistry , Magnoliopsida/metabolism , Peptides/chemistry , Amino Acid Sequence , Carrier Proteins/genetics , Cloning, Molecular/methods , Escherichia coli/genetics , Magnoliopsida/genetics , Molecular Sequence Data , Peptides/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , SolubilityABSTRACT
BACKGROUND: Allergens are mostly composed of glycoprotein structures. It is believed that glycan-specific antibodies may lead to false-positive reactions in immunoassays. In this study we investigated the glycosylation state of grape allergens as well as the presence of antibodies to cross-reactive carbohydrate determinants (anti-CCDs) in sera from grape-sensitive individuals. METHODS: Grape extract proteins were electrotransferred onto PVDF membranes and their glycosylation states were analyzed by blotting methods. To assess the presence of anti-CCDs, natural and mildly deglycosylated proteins were immunoblotted with grape-allergic subjects' sera. We also measured the IgE reactivity of each subject's sera with other fruit extracts via an indirect ELISA. RESULTS: Immunoblotting studies showed that mildly deglycosylated grape proteins had lower IgE-binding capacity than their intact natural counterparts, which could be due to the presence of anti-CCDs. Biotinylation studies confirmed that the glycosylation levels of the 24, 32, and 60 kDa IgE-reactive proteins were higher than those of the 38 and 45 kDa ones. Lectin blotting showed that the 24 and 60 kDa bands were highly mannosylated, with the highest level of mannosylation on the 24 kDa allergen. CONCLUSION: This study showed that some grape allergens are glycosylated and that anti-CCD antibodies may cause weakly false-positive results during assessment of IgE reactivity to grape allergens.
