Identification, characterization, and cloning of a complementary DNA encoding a 60-kd house dust mite allergen (Der f 18) for human beings and dogs.

BACKGROUND: House dust mites of the Dermatophagoides genus are the most important cause of perennial allergic disease in both humans and companion animals. Although the major mite allergens for humans are proteins of relatively low molecular weight, this is not the case for dogs. Western blotting shows that canine anti-mite IgE responses are directed primarily toward proteins in the molecular weight range of 50 to 120 kd. OBJECTIVE: The objectives of this study were to characterize a D farinae allergen with a molecular weight of approximately 60 kd and to isolate the cDNA coding for this allergen. METHODS: A protein of apparent molecular weight of 60 kd was identified by Western blotting by using canine serum IgE from house dust mite-sensitized atopic dogs. The protein was purified from homogenized D farinae mite bodies by ammonium sulfate precipitation, followed by gel filtration and cation exchange HPLC. The presence of IgE directed to the 60-kd protein in sera from humans and dogs with dust mite allergy was measured by FcepsilonRIalpha-based ELISA. A cDNA encoding a full-length 60-kd protein was isolated from a D farinae cDNA library by a combination of both PCR amplification and hybridization screening. A panel of mAbs specific for the 60-kd protein was generated and used to localize the protein in whole body sections of D farinae mites. RESULTS: ELISA showed that the purified protein bound IgE in 54% of the sera from patients with D farinae allergy. In addition, the 60-kd protein was able to bind IgE in 57% to 77% of D farinae -sensitized dogs. A cDNA was isolated that encoded a protein of 462 amino acids, consisting of a 25 amino acid signal sequence and a 437 amino acid mature protein. The calculated molecular weight of the mature protein is 50 kd, and the amino acid sequence contains a single N-glycosylation site. A protein database search showed homology with multiple chitinases. A mAb specific for the 60-kd chitinase recognized the allergen in the mite digestive system, but fecal pellets did not stain positively for this allergen. CONCLUSIONS: A 60-kd D farinae protein (Der f 18), with homology to chitinase, is a major allergen for humans and dogs sensitive to house dust mites.

Expression and characterization of recombinant canine IL-13 receptor alpha2 protein and its biological activity in vitro.

Our previous study showed that recombinant canine IL-13 (rcaIL-13) stimulated production of allergen-specific IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs. This has also been demonstrated using human IL-13 (huIL-13) and PBMC isolated from human allergy patients. The stimulatory activity of rcaIL-13 was specifically inhibited by a fusion protein of the extracellular domain of canine IL-13Ralpha2 and the Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). In this communication, we report the construction and expression of a non-fused recombinant extracellular domain of canine IL-13Ralpha2 (rcaIL-13Ralpha2) in an E. coli expression system. The E. coli expressed rcaIL-13Ralpha2 was isolated in inclusion bodies, then solubilized in buffer containing denaturants and reducing agents. After refolding and purification, the biological activity of rcaIL-13Ralpha2 was found in the monomer fraction resulting from gel filtration and ion exchange chromatographies. Biological activity of purified rcaIL-13Ralpha2 was demonstrated by the specific inhibition of rcaIL-13 activity in a TF-1 cell proliferation assay. Additionally, rcaIL-13Ralpha2 was found to be active in neutralizing rcaIL-13 induced upregulation of IgE mRNA levels in PBMCs of "high IgE" dogs, which have been bred to exhibit a predisposition for high IgE production and are used as a model for allergic asthma. The data confirm our previous report that the regulatory effects of IL-13 on IgE production in canine PBMCs are similar to those reported in humans. Thus, allergic dogs, such as the "high IgE" producing dogs, may be excellent models for research on IgE-mediated diseases in humans.