Abstract
An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10(-3)-10(-5) micrograms/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 microgram/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: beta-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (approximately 20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.
Highlights
Inhalation of environmental allergens, derived from pollens, dust mites, animal danders, insects, and fungi, is the most common cause of IgE antibody responses in humans
We report that a 21-kDa CR allergen, Bla g 4, is a ligand binding protein, or calycin, and show that this family contains several allergens that are a common cause of IgE antibody responses, including mouse and rat urinary proteins, and -lactoglobulin
The allergen encoded by clone bg12 A was provisionally designated B. germanica allergen 4, Bla g 4, in keeping with the revised WHO/IUIS allergen nomenclature [16]
Summary
CDNA Cloning—Total RNA was isolated from 6-g adult B. germanica, of mixed sexes, using 5 M guanidinium isothiocyanate, 0.01 M EDTA, 5% 2-mercaptoethanol, 0.05 M Tris-HCl, pH 7.5 [14]. The B. germanica cDNA library was plated on NZY agar and screened using IgE antibodies. Immunoassay for IgE Antibodies to rBla g 4—IgE anti-Bla g 4 ab were measured in sera from 73 CR-allergic asthmatic patients, using an antigen-binding radioimmunoassay [19]. Serum dilutions of 1:2 and 1:10 were incubated with 125I-rBla g 4 (ϳ100,000 cpm added) for 4 h at room temperature and precipitated overnight at 4 °C with 50 l of sheep anti-human IgE (The Binding Site, San Diego, CA). Immediate Skin Testing—Quantitative intradermal skin tests were performed using serial 10-fold dilutions of B. germanica extract (1/20 w/v, Allergy Laboratories of Ohio, Columbus, OH) or purified recombinant Bla g 4 from 100–10Ϫ5 g/ml, as described previously [12, 19]. The Southern blot was hybridized with [␣-32P]dCTPlabeled 650-bp Bla g 4 cDNA probe at 37 °C and autoradiographed following a 3-h exposure to Kodak XAR film
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