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Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the development of asthma in genetically predisposed individuals. We compared the allergenic importance of two cockroach (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid sequence of the major 36-kDa allergen, Bla g2. A survey of 106 sera from CR allergic patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2 to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gave positive reactions using 10(-3) micrograms/ml either allergen, whereas controls showed no response to 10 micrograms/ml. Natural Bla g 2 was purified and the sequence of the NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and a 328-amino acid mature protein, which showed the highest degree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%), with similar identity to pepsin, cathepsins D and E, renin, and chymosin. Bla g 2 mRNA and protein were detected in B. germanica, but not in Periplaneta americana, the other principal domiciliary CR species in the U.S. High concentrations of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventriculus). The results show that Bla g 2 is a major species-specific allergen of B. germanica and suggest that the allergen functions as a digestive enzyme in the cockroach.

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We report that a major 23-kDa allergen from German cockroach (Blattella germanica) is a glutathione S-transferase (EC 2.5.1.18; GST). Natural B. germanica GST, purified from cockroach body extracts by glutathione affinity chromatography, and recombinant protein expressed in Escherichia coli using the pET21a vector, showed excellent IgE antibody binding activity. B. germanica GST caused positive immediate skin tests in cockroach-allergic patients using as little as 3 pg of recombinant protein. The NH 2 -terminal sequence of the natural protein and the deduced amino acid sequence from cDNA were identical except for one substitution (Phe 9 3 Cys). Assignment of this protein to the GST superfamily was based on binding to glutathione and sequence identity (42-51%) to the GST-2 subfamily from insects, including Anopheles gambiae and Drosophila melanogaster. B. germanica GST contained 18 of the 26 invariable residues identified in mammalian GST by xray crystallography and exhibited enzymic activity against a GST substrate. Our results show that cockroach GST causes IgE antibody responses and is associated with asthma. The data strongly support the view that the immune response to GST plays an important role in allergic diseases.Cockroaches produce potent allergens, which give rise to IgE antibody (Ab) 1 responses in genetically predisposed individuals living in cockroach-infested housing (1-8). IgE Ab to cockroach are strongly associated with asthma, and sensitization to cockroach allergens is a major risk factor for hospital emergency room visits for asthma (5,9,10). Although cockroach may carry viral and bacterial pathogens, asthma is the only disease for which an unequivocal causal relationship with cockroach exposure has been established (9 -11). Over the last few years, several allergens from the principal domiciliary cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), have been cloned and insights into their biological function have been obtained (12-15). B. germanica allergen Bla g 2 shows sequence homology to aspartic proteases, whereas Bla g 4 is a ligand-binding protein or calycin (12, 13). In Taiwan, P. americana is an important cause of asthma, and a 72-kDa allergen, Per a 3, has recently been cloned, which shows homology to insect hemolymph proteins (arylphorins) (14).We have identified an important B. germanica allergen with sequence homology to the glutathione S-transferase (GST) superfamily. These enzymes are involved in the detoxification of endogenous and xenobiotic toxic compounds and are widely distributed in most forms of life (16 -18). In molecular biology, plasmid vectors have been constructed to express foreign proteins in Escherichia coli, as fusion proteins with the COOH terminus of GST from Schistosoma japonicum. These high level expression systems (pGEX vectors) have been widely used for purification of recombinant proteins by glutathione affinity chromatography (19).Here, we report the sequence, purification, and recombinant expression...

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 g/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 g/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: ␤-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (ϳ20%), 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.

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Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of ϳ100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.