Abstract
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 NH2-terminal sequence of the natural protein and the deduced amino acid sequence from cDNA were identical except for one substitution (Phe9 --> 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 x-ray 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.
Highlights
Cockroaches produce potent allergens, which give rise to IgE antibody (Ab)1 responses in genetically predisposed individuals living in cockroach-infested housing [1,2,3,4,5,6,7,8]
18 of the 26 invariable residues identified in mammalian glutathione S-transferase (GST) by x-ray crystallography were present in B. germanica GST, including Tyr-8, Gln-63, and Asp-97, which are involved in glutathione binding at the G-site of the molecule [17, 18, 31,32,33,34,35]
We have reported the identification, purification, molecular cloning, and sequencing of a glutathione S-transferase from the cockroach Blattella germanica
Summary
Antibody; GST, glutathione S-transferase; Bla g 5, Blattella germanica allergen 5; PAGE, polyacrylamide gel electrophoresis; RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; CDNB, 1-chloro-2,4-dinitrobenzene. 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). 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]. Homologous GST allergens were identified in the house dust mite, Dermatophagoides pteronyssinus, and in the helminth parasite Schistosoma mansoni [20, 21], defining the importance of the GST superfamily in causing IgE antibody responses
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