Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cells

Authors

  • Jacquet,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Haumont,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Massaer,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Daminet,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Garcia,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Mazzu,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Jacobs,

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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  • Bollen

    1. Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
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Jacquet Universite Libre de Bruxelles, Rue des Professeurs Jeener et Brachet, 12, B-6041 Gosselies, Belgium.

Abstract

Background

The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy.

Objective

This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells.

Methods

The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity.

Results

Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive.

Conclusions

The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy.

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