What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?
Article first published online: 3 APR 2002
Clinical & Experimental Allergy
Volume 32, Issue 3, pages 448–454, March 2002
How to Cite
Dìaz-Perales, A., Sánchez-Monge, R., Blanco, C., Lombardero, M., Carillo, T. and Salcedo, G. (2002), What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?. Clinical & Experimental Allergy, 32: 448–454. doi: 10.1046/j.1365-2222.2002.01306.x
- Issue published online: 3 APR 2002
- Article first published online: 3 APR 2002
- Submitted 19 April 2001; revised 9 July 2001; accepted 31 August 2001
- class I chitinases;
- hevein-like domain;
- latex hevein;
- latex–fruit syndrome;
- plant panallergens
Background Class I chitinases are the major panallergens in fruits associated with the latex–fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial.
Objective We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model.
Methods Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed.
Results Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex–fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70–90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results.
Conclusions Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens.