Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity

Abstract

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1-specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross-reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut-specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.Keywords: IgE; protein engineering; allergy; immunotherapy Specific immunotherapy is the only curative treatment of IgE mediated allergy. It is based on repeated injections of increasing doses of crude allergen extracts and exhibits variable efficacy. Crude allergen extracts have the major drawback of being highly allergenic. They present the risk of inducing severe anaphylactic side effects upon injection. Thus, there is a need for new molecules derived from allergens to perform safer immunotherapy. Recent advances in the understanding of immunotherapy have underlined the major role played by the allergen-specific CD4 + T lymphocytes in the control of the allergic response and the induction of tolerance (Akdis and Blaser 1999). These cells recognize the allergens as 13 to 25 amino acid peptides, called T cell epitopes. These peptides are produced by the degradation of the allergen in the antigen presenting cells and are presented by HLA Class II molecules to CD4 + T cells. Their injection in mice in saline buffer and by different routes (Briner et al. 1993;Burkhart et al. 1999;Sundstedt et al. 2003) leads to T cell tolerance as a result of IL-10 secretion by antigen-specific CD4 + T lymphocytes (Burkhart et al. 1999;Sundstedt et al. 2003).IL-10 is an immunosuppressive cytokine, which diminishes the T cell activation and IgE secretion by B lympho- Article published online ahead of print. Article and publication date are at

Relevance of IgE binding to short peptides for the allergenic activity of food allergens

“…This finding suggests that not only conformational epitopes but also continuous determinants may require a structural context for proper epitope presentation, corroborating the view that stability of a proteins' 3-dimensional structure-especially the in case of food allergens-seems to play an important role in terms of immunogenicity and allergenicity. 41,47,48 The findings further serve to illustrate how dramatically different results may be obtained, depending on the experimental approach used for epitope identification and the method used for detection of antibody binding. Assuming that fluid phase binding of IgE antibodies is more relevant in relation to in vivo allergenicity, it is possible that solid phase approaches for epitope identification may generate partially misleading results.…”

Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening

“…Similar to the Api m 1 mutations, 25 as described in the Results section in this article's Online Repository at www.jacionline.org, some of the single mutations even indicated stabilization of the fold (combined scores <29.18). The highest combined score of 29.07 would rank on position 880 in the list of all possible 3021 single point mutations for Bet v 1, and therefore none of these mutations were considered by the algorithm used in the present study.…”

Conformational studies on Arabidopsis sulfurtransferase AtStr1 with spectroscopic methods

“…Due to the removal of the linker elongation, the protein loses tight packing of the tertiary structure and thus adopts the structural features of the so-called molten globule state. It is noteworthy that although no tight tertiary structure was detected, the percentage secondary structure of the protein often remains comparable to the wild-type protein (Buhot et al, 2004), as in the case of AtStr1wlink.…”

“…The spectra recorded between 190 and 260 nm revealed differences in the curve shape of AtStr1wlink in comparison to the three other proteins (Figure 3). Spectra of these three proteins were characterized by a positive signal below 200 nm, a minimum at 208 nm, and a shoulder at 225 nm, indicating that they adopted an ordered structure according to Buhot et al (2004), whereas the spectrum of AtStr1wlink did not show a minimum at 208 nm nor a shoulder at 225 nm.…”