Molecular Immunology 46 (2008) 294–303 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Mutational analysis of amino acid residues involved in IgE-binding to the Malassezia sympodialis allergen Mala s 11 Monica Vilhelmsson a,b,c , Andreas G. Glaser b , Daniel Badia Martinez c , Margit Schmidt d , Catharina Johansson a , Claudio Rhyner b , Kurt D. Berndt e,f , Annika Scheynius a , Reto Crameri b , Adnane Achour c,*,1 , Arezou Zargari g,1 a Clinical Allergy Research Unit, Department of Medicine Solna, Karolinska Institutet and Karolinska University Hospital Solna, 171 76 Stockholm, Sweden b Swiss Institute of Allergy and Asthma Research (SIAF), Oberestrasse 22, CH-7270 Davos, Switzerland c Centre for Infectious Medicine, F59, Department of Medicine Huddinge, Karolinska Institutet and Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden d Department of Biology, East Carolina University, Greenville, NC 27858, USA e Department of Biosciences and Nutrition, Karolinska Institutet,141 57 Huddinge, Sweden f Department of Life Sciences, Södertörns Högskola, 141 89 Huddinge, Sweden g InDex Pharmaceutical AB, Scheeles väg 2, 171 77 Stockholm, Sweden article info Article history: Received 24 June 2008 Received in revised form 22 July 2008 Accepted 23 July 2008 Available online 14 October 2008 Keywords: IgE Cross-reactivity MnSOD Atopic eczema Allergy Malassezia sympodialis abstract The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSOD we identified four regions each composed of 4–5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Atopic eczema (AE) is a chronic inflammatory skin disease char- acterised by pruritic inflammatory skin lesions (Bieber, 2008). Although the pathogenic mechanisms underlying AE remain largely unknown several factors such as genetic predisposition, exposure to environmental allergens and skin colonization with microor- ganisms appear to be of importance (Akdis et al., 2006). The * Corresponding author. Tel.: +46 8 5248 6232; fax: +46 8 30 42 76. E-mail address: adnane.achour@ki.se (A. Achour). 1 Shared last authorship. opportunistic yeast Malassezia sympodialis is a member of the nor- mal cutaneous flora (Scheynius et al., 2002). Approximately 50% of adult AE patients have serum IgE specific for M. sympodialis aller- gens or show immediate-type skin reactions against crude extracts of this yeast (Schmid-Grendelmeier et al., 2006) while such reactiv- ity is very rare in other allergic diseases, (Casagrande et al., 2006) suggesting an association between M. sympodialis sensitization and AE. One contributing factor to the special host–microbe interaction between AE and M. sympodialis is likely to be the dysfunctional skin barrier characteristic for AE as a result of dryness and scratching (Leung et al., 2004) which facilitates the interaction between the yeast and antigen presenting cells in the skin. In addition, the higher pH associated with AE skin induces an increased release of allergens 0161-5890/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2008.07.036