Structural and functional studies of Malassezia sympodialis-derived allergens

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medicine

Sammanfattning: Atopic eczema (AE) is a chronic inflammatory skin disease characterised by pruritic (itchy) skin lesions. The pathogenic mechanisms underlying AE are still unclear, although several factors such as genetic predisposition, a dysfunctional skin barrier, exposure to environmental allergens and skin colonisation with microorganisms appear to be of importance. Malassezia sympodialis is a yeast which is part of our normal cutaneous flora. Approximately 50% of adult AE patients have serum IgE specific for M. sympodialis allergens or display immediate-type skin reactions against crude extracts of this yeast, while such reactivity is rarely observed in other allergic diseases, indicating that sensitization to this yeast is associated with AE. Ten allergens from M. sympodialis have been cloned to date, designated Mala s 1 and Mala s 5-13. Six of these exhibit sequence homology to known proteins whereas four do not. The aim of this thesis has been to gain knowledge of host-microbe interactions of allergens from M. sympodialis in the pathogenic mechanisms of AE and in healthy individuals. This has been accomplished by studying structural properties and cellular interactions, with a special focus on the two allergens Mala s 1 and Mala s 11. Mala s 1 is a major allergen mainly localized in the yeast cell wall and exposed on the cell surface. Interestingly, Mala s 1 does not exhibit any significant sequence homology to known proteins. We have solved the crystal structure of Mala s 1 by singlewavelength anomalous dispersion techniques using selenomethionine-substituted Mala s 1. Mala s 1 folds into a six-fold beta-propeller, a novel fold among allergens. The putative active site of Mala s 1 overlaps structurally with putative active sites in potential homologues, Q4P4P8 and Tri 14, from the plant parasites Ustilago maydis and Gibberella zeae, respectively. This resemblance suggests that Mala s 1 and the parasite proteins may have similar functions. In addition, we demonstrate that Mala s 1 binds to phosphatidylinositol (PtdIns) (3)-phosphate (P), PtdIns(4)P, and PtdIns(5)P, lipids possibly playing a role in the localization of Mala s 1 to the cell surface. Mala s 11 displays a high degree of sequence homology to human manganese superoxide dismutase (hMnSOD). In AE patients sensitized to M. sympodialis, hMnSOD can elicit positive skin prick tests or atopy patch tests along with binding IgE, suggesting an autoimmune response. We report that Mala s 11 is able to inhibit IgE-binding to hMnSOD and vice versa, indicating that these two homologues share common IgE epitopes. We have also identified residues possibly involved in such cross-reactivity. In addition, we compared the influence of Mala s 11 and hMnSOD on human dendritic antigen presenting cells. Whereas rhMnSOD did not affect the phenotype of monocytederived dendritic cells (DCs), rMala s 11 up-regulated maturation markers and induced significantly higher levels of inflammatory cytokines in the culture supernatants. This suggests that DCs from healthy individuals possess the ability to distinguish between Mala s 11 and hMnSOD despite their high homology. Whether this is also the case for DCs in AE patients remains to be clarified. In summary, we have determined a novel three dimensional structure not previously demonstrated among allergens. We demonstrate the ability of DCs to distinguish between proteins with high sequence homology and we provide a structural basis underlying the autoimmune response to hMnSOD in AE based on IgE-mediated cross-reactivity.

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