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- Kaiser, L, et al.
(författare)
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Structural characterization of the tetrameric form of the major cat allergen Fel d 1
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 370:4, s. 714-727
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Tidskriftsartikel (refereegranskat)abstract
- Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.
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- Saarne, T, et al.
(författare)
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Cloning and characterisation of two IgE-binding proteins, homologous to tropomyosin and alpha-tubulin, from the mite Lepidoglyphus destructor
- 2003
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Ingår i: International archives of allergy and immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 130:4, s. 258-265
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Tidskriftsartikel (refereegranskat)abstract
- <i>Background:</i> The dust mite <i>Lepidoglyphus destructor</i> is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from <i>L. destructor</i> (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from <i>L. destructor</i>, and to evaluate their IgE-binding reactivities. <i>Methods:</i> PCR and screening with sera from <i>L. destructor</i>-sensitised individuals were used to isolate new clones from a phage display <i>L. destructor</i> cDNA library. The complete coding sequences of the clones were determined and expressed as His<sub>6</sub>-tagged recombinant proteins in <i>Escherichia coli</i>. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. <i>Results:</i> Two new clones, showing homology to tropomyosin and α-tubulin in several species, were isolated from the phage display <i>L. destructor</i> cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and α-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. <i>Conclusions:</i> Two new allergens from <i>L. destructor</i> have been identified and can now be added to the repertoire of recombinant <i>L. destructor</i> allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.
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