Crystal Structure of the Allergen Equ c 1

Abstract

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 Å resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a ␤-barrel flanked by a C-terminal ␣-helix. The space between the two ␤-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.The incidence of allergic diseases is increasing in developed countries resulting from growing exposure to allergens, altered stimulation of the immune system during development, and probably facilitating an adjuvant effect of the environment. Allergy to animals is distinguishable by its intensity and the possibility of sensitization by a limited contact with danders or hair. The animals responsible for allergy are obviously familiar domestic cats and dogs, but also the horse, which is often incriminated. Five main horse allergens have been isolated and purified (1, 2). Among these, the Equ c 1 protein (molecular mass 21.5 kDa, 187 amino acids, pI ϭ 4.5) has been defined as a major allergen, because it induces an IgE-mediated type I allergic reaction in a majority of the patients allergic to horses.Equ c 1 belongs to the lipocalin family (3, 4). Although members of this family display low sequence similarity (often lower than 20% of amino acid identities), all share a conserved folding pattern, an 8-stranded ␤-barrel flanked by an ␣-helix at the C-terminal end of the polypeptide chain. The lipocalin ␤-barrel often defines a central apolar cavity that serves for the binding and transport of small hydrophobic molecules such as retinol (retinol-binding protein (5, 6)), odorant molecules (bovine odorant-binding protein (7, 8)), or pheromones (mouse major urinary protein, mMUP 1 (9), and rat urinary ␣2-globulin (10)). Several lipocalins have been described as allergenic proteins, among which the mouse major urinary protein mMUP (11), rat urinary ␣2-globulin rat urinary ␣2-globulin (12), bovine ␤-lactoglobulin (13), cockroach allergen Bla g 4 (14), dog allergens Can f 1 and Can f 2 (15), and bovine allergen Bos d 2 (16).Whether a protein exhibits special structural characteristics that are responsible for its allergenic properties is an issue that remains poorly understood. Although the three-dimensional structures of some allergenic lipocalins are known (mMUP (9), rat urinary ␣2-globulin (10), bovine ␤-lactoglobulin (17, 18), and Bos d 2 (19)), little information is availa...

Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis

“…This organization is unlikely to be a crystallization artifact, because the monomer-monomer interfaces are larger for VDE than for other lipocalins well known to be dimers (22,43,44). Our docking experiments support the identification of the violaxanthinbinding site in a VDE dimer, and mutational analysis is consistent with in silico calculations.…”

Structures of Two Major Allergens, Bla g 4 and Per a 4, from Cockroaches and Their IgE Binding Epitopes

“…The 9th ␤-strand (␤I) can be found in many lipocalins but was absent in Bla g 4 and Per a 4. In Bla g 4, a short helical segment preceding the ␤A strand crossed and closed the bottom of the ␤-barrel, whereas the CD loop and three 3 10 helices from the AB loop, EF loop, and GH loop surrounded the entrance of the cavity on top of the ␤-barrel (Fig. 1, B and C).…”

“…There is no significant hydrophobic interaction involved in the dimeric interface, a case that is very different for other lipocalins. For instance, Equ c 1 has an extended hydrophobic patch at the center of the dimeric interface (10). The dimeric interface of Per a 4 buried an area of 858 Å 2 , which is ϳ10% of the total solvent accessible surface.…”

“…Generally, structures of lipocalins are made up of eight anti-parallel ␤-strands forming a ␤-barrel, which contains a ligand binding cavity (7). Several allergenic lipocalin proteins have been characterized so far, such as Bos d 2 (8), Bos d 5 or bovine ␤-lactoglobulin (9), Equ c 1 (10), Mus m 1 (11), and rat urinary ␣2 globulin (12). Although several crystal structures of these lipocalins have been determined, exact IgE epitope residues of these allergens are not well understood.…”

NMR Solution Structure of Ole e 6, a Major Allergen from Olive Tree Pollen

“…Structural comparison of proteins that induce an allergenic response can be used to predict allergenic cross-responses (52), eventually to determine possible characteristics of IgE recognition (50), and to predict the regions recognized by the immunoglobulins (10,53,54). Comparison of the whole sequence of Ole e 6 with the Swiss-Prot data base had shown only homology with a cysteine-rich putative protein from N. tabacum (55) of unknown three-dimensional structure.…”