Crystallization and preliminary crystallographic analysis of the major horse allergen Equ c 1

Grégoire, Christophe; Tavares, Gisele; Lorenzo, Hans K.; Dandeu, J P; David, B.; Alzari, Pedro M.

doi:10.1107/s0907444998015510

Cited by 19 publications

(15 citation statements)

References 13 publications

(4 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Equ c 1 Dimer-Equ c 1 is found to form a dimer in the crystal, in agreement with gel filtration experiments in solution at neutral pH (4). The crystallographic dimer is formed by the side-to-side packing of the ␤-barrels through the interaction of strands ␤F, ␤G, and ␤H from each monomer, with the two ␣-helices on the same side of the dimer and running antiparallel to each other (Fig.…”

supporting

confidence: 60%

“…Briefly, rSLG Equ c 1 was expressed as a His-tagged protein in Escherichia coli, purified by metalion affinity chromatography, treated with factor Xa to excise the His tag, and concentrated to 6.0 mg/ml for crystallization. Tetragonal bipyramidal crystals, space group P4 1 2 1 2, were grown at 291 K using the hanging drop vapor diffusion technique (4). A first diffraction data set (with an overall R-merge value of 8.9%) was collected at 2.9 Å resolution from a flash-frozen (110 K) crystal using synchrotron radiation at EMBL/DESY (Hamburg).…”

Section: Methodsmentioning

confidence: 99%

“…Crystallization and Data Collection-Protein expression, purification, and crystallization of recombinant sublingual gland (rSLG) Equ c 1 have been previously described (3,4). Briefly, rSLG Equ c 1 was expressed as a His-tagged protein in Escherichia coli, purified by metalion affinity chromatography, treated with factor Xa to excise the His tag, and concentrated to 6.0 mg/ml for crystallization.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Crystal Structure of the Allergen Equ c 1

Lascombe¹,

Grégoire²,

Poncet³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

supporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Crystal Structure of the Allergen Equ c 1

Lascombe¹,

Grégoire²,

Poncet³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The occurrence of Bet v 1-dimers in pollen extracts as well as in recombinant allergen preparations has previously been observed in SDS-PAGE gels and Western blots (9 -13). Furthermore, many important allergens are known to appear as homodimers or -oligomers in nature, e.g., Phl p 1 (14), Phl p 5b (15), Phl p 7 (16), and Phl p 11 (17) from grass pollen; Fel d 1 from cat dander (18); parvalbumin from cod (19); the panallergen tropomyosin (20,21); Api m 4 from bee venom (22); Equ c 1 from horse (23,24); Sol i II from fire ant (25); Ara h 1 (26,27) and Ara h 2 (28) from peanut; ABA-1 from Ascaris (helminth) (29,30); bovine ␤-lactoglobulin (31-34) and bovine dander allergen (35); BGP-2 from Bermuda grass pollen (36); or Ves v 5 from wasp venom (37).…”

mentioning

confidence: 99%

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

Schöll¹,

Kalkura

Shedziankova

et al. 2005

The Journal of Immunology

View full text Add to dashboard Cite

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

show abstract