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

International audienceThe secreted protein Equ c 1 is the major component responsible for the induction of specific IgE antibodies in patients sensitized to horse allergens. Equ c 1 belongs to the lipocalin superfamily of hydrophobic ligand-binding proteins, which also includes other known allergens. Equilibrium sedimentation and gel-filtration studies demonstrate that both the glycosylated form of Equ c 1 purified from horse salivary glands and the non-glycosylated recombinant form expressed in bacteria exist predominantly as dimers in solution. As observed for other dimeric lipocalins, acidic pH and low protein concentration favour dimer dissociation. The recombinant form of Equ c 1 has been crystallized using ammonium sulfate as a precipitant. The crystals belong to the tetragonal space group P41212 with cell parameters a = b = 84.0, c = 56.1 A, and contain a single molecule in the asymmetric unit. A complete data set from native crystals was collected at the synchrotron source in Hamburg to 2.9 A resolution using a frozen crystal, and structure determination is in progress

cDNA Cloning and Sequencing Reveal the Major Horse Allergen Equ c1 to Be a Glycoprotein Member of the Lipocalin Superfamily

International audienceThe gene encoding the major horse allergen, designated Equus caballus allergen 1 (Equ c1), was cloned from total cDNA of sublingual salivary glands by reverse transcription-polymerase chain reaction using synthetic degenerate oligonucleotides deduced from N-terminal and internal peptide sequences of the glycosylated hair dandruff protein. A recombinant form of the protein, with a polyhistidine tail, was expressed in Escherichia coli and purified by immobilized metal affinity chromatography. The recombinant protein is able to induce a passive cutaneous anaphylaxis reaction in rat, and it behaves similarly to the native Equ c1 in several immunological tests with allergic patients' IgE antibodies, mouse monoclonal antibodies, or rabbit polyclonal IgG antibodies. Amino acid sequence identity of 49-51% with rodent urinary proteins from mice and rats suggests that Equ c1 is a new member of the lipocalin superfamily of hydrophobic ligand-binding proteins that includes several other major allergens. An RNA blot analysis demonstrates the expression of mRNA Equ c1 in liver and in sublingual and submaxillary salivary glands