RESUMO
BACKGROUND: Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model. METHODS: Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines. RESULTS: Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines. CONCLUSION: Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered.
Assuntos
Alérgenos/imunologia , Reações Antígeno-Anticorpo/imunologia , Imunoglobulina E/imunologia , Lisina/imunologia , Proteínas de Plantas/imunologia , Ribonucleases/imunologia , Alérgenos/biossíntese , Alérgenos/genética , Sequência de Aminoácidos , Reações Antígeno-Anticorpo/genética , Basófilos/imunologia , Basófilos/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Dicroísmo Circular , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Pirofosfatases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Ribonucleases/biossíntese , Ribonucleases/genética , Testes CutâneosRESUMO
The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formosensis (Taiwan viper). It is a heterodimer of two highly homologous (65% identity) but oppositely charged subunits: a basic and neurotoxic PLA(2) (RV-4) and an acidic non-toxic component with a very low enzymatic activity (RV-7). The crystal structure of the complex has been determined by molecular replacement and refined to 1.9 A resolution and an R factor of 22.3% with four RV-4/RV-7 complexes in the asymmetric unit, which do not exhibit any local point-group symmetry. The complex formation decreases the accessible surface area of the two subunits by approximately 1425 A(2). Both PLA(2)s are predicted to have very low, if any, anticoagulant activity. The structure of viperotoxin F is compared with that of the heterodimeric neurotoxin vipoxin from the venom of another viper, V. ammodytes meridionalis. The structural basis for the differences between the pharmacological activities of the two toxins is discussed. The neutralization of the negative charge of the major ligand for Ca(2+), Asp49, by intersubunit salt bridges is probably a common mechanism of self-stabilization of heterodimeric Viperinae snake-venom neurotoxins in the absence of bound calcium.