Assuntos
Antígenos de Neoplasias/imunologia , Epitopos/imunologia , Genes de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Sequência de Aminoácidos , Anticorpos Antineoplásicos/imunologia , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/genética , Comunicação Autócrina , Sequência Consenso , Epitopos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Ativação Linfocitária , Dados de Sequência Molecular , Proteínas do Mieloma/imunologia , Biblioteca de Peptídeos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.