title: Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1.
abstract: 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.
authors.affiliation: Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria.
authors.forename: Tanja
authors.initials: T
authors.lastname: Ball
authors.forename: William
authors.initials: W
authors.lastname: Edstrom
authors.forename: Ludwig
authors.initials: L
authors.lastname: Mauch
authors.forename: Jacky
authors.initials: J
authors.lastname: Schmitt
authors.forename: Bernd
authors.initials: B
authors.lastname: Leistler
authors.forename: Helmut
authors.initials: H
authors.lastname: Fiebig
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Sperr
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Hauswirth
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Dietrich
authors.initials: D
authors.lastname: Kraft
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D013058:Mass Spectrometry; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D018411:Spodoptera
chemical_list: D000485:Allergens; D000939:Epitopes; D010940:Plant Proteins; D011994:Recombinant Proteins; C090396:PHLPI protein, Phleum pratense; D007073:Immunoglobulin E