Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1.
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.
Ball
Tanja
T
Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria.
Edstrom
William
W
Mauch
Ludwig
L
Schmitt
Jacky
J
Leistler
Bernd
B
Fiebig
Helmut
H
Sperr
Wolfgang R
WR
Hauswirth
Alexander W
AW
Valent
Peter
P
Kraft
Dietrich
D
Almo
Steven C
SC
Valenta
Rudolf
R
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
D000485:Allergens; D000939:Epitopes; D010940:Plant Proteins; D011994:Recombinant Proteins; C090396:PHLPI protein, Phleum pratense; D007073:Immunoglobulin E