title: A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.
abstract: Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.
authors.affiliation: Department of Pathophysiology, University of Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Flicker
authors.forename: S
authors.initials: S
authors.lastname: Vrtala
authors.forename: P
authors.initials: P
authors.lastname: Steinberger
authors.forename: L
authors.initials: L
authors.lastname: Vangelista
authors.forename: A
authors.initials: A
authors.lastname: Bufe
authors.forename: A
authors.initials: A
authors.lastname: Petersen
authors.forename: M
authors.initials: M
authors.lastname: Ghannadan
authors.forename: W R
authors.initials: WR
authors.lastname: Sperr
authors.forename: P
authors.initials: P
authors.lastname: Valent
authors.forename: L
authors.initials: L
authors.lastname: Norderhaug
authors.forename: B
authors.initials: B
authors.lastname: Bohle
authors.forename: H
authors.initials: H
authors.lastname: Stockinger
authors.forename: C
authors.initials: C
authors.lastname: Suphioglu
authors.forename: E K
authors.initials: EK
authors.lastname: Ong
authors.forename: D
authors.initials: D
authors.lastname: Kraft
authors.forename: R
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity / Q000235:genetics; D001491:Basophils / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D002942:Circular Dichroism; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006109:Poaceae / Q000737:chemistry / Q000276:immunology*; D011058:Pollen / Q000737:chemistry* / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D003313:Zea mays / Q000737:chemistry / Q000276:immunology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C095238:PHLPVI protein, Phleum pratense; D010446:Peptide Fragments; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E