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.
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.
Flicker
S
S
Department of Pathophysiology, University of Vienna, Austria.
Vrtala
S
S
Steinberger
P
P
Vangelista
L
L
Bufe
A
A
Petersen
A
A
Ghannadan
M
M
Sperr
W R
WR
Valent
P
P
Norderhaug
L
L
Bohle
B
B
Stockinger
H
H
Suphioglu
C
C
Ong
E K
EK
Kraft
D
D
Valenta
R
R
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
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