Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition.
Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.
Hayek
B
B
Department of General and Experimental Pathology, AKH, University of Vienna, Austria.
Vangelista
L
L
Pastore
A
A
Sperr
W R
WR
Valent
P
P
Vrtala
S
S
Niederberger
V
V
Twardosz
A
A
Kraft
D
D
Valenta
R
R
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D052179:Antigens, Plant; D001059:Apoproteins / Q000737:chemistry; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics; D017449:Dermatitis, Allergic Contact / Q000276:immunology / Q000628:therapy; D003888:Desensitization, Immunologic; D004926:Escherichia coli; D018257:Helix-Loop-Helix Motifs; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D011058:Pollen / Q000276:immunology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014197:Trees
D000485:Allergens; C116573:Aln g 4 protein, Alnus glutinosa; D052179:Antigens, Plant; D001059:Apoproteins; D018076:DNA, Complementary; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E; D002118:Calcium