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

J Immunol. 1998 Dec 15;161(12):7031-9.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / chemistry
  • Allergens / genetics
  • Allergens / immunology*
  • Amino Acid Sequence
  • Animals
  • Antigen-Antibody Reactions / drug effects
  • Antigens, Plant
  • Apoproteins / chemistry
  • Base Sequence
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Circular Dichroism
  • Cross Reactions
  • DNA, Complementary / genetics
  • Dermatitis, Allergic Contact / immunology
  • Dermatitis, Allergic Contact / therapy
  • Desensitization, Immunologic
  • Escherichia coli
  • Helix-Loop-Helix Motifs*
  • Histamine Release
  • Humans
  • Immunoglobulin E / immunology*
  • Models, Molecular
  • Molecular Sequence Data
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / immunology*
  • Plant Proteins / therapeutic use
  • Pollen / immunology*
  • Protein Conformation
  • Protein Structure, Secondary
  • Rabbits
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / immunology
  • Rhinitis, Allergic, Seasonal / immunology
  • Rhinitis, Allergic, Seasonal / therapy
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Structure-Activity Relationship
  • Trees

Substances

  • Allergens
  • Aln g 4 protein, Alnus glutinosa
  • Antigens, Plant
  • Apoproteins
  • DNA, Complementary
  • Plant Proteins
  • Recombinant Fusion Proteins
  • Immunoglobulin E
  • Calcium