Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens

Background: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen. Objectives: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis. Methods: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens. Results: 2-D immunolabeling with individual patients’ sera showed the distinguishable IgE-binding patterns inlaid with 4–87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5–75% of IgE-binding frequency) and 3 Cry j 2 (32.5–40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2. Conclusions: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.