CHARACTERIZATION OF A NEW IGE-BINDING 35-KDA PROTEIN FROM BIRCH POLLEN WITH CROSS-REACTING HOMOLOGS IN VARIOUS PLANT FOODS

The present investigation was undertaken to obtain molecular data of a new immunoglobulin (Ig)E-binding birch pollen protein with a mass of 35 kDa. In a previous study, this protein showed IgE cross-reactivity with 34- and 35-kDa proteins in apples, pears, carrots, bananas and ot her exotic fruits. Since the protein was N-terminally blocked, it was purified by preparative SDS-PAGE, and multiple proteolytic fragments w ere subsequently generated by in-gel digestion with the endoproteinase s Glu C, Lys C and Clostripain. After electrophoretic separation and b lotting onto polyvinylidene difluoride (PVDF), the resulting polypepti des were subjected to N-terminal amino acid microsequencing. The inter nal sequences obtained showed a high degree of sequence identity to is oflavone reductases (IFR) and isoflavone reductase-like proteins (LRL) from several plants which also had a similar size. For a stretch of 2 5 consecutive residues this identity ranged from 56% for IFR from peas and chick peas and an IRL from maize, to 80% for a tobacco IRL. A 453 bp fragment was amplified from total birch pollen RNA by polymerase c hain reaction (PCR) using primers derived from the nucleotide sequence of the tobacco IRL. The deduced 151 amino acid sequence represented a pproximate to 50% of the protein and confirmed the sequence identities obtained by Edman degradation. Moreover, the 25 amino acid sequence w as included in the cloned fragment. Deduced and determined amino acids showed only one mismatch, which was due to a single nucleotide exchan ge. At the antibody level, the immunological relationship of the birch pollen protein to LRL and IFR was demonstrated by immunoblotting with a rabbit antiserum against a pea IFR which recognized the same birch protein as patients' IgE. The rabbit antiserum also reproduced the cro ss-reactivity pattern previously observed with patients' IgE by recogn izing related proteins in specific plant foods, including some exotic fruits. We therefore suggest that the 35-kDa birch pollen protein belo ngs to the IFR/IRL family and represents a minor allergen, possibly be ing responsible for less common pollen-related food allergies in patie nts allergic to birch pollen.