BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food. OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins. METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA. RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin. CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food. Copyright 2003 S. Karger AG, Basel
BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food. OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins. METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergicpatients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA. RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomatoprofilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergicpatients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin. CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food. Copyright 2003 S. Karger AG, Basel