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Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum).
Int Arch Allergy Immunol. 2003 Aug; 131(4):245-55.IA

Abstract

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.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Salzburg, Salzburg, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12915767

Citation

Willerroider, M, et al. "Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc E 1, Profilins From Bell Pepper (Capsicum Annuum) and Tomato (Lycopersicon Esculentum)." International Archives of Allergy and Immunology, vol. 131, no. 4, 2003, pp. 245-55.
Willerroider M, Fuchs H, Ballmer-Weber BK, et al. Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). Int Arch Allergy Immunol. 2003;131(4):245-55.
Willerroider, M., Fuchs, H., Ballmer-Weber, B. K., Focke, M., Susani, M., Thalhamer, J., Ferreira, F., Wüthrich, B., Scheiner, O., Breiteneder, H., & Hoffmann-Sommergruber, K. (2003). Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). International Archives of Allergy and Immunology, 131(4), 245-55.
Willerroider M, et al. Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc E 1, Profilins From Bell Pepper (Capsicum Annuum) and Tomato (Lycopersicon Esculentum). Int Arch Allergy Immunol. 2003;131(4):245-55. PubMed PMID: 12915767.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). AU - Willerroider,M, AU - Fuchs,H, AU - Ballmer-Weber,B K, AU - Focke,M, AU - Susani,M, AU - Thalhamer,J, AU - Ferreira,F, AU - Wüthrich,B, AU - Scheiner,O, AU - Breiteneder,H, AU - Hoffmann-Sommergruber,K, PY - 2002/10/23/received PY - 2003/05/21/accepted PY - 2003/8/14/pubmed PY - 2003/9/17/medline PY - 2003/8/14/entrez SP - 245 EP - 55 JF - International archives of allergy and immunology JO - Int Arch Allergy Immunol VL - 131 IS - 4 N2 - 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. SN - 1018-2438 UR - https://www.unboundmedicine.com/medline/citation/12915767/Cloning_and_molecular_and_immunological_characterisation_of_two_new_food_allergens_Cap_a_2_and_Lyc_e_1_profilins_from_bell_pepper__Capsicum_annuum__and_Tomato__Lycopersicon_esculentum__ L2 - https://www.karger.com?DOI=10.1159/000072136 DB - PRIME DP - Unbound Medicine ER -