Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3) |
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Authors: | Ramazzina Ileana Amato Stefano Passera Elisabetta Sforza Stefano Mistrello Gianni Berni Rodolfo Folli Claudia |
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Affiliation: | a Department of Biochemistry and Molecular Biology, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italyb Lofarma SpA, Viale Cassala 40, 20143 Milan, Italyc Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy |
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Abstract: | Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy. |
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Keywords: | A, absorbance ATP, adenosine 5&prime -triphosphate bp, base pair(s) CD, circular dichroism cDNA, DNA complementary to RNA C-terminal, carboxyl-terminal CTP, cytidine 5&prime -triphosphate d, deoxyribo Da, dalton(s) DEPC, diethyl pyrocarbonate DIECA, diethyldithiocarbamate EDTA, ethylenediaminetetraacetic acid ELISA, enzyme-linked immunosorbent assay ESI-MS, electrospray mass spectrometry GTP, guanosine 5&prime -triphosphate ID, identification Ig, immunoglobulin(s) IPTG, isopropyl β-D-thiogalactopyranoside kDa, kilodalton(s) LDS, lithium dodecyl sulfate LTP, lipid transfer protein(s) Lyso C16, 1-palmitoil-2-lysophosphatidylcholine Mal d, Malus domestica M-MLV, Moloney Murine Leukemia Virus MOPS, morpholinopropanesulfonic acid mRNA, messenger RNA nt, nucleotide(s) N-terminal, amino-terminal OD, optical density units OAS, oral allergy syndrome PAGE, polyacrylamide-gel electrophoresis PBS, phosphate buffered saline PCR, polymerase chain reaction PDB, protein data bank Pro, proline Pru ar, Prunus armeniaca Pru av, Prunus avium Pru d, Prunus domestica Pru p, Prunus persica PVPP, polyvinylpyrrolidone Pyr c, Pyrus communis RAST, radioallergosorbent test SDS, sodium dodecyl sulfate SSC, 0.15 M NaCl/0.015 M sodium citrate pH 7 SYS, systemic symptoms Tm, melting temperature Tris, tris(hydroxymethyl)aminomethane TTP, thymidine 5&prime -triphosphate UTP, uridine 5&prime -triphosphate UV, ultraviolet |
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