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Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3)
Authors:Ramazzina Ileana  Amato Stefano  Passera Elisabetta  Sforza Stefano  Mistrello Gianni  Berni Rodolfo  Folli Claudia
Affiliation:
  • a Department of Biochemistry and Molecular Biology, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy
  • b Lofarma SpA, Viale Cassala 40, 20143 Milan, Italy
  • c Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
  • 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.
    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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