Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein.
Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.
Krebitz
Monika
M
Department of Pathophysiology, University of Vienna, AKH-EBO-3Q, Waehringer Guertel 18-20, 1090, Vienna, Austria.
Wagner
Birgit
B
Ferreira
Fatima
F
Peterbauer
Clemens
C
Campillo
Nuria
N
Witty
Michael
M
Kolarich
Daniel
D
Steinkellner
Herta
H
Scheiner
Otto
O
Breiteneder
Heimo
H
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005512:Food Hypersensitivity / Q000097:blood / Q000209:etiology / Q000276:immunology*; D005670:Fusarium / Q000187:drug effects*; D018506:Gene Expression Regulation, Plant; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism*; D027845:Malus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010407:Penicillium / Q000187:drug effects*; D010446:Peptide Fragments / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013549:Sweetening Agents / Q000737:chemistry / Q000494:pharmacology*; D014026:Nicotiana / Q000737:chemistry; D014027:Tobacco Mosaic Virus / Q000235:genetics; D014162:Transfection
D000485:Allergens; D000935:Antifungal Agents; D018076:DNA, Complementary; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D013549:Sweetening Agents; D007073:Immunoglobulin E; C003427:thaumatin protein, plant