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

J Mol Biol. 2003 Jun 13;329(4):721-30. doi: 10.1016/s0022-2836(03)00403-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / chemistry
  • Allergens / genetics
  • Allergens / pharmacology*
  • Antifungal Agents / chemistry
  • Antifungal Agents / pharmacology*
  • Cloning, Molecular
  • DNA, Complementary
  • Food Hypersensitivity / blood
  • Food Hypersensitivity / etiology
  • Food Hypersensitivity / immunology*
  • Fusarium / drug effects*
  • Gene Expression Regulation, Plant
  • Humans
  • Immunoblotting
  • Immunoglobulin E / metabolism*
  • Malus*
  • Models, Molecular
  • Molecular Sequence Data
  • Penicillium / drug effects*
  • Peptide Fragments / metabolism
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / pharmacology*
  • Recombinant Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Sweetening Agents / chemistry
  • Sweetening Agents / pharmacology*
  • Tobacco / chemistry
  • Tobacco Mosaic Virus / genetics
  • Transfection

Substances

  • Allergens
  • Antifungal Agents
  • DNA, Complementary
  • Peptide Fragments
  • Plant Proteins
  • Recombinant Proteins
  • Sweetening Agents
  • Immunoglobulin E
  • thaumatin protein, plant

Associated data

  • GENBANK/AJ243427