The C-terminal segment of the 1,3-beta-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization.

Abstract

Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses. Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries. Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris. The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC. Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure. Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48. The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen. These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy. Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.

• Authors: Oscar Palomares, Mayte Villalba, Rosalía Rodríguez
• Journal: The Biochemical journal (Biochem J) Vol. 369 Issue Pt 3 Pg. 593-601 (Feb 01 2003) ISSN: 0264-6021 [Print] England
• PMID: 12392450 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Antigens, Plant
• Disulfides
• Immune Sera
• Peptide Fragments
• Plant Proteins
• Recombinant Proteins
• Immunoglobulin E
• OLE9 protein, Olea europaea
• beta-Glucosidase

Topics

• Allergens (chemistry, genetics, immunology, metabolism, physiology)
• Amino Acid Sequence
• Antigens, Plant
• Chromatography, High Pressure Liquid
• Circular Dichroism
• Disulfides
• Enzyme-Linked Immunosorbent Assay (methods)
• Gene Expression Regulation
• Hypersensitivity (immunology)
• Immune Sera
• Immunoblotting
• Immunoglobulin E (metabolism)
• Molecular Sequence Data
• Olea
• Peptide Fragments (genetics, immunology, metabolism)
• Pichia (genetics)
• Plant Proteins (chemistry, physiology)
• Pollen (chemistry, genetics)
• Protein Structure, Tertiary
• Recombinant Proteins (genetics, isolation & purification, metabolism)
• beta-Glucosidase (chemistry, physiology)