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.
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.
Palomares
Oscar
O
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain.
Villalba
Mayte
M
Rodríguez
Rosalía
R
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005786:Gene Expression Regulation; D006967:Hypersensitivity / Q000276:immunology; D007106:Immune Sera; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D008969:Molecular Sequence Data; D031658:Olea; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000502:physiology*; D011058:Pollen / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000502:physiology*
D000485:Allergens; D052179:Antigens, Plant; D004220:Disulfides; D007106:Immune Sera; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C433244:OLE9 protein, Olea europaea; D001617:beta-Glucosidase