PubMed · 11289338
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R W Weber. 2001. Red oak.. https://doi.org/10.1016/s1081-1206(10)63292-9
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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.
Specific allergy vaccination (SAV) is associated with increased levels of allergen specific IgG in serum. It is not clear, however, to what extent qualitative changes in allergen binding to IgG may be induced as well. We therefore analyzed the binding of the major allergen in pollen of birch (Betula verrucosa) (Bet v 1), the major allergen in birch pollen, to serum IgG and IgE, separately and in competition. Sera from six birch pollen-allergic patients were obtained before and after 5 years of SAV, and binding was assessed with 125I-Bet v 1. Before SAV, IgG bound more than eight times the amount of Bet v 1 compared with IgE, and together they accounted for more than 85% of the serum binding capacity. While SAV induced minimal changes in IgE binding, the IgG binding capacities increased 6-32 times. In contrast, the binding avidities (K(d) 28-40pM) changed less than 20%, pre- and post-SAV IgG provided similar inhibition of Bet v 1 binding to IgE at equimolar levels, and cross inhibition studies between IgG and IgE showed low inter-individual differences. Following SAV, all sera reduced Bet v 1 binding to CD23(+) cells, correlating with reduced binding of Bet v 1 to IgE (P<0.001). These results show that high avidity IgG of low inter-individual difference in Bet v 1 binding quality is the dominant binding factor of Bet v 1 in sera of birch pollen-allergic patients, and that SAV-induced inhibition of binding of Bet v 1 to IgE can be explained mainly or solely by increased amounts of IgG.
The major allergen (parvalbumin) from cod, designated Allergen M Gad c 1, has been intensively studied both from the structural and immunological sides. In the present study, transcripts of two isotypic parvalbumin genes in Atlantic cod were identified and characterized. Subsequently, subfragments were inserted into the expression vector pET-19b, generating plasmids with coding capacity for complete parvalbumin polypeptides fused to an N-terminal his(10) tag. Most of the recombinant products were found in the soluble fraction of the expression host Escherichia coli. The target proteins showed to react with polyclonal antibodies raised against Allergen M and demonstrated binding to specific IgE from 12 sera of patients allergic to cod in ELISA inhibition experiments. Sera with classes 4 and 5 CAP FEIA exhibited also strong binding to recombinant parvalbumins in immunoblots.