This thesis describes the molecular and immunological characterization of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. A Bermuda grass (Cynodon dactylon) pollen cDNA library was constructed and a full length cDNA encoding Cyn d 1 gene was selected for further molecular and immunological characterization. Earlier studies had shown that bacterially expressed recombinant Cyn d 1 protein was non-IgE reactive due to lack of post-translational glycosylation in prokaryotic expression system. But in the present study, the expression of biologically active recombinant Cyn d 1 in E. coli was found to be reactive with serum IgE from Bermuda grass pollen sensitive patients. Further studies were undertaken to determine the IgE-binding regions of Cyn d 1 by generating a series of overlapping fragments of varying lengths covering the entire allergen molecule. These fragments were individually expressed in E. coli and the purified proteins were analysed to compare their IgE reactivity using sera pool from grass pollen allergic patients. This study led to the identification of two major IgE-binding regions and, deletion of either or both regions resulted in a significant reduction in the IgE reactivity. This information gathered from epitope mapping along with modern computational tools provided important clues to select residues to target for site directed mutagenesis for producing low IgE binding variants of Cyn d 1.
To characterize the amino acid residues from Cyn d 1 which interact with IgE, the sequence was scanned by site-directed mutagenesis taking into account the sequence of homologous non allergenic 0-expansin gene, EXPB1, from Arabidopsis. Cyn d 1 hypoallergenic mutant forms with reduced IgE binding have been engineered based on the structural homology of non allergenic Arabidopsis beta expansin protein EXPB1 as a model. Nine potential IgE-interacting residues were identified and characterized, and based on these residues eight mutant Cyn d 1 variants with different combinations of mutated residues were designed and produced. Three out of the eight mutants showed reduced IgE-binding capacity as studied in vitro by immunodot blot and direct ELISA analysis. Hypoallergenic forms of the Cyn d 1 allergen can aid in the development of immunotherapeutic reagents for treatment of grass pollen allergy.
Due to IgE-cross-reactivity, allergic patients exhibit broad sensitization to pollens of most grass species. Allergens from different sources can show immunological cross reactivity, often resulting from conservation of IgE epitopes. Therefore, immunoblot inhibition assays were performed using recombinant proteins to test the specific pattern of cross reactivity between allergens from Bermuda grass pollen (Chloridoedeae sub family) and Rye grass pollen (Pooideae sub family). The allergen profile of Rye grass pollen comprises Lol p 1 (group 1) and Lol p 5 (group 5), the two major allergen groups, Bermuda grass pollen on the other hand contains only Cyn d 1 (group 1) allergen. The group 1 allergens show very high amino acid sequence identity (80 %) and share common epitopes. The inhibition studies revealed a high degree of cross reactivity between the group 1 allergens from both grasses but none was observed between group 1 allergen from Bermuda grass and group 5 allergen from rye grass pollen. Rye grass pollen extract completely inhibited Bermuda grass pollen extract but not vice versa. These results suggest that basis of overall low level of cross reactivity between the Pooids and Chloridooids as observed by RAST can be attributed to absence of group 5 allergen from Chloridoids.
Sequence comparison has identified a homologue ((3-expansin clone At2g20750 or EXPB1) in Arabidopsis of the Cyn d 1 gene. The EXPB1 protein is 42% similar to the Cyn d 1 protein. The function of some pollen allergen genes has been suggested based on their homologies with genes of known sequence. Expansins are a family of proteins that are known to induce extension of plant cell walls. A study was undertaken to determine the expression pattern of EXPB1 in Arabidopsis. RT-PCR analysis, in situ hybridization and GFP-promoter construct were used to study the pattern of gene expression of EXPB1 in Arabidopsis. RT-PCR analysis revealed EXPB1 transcripts only in floral tissue and the EXPB1 transcripts were localized in pollens by in situ hybridization. The absence of EXPB1 transcripts in any other tissues like leaf, stem and root indicates that the EXPB1 gene has pollen specific expression. GFP-promoter fusions showed high level of expression in tri-cellular pollen
