Cloning, expression and immunological characterisation of Coc n 1, the first major allergen from Coconut pollen.

Coconut pollen has been documented to be a major contributor to the aeroallergen load in India, causing respiratory allergy in a large cohort of susceptible individuals. Here, we report the identification of the first major allergen from Coconut pollen, Coc n 1. The full-length sequence of the allergen was determined from previously identified peptides and overexpressed in E. coli. Recombinant Coc n 1 folded into a trimer and was found to possess allergenicity equivalent to its natural counterpart. Proteolytic processing of Coc n 1 led to the formation of an immunodominant ∼20 kDa C-terminal subunit and the site of cleavage was determined by amino acid microsequencing. Five linear IgE binding epitopes were predicted and mapped on the homology modelled structure of Coc n 1. Amongst three immunodominant epitopes, two were present towards the C-terminal end. Coc n 1 was found to belong to the highly diverse cupin superfamily and mimics its structure with known 7S globulin or vicilin allergens but lacks sequence similarity. Using sequence similarity networks, Coc n 1 clustered as a separate group containing unannotated cupin domain proteins and did not include known vicilin allergens except Gly m Bd 28 kDa, a Soybean major allergen. 7S globulins are major storage proteins and food allergens, but presence of such protein in pollen grains is reported for the first time. Further study on Coc n 1 may provide insights into its function in pollen grains and also in the development of immunotherapy to Coconut pollen allergy.

Charting novel allergens from date palm pollen (Phoenix sylvestris) using homology driven proteomics.

Pollen grains from Phoenix sylvestris (date palm), a commonly cultivated tree in India has been found to cause severe allergic diseases in an increasing percentage of hypersensitive individuals. To unearth its allergenic components, pollen protein were profiled by two-dimensional gel electrophoresis followed by immunoblotting with date palm pollen sensitive patient sera. Allergens were identified by MALDI-TOF/TOF employing a layered proteomic approach combining conventional database dependent search and manual de novo sequencing followed by homology-based search as Phoenix sylvestris is unsequenced. Derivatization of tryptic peptides by acetylation has been demonstrated to differentiate the 'b' from the 'y' ions facilitating efficient de novo sequencing. Ten allergenic proteins were identified, out of which six showed homology with known allergens while others were reported for the first time. Amongst these, isoflavone reductase, beta-conglycinin, S-adenosyl methionine synthase, 1, 4 glucan synthase and beta-galactosidase were commonly reported as allergens from coconut pollen and presumably responsible for cross-reactivity. One of the allergens had IgE binding epitope recognized by its glycan moiety. The allergenic potency of date palm pollen has been demonstrated using in vitro tests. The identified allergens can be used to develop vaccines for immunotherapy against date palm pollen allergy. THE SIGNIFICANCE OF THE STUDY: Identification of allergenic proteins from sources harboring them is essential in developing therapeutic interventions. This is the first comprehensive study on the identification of allergens from Phoenix sylvestris (date palm) pollen, one of the major aeroallergens in India using a proteomic approach. Proteomic methods are being increasingly used to identify allergens. However, since many of these proteins arise from species which are un-sequenced, it becomes difficult to interpret those using conventional proteomics. Date palm being an unsequenced species, the IgE-reactive proteins have been identified using a stratified proteomic workflow incorporating manual de novo sequencing and homology-based proteomics. This study also gives an insight into the presence of glycan nature of the IgE binding epitopes. Five proteins have been found to be common with coconut pollen allergens and presumably responsible for cross-reactivity. These can be used in diagnostics to differentiate patient cohorts allergic to both coconut and date palm pollen from true date palm pollen allergic subjects. This would also determine better specific immunotherapy regimes between the two cohorts. The allergens identified herein have potential towards vaccine development in date palm pollen allergy as well as in enriching the existing catalogue of allergenic proteins.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients.

BACKGROUND: Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. METHODOLOGY: Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry. RESULTS: Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease. CONCLUSION: Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.