Comprehensive characterization and molecular insights into the salt tolerance of a Cu, Zn-superoxide dismutase from an Indian Mangrove, Avicennia marina.

Superoxide dismutases are important group of antioxidant metallozyme and play important role in ROS homeostasis in salinity stress. The present study reports the biochemical properties of a salt-tolerant Cu, Zn-superoxide from Avicennia marina (Am_SOD). Am_SOD was purified from the leaf and identified by mass-spectrometry. Recombinant Am_SOD cDNA was bacterially expressed as a homodimeric protein. Enzyme kinetics revealed a high substrate affinity and specific activity of Am_SOD as compared to many earlier reported SODs. An electronic transition in 360-400 nm spectra of Am_SOD is indicative of Cu2+-binding. Am_SOD activity was potentially inhibited by diethyldithiocarbamate and H2O2, a characteristic of Cu, Zn-SOD. Am_SOD exhibited conformational and functional stability at high NaCl concentration as well in alkaline pH. Introgression of Am_SOD in E. coli conferred tolerance to oxidative stress under highly saline condition. Am_SOD was moderately thermostable and retained functional activity at ~ 60 °C. In-silico analyses revealed 5 solvent-accessible N-terminal residues of Am_SOD that were less hydrophobic than those at similar positions of non-halophilic SODs. Substituting these 5 residues with non-halophilic counterparts resulted in > 50% reduction in salt-tolerance of Am_SOD. This indicates a cumulative role of these residues in maintaining low surface hydrophobicity of Am_SOD and consequently high salt tolerance. The molecular information on antioxidant activity and salt-tolerance of Am_SOD may have potential application in biotechnology research. To our knowledge, this is the first report on salt-tolerant SOD from mangrove.

Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Allergenicity assessment of Delonix regia pollen grain and identification of allergens by immunoproteomic approach.

BACKGROUND: Plantation of road-side avenue trees has become a major part of urbanization programme for megacity beautification and environmental management. Due to evergreen habit and vibrant flower colour, Delonix regia (Gulmohor/Flamboyant) is frequently selected as ornamental tree for plantation programme. However, D. regia pollen is related to IgE mediated allergic reactions and no allergen has been reported so far. OBJECTIVE: Measuring the prevalence of D. regia pollen sensitivity among the local atopic individuals and identifying the allergens using immunoproteomic tools. METHODS: Aerobiological study was conducted for a period of two years to record the D. regia pollen concentration in the outdoor ambient air. Clinico-immunological tests were performed on atopic individuals to check the prevalence of sensitivity against D. regia pollen. Allergens were detected in the pollen proteome, fractionated in 1D and 2D gel by IgE serology and finally identified by mass spectrometry. RESULT: In the study area D. regia pollen grains were present in ample amount in the air during May to July. About 38% of atopic individuals displayed positive Skin Prick Test (SPT) against D. regia pollen along with elevated level of specific IgE and histamine in the sera. Immunoproteomic analyses revealed the presence of 14 IgE reactive proteins in the 2D pollen proteome, of which 8 IgE reactive proteins were identified by MALDI TOF/TOF using homology driven proteomic approach. CONCLUSION: This study demonstrated pollen related allergy symptoms by D. regia and gave significant message regarding the plantation programme to avoid the unnecessary load of allergic pollen. Also, a panel of 8 allergens were identified for the first time from D. regia pollen. Detailed study of these allergens would help to design immunotherapeutic strategies for pollinosis management.

Identification, cloning, and immunological studies on a major eggplant (Solanum melongena L.) allergen Sola m 1: A new member of profilin allergen family.

Eggplant or brinjal (Solanum melongena L.) is widely consumed worldwide and thought to trigger allergic reactions in sensitive individuals. So far, no molecular information is available on the allergy-eliciting components of eggplant. In this study, a 17 kDa profilin, Sola m 1, was identified from eggplant by employing an immunoproteomic approach. Based on MALDI-TOF/TOF derived sequences, the full-length cDNA of Sola m 1 was PCR amplified and then cloned. Recombinant (r) Sola m 1 was expressed in E. coli and then purified by metal affinity and gel filtration. rSola m 1 reacted with IgE-antibodies in the sera from all eggplant allergic patients. rSola m 1 also displayed allergenic activity by stimulating histamine release. rSola m 1 was monomeric, and the CD spectra revealed it to be folded with a mixture of α-helices and ß-strands. In the melting curve, rSola m 1 exhibited an irreversible denaturation where no refolding took place. Sola m 1 was found to share >80 % sequence identity with Bet v 2, which was further validated by confirming the presence of significant cross-reactivity with Bet v 2 in IgE-inhibition assay. IgE-cross reactivity was also observed between rSola m 1 and profilins from six other foods. In SGF assay, no rSola m 1-derived fragments exhibited IgE-reactivity after prolonged digestion suggesting the association of rSola m 1 with the oral allergy syndromes. Immunofluorescence localization revealed a high abundance of Sola m 1 allergen in eggplant seeds as compared to other edible parts. Taken together, Sola m 1 is the first major eggplant allergen reported in this study, which has the potential of being used as a candidate antigen in component-resolved diagnosis and immunotherapy.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma.

BACKGROUND: The pathobiology of atopic asthma is complex and the symptoms similar to other respiratory diseases. As such, identification of biomarkers of atopic asthma is of prime importance for better diagnosis and control of the disease. OBJECTIVES: We sought to study the changes in plasma proteome and cytokine-expression profile across healthy and atopic asthmatics for identifying biomarkers and exploring aberrant pathways for atopic asthma. METHODS: A pilot-scale study in humans was performed to identify differentially expressed proteins in blood plasma of healthy controls (n=5) and treatment-naïve atopic asthma patients (n=5) using quantitative label-free liquid chromatography-tandem mass spectrometry proteomics and ELISA. RESULTS: Mass spectrometry-based proteomic analysis revealed ApoE to be significantly downregulated in atopic asthmatics compared to healthy volunteers. Decreased expression of ApoE in atopic asthmatics was validated by immunoblotting (50.74% decrease). Comparison with atopic asthmatics and COPD patients showed that ApoE was decreased (36.33%) in atopic asthma compared to COPD. IL33 was significantly upregulated in atopic asthmatics compared to healthy subjects (3.84-fold). CONCLUSION: ApoE was downregulated and IL33 upregulated in atopic asthma patients compared to healthy volunteers. These two proteins' profiles were distinct in atopic asthma from healthy and COPD plasma samples. Differential expression of these proteins could serve as a probable candidate for a two-protein classifier-based prognostic biomarker of atopic asthma.

Cobbia bengalensis sp. nov. (Xyalidae: Monhysterida) from an eroding island of Sundarban, India.

A new species, Cobbia bengalensis sp. nov., of free-living marine nematodes is described from an island of Sundarban, Indian coast and compared with other eight valid species of the genus. Cobbia bengalensis sp. nov. is characterized and differentiated from the others by a unique combination of sexually dimorphic tail, arrangement of anterior sensilla, amphid position, presence of four subcephalic sensilla at the level of amphid, buccal cavity with one dorsal and two subventral teeth, elongated cardia and the shape of gubernaculum. Some previously recorded taxa thought to be the species of Cobbia has also been discussed. Wrongly recorded data for nematofaunal inventory from Indian water has also been criticized.