title: Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae.
abstract: BACKGROUND
  Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation 'Rhi o 1'.
  
  
  METHODS
  The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.
  
  
  RESULTS
  The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens.
  
  
  CONCLUSIONS
  The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.
authors.affiliation: Division of Plant Biology, Bose Institute (Main campus), 93/1 Acharya Prafulla Chandra Road, Kolkata- 700009, West Bengal, India.
authors.forename: Gaurab
authors.initials: G
authors.lastname: Sircar
authors.affiliation: Division of Plant Biology, Bose Institute (Main campus), 93/1 Acharya Prafulla Chandra Road, Kolkata- 700009, West Bengal, India.
authors.forename: Bodhisattwa
authors.initials: B
authors.lastname: Saha
authors.affiliation: Biomedical Informatics Center, National Institute of Cholera and Enteric Diseases, Kolkata-700010, West Bengal, India.
authors.forename: Rahul Shubhra
authors.initials: RS
authors.lastname: Mandal
authors.affiliation: Department of Allergy and Asthma, Belle Vue Clinic, 9, Dr. U. N. Brahmachari Street, Kolkata-700001, West Bengal, India.
authors.forename: Naren
authors.initials: N
authors.lastname: Pandey
authors.affiliation: Bioinformatics Centre, Bose Institute (Centenary Building), P 1/12, C. I. T. Road, Scheme-VIIM, Kolkata- 700054, West Bengal, India.
authors.forename: Sudipto
authors.initials: S
authors.lastname: Saha
authors.affiliation: Division of Plant Biology, Bose Institute (Main campus), 93/1 Acharya Prafulla Chandra Road, Kolkata- 700009, West Bengal, India.
authors.forename: Swati
authors.initials: S
authors.lastname: Gupta Bhattacharya
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D003429:Cross Reactions / Q000276:immunology; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009091:Mucormycosis / Q000276:immunology / Q000382:microbiology; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins; D012233:Rhizopus / Q000235:genetics* / Q000276:immunology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D055815:Young Adult
chemical_list: D000485:Allergens; D000946:Antigens, Fungal; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D057055:Aspartic Acid Proteases