title: Immunoproteomic characterization of Ambrosia artemisiifolia pollen allergens in canine atopic dermatitis.
abstract: Canine atopic dermatitis (CAD) is an immune system disorder that affects 10-15% of the canine population. Short ragweed (Ambrosia artemisiifolia) pollen represents one of the major seasonal sources of allergenic pollen proteins in Europe, particularly in the Pannonian valley of the Balkan region. In Serbia, about 66% of atopic dogs showed a positive intradermal skin test with its pollen extract, which is second to house dust mites. Therefore, characterization of Ambrosia artemisiifolia pollen components, in terms of defining major and minor allergens that induce clinically manifested allergic reaction in dogs, is important for valid diagnosis and efficient therapy. This study has, for the first time, characterized and identified major Ambrosia artemisiifolia allergens in CAD, using an immunoproteomic approach. To assess the prevalence of specific IgE in electrophoretically separated ragweed pollen proteins, individual reactivity of sera from dogs with CAD was analyzed and compared to the reactivity of sera from healthy dogs in the non-reducing conditions, which were found optimal for specific canine IgE detection. A specific IgE band (38 kDa) was recognized as the most dominant allergen in CAD, occurring in 81% of positive dog's sera. 2-D immunoblotting followed by a mass spectrometry peptide fingerprint analyses with pooled canine and human atopic sera, revealed that 38 kDa major Ambrosia atremisiifolia allergens in CAD were all five isoallergens of the Amb a 1 group (antigen E), including the previously named Amb a 2 (antigen K). In contrast to canine sera, human atopic sera also recognized lower mass allergens such as the β fragment of Amb a 1 and profilins (Amb a 8 variants). The most prominent ragweed proteins in CAD, represent, as in humans, variants of all five isoallergens of the Amb a 1 group (pectate lyase): Amb a 1.0101 and its natural variant E1XUL2, Amb a 1.0202, 1.0304, 1.0402 and the natural variant of Amb a 1.0501, E1XUM0, as well as the α fragment of pollen allergen Amb a 1.0201.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia.
authors.forename: Jana
authors.initials: J
authors.lastname: Ognjenovic
authors.forename: Natalija
authors.initials: N
authors.lastname: Milcic-Matic
authors.forename: Katarina
authors.initials: K
authors.lastname: Smiljanic
authors.forename: Olga
authors.initials: O
authors.lastname: Vuckovic
authors.forename: Lidija
authors.initials: L
authors.lastname: Burazer
authors.forename: Nikola
authors.initials: N
authors.lastname: Popovic
authors.forename: Dragana
authors.initials: D
authors.lastname: Stanic-Vucinic
authors.forename: Tanja Cirkovic
authors.initials: TC
authors.lastname: Velickovic
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D031164:Ambrosia / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D052179:Antigens, Plant / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003876:Dermatitis, Atopic / Q000276:immunology / Q000662:veterinary*; D004283:Dog Diseases / Q000276:immunology*; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology / Q000662:veterinary*; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D040901:Proteomics; D017386:Sequence Homology, Amino Acid; D055771:Serbia; D053719:Tandem Mass Spectrometry
chemical_list: D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D010936:Plant Extracts; D010940:Plant Proteins; C433085:ragweed pollen; D007073:Immunoglobulin E
keywords: AD; Amb a 1 allergens; Ambrosia artemisiifolia pollen; CAD; CBB; Canine atopic dermatitis; Coomassie brilliant blue; Dog; ID; IDST; Immunoproteomics; LTQ; MS; PAA; PNU; Short ragweed; TTBS; Tris buffered saline with 0.1% Tween 20 (pH 7.4); atopic dermatitis; canine atopic dermatitis; internal diameter; intradermal skin test; linear ion trap with quadrupole mass filter; mass spectroscopy; nLC–MS/MS; nano-liquid chromatography coupled to tandem mass spectroscopy; polyacrylamide; protein nitrogen unit