title: An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.
abstract: Endo-β-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic β-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that β-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen β-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long β-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of β-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.
authors.affiliation: Biochemistry and Molecular Biology I Department Complutense, University of Madrid, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Torres
authors.affiliation: Biochemistry and Molecular Biology I Department Complutense, University of Madrid, Madrid, Spain.
authors.forename: Oscar
authors.initials: O
authors.lastname: Palomares
authors.affiliation: Virgen del Rocío University, Hospital of Seville, Seville, Spain.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Quiralte
authors.affiliation: Hôpital Lyautey, Hopitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Pauli
authors.affiliation: Biochemistry and Molecular Biology I Department Complutense, University of Madrid, Madrid, Spain.
authors.forename: Rosalía
authors.initials: R
authors.lastname: Rodríguez
authors.affiliation: Biochemistry and Molecular Biology I Department Complutense, University of Madrid, Madrid, Spain.
authors.forename: Mayte
authors.initials: M
authors.lastname: Villalba
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031661:Fraxinus / Q000737:chemistry; D015870:Gene Expression; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007073:Immunoglobulin E / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D031658:Olea / Q000737:chemistry; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011058:Pollen / Q000737:chemistry* / Q000201:enzymology / Q000276:immunology; D011485:Protein Binding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012130:Respiratory Hypersensitivity / Q000097:blood / Q000276:immunology / Q000503:physiopathology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D007106:Immune Sera; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C433244:OLE9 protein, Olea europaea; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase