An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family

PLOS ONE, Dec 2019

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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0133066&type=printable

An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family

July An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family María Torres 0 1 Oscar Palomares 0 1 Joaquín Quiralte 0 1 Gabrielle Pauli 0 1 Rosalía Rodríguez 0 1 Mayte Villalba 0 1 0 1 Biochemistry and Molecular Biology I Department Complutense, University of Madrid , Madrid , Spain , 2 Virgen del Rocío University, Hospital of Seville , Seville, Spain, 3 Hôpital Lyautey , Hopitaux Universitaires de Strasbourg , Strasbourg , France 1 Editor: Wei Wang , Henan Agricultural Univerisity, CHINA 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-1,3glucanase 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 carbohydratebinding 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. - Competing Interests: The authors have declared that no competing interests exist. Beta-1,3-glucanases [glucan endo-1,3-β-D-glucosidases (EC 3.2.1.39)] constitute a ubiquitous family of proteins widely distributed among higher plants, fungi and bacteria. The biochemical activity of these enzymes is based on the hydrolytic cleavage of 1,3-D-glucosidic linkages between β-1,3-glucans, which are major components of the cell wall surrounding fungi and plants. The enzymatic activity of β-1,3-glucanases is crucial in the chemical changes of the glucan composition and in the remodeling of the cell wall. In fungi, these enzymes play a role in cell expansion, cell-cell fusion and spore release [1]; in bacteria, assimilating fungal cell wall as food [2] and developing metabolic functions [3], and in plants, participating in specialized stages of their development such as cell division [4], microsporogenesis [5], pollen tube growth or seed germination [6]. Beta-1,3-glucanases from higher plants belong to the glycosyl hydrolase family 17 (GHF 17) [7, 8]. They exist in multiple forms differing on their molecular properties (molecular mass, isoelectric point, primary structure, and glycosylation) [9], cellular localization and expression pattern, supporting the diverse roles for these enzymes in plants[10]. Protective responses against pathogen attack [6, 11, 12] have been attributed to the expression of these enzymes that belong to the group 2 of pathogenesis-related (PR) proteins [11, 12]. Their antifungal activity in plants has made these enzymes important tools for strategies to develop resistance in crop plants against fungal pathogens [13]. Beta-1,3-glucanases are also constitutively expressed in several tissues of plants and have been implicated in physiological and developmental processes in organs such as bud, style of flower, anther, seed from uninfected plants, e.g. cell division or pollen development. An allergenic character has been associated to several members of this protein family, either from pollens [14], fruits [15] or natural rubber latex [16]. The crucial roles of these molecules acting as allergens and triggering the symptoms among allergic patients make their characterization relevant for the diagnosis of such hypersensitivity disorder. Poll (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0133066&type=printable

María Torres, Oscar Palomares, Joaquín Quiralte, Gabrielle Pauli, Rosalía Rodríguez, Mayte Villalba. An Enzymatically Active β-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family, PLOS ONE, 2015, Volume 10, Issue 7, DOI: 10.1371/journal.pone.0133066