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.

Fig 1. Detection of a β-1,3-glucanase homolog to Ole e 9 in ash pollen.

Coomassie Blue staining (CBS), IgG reactivity of pAbs obtained agaisnt rNtD and rCtD of Ole e 9 and IgE reactivity of a pool of sera from patients allergic to olive pollen, to olive and ash pollen extracts (40 μg of total protein). Molecular mass markers are indicated.

Fig 2. Nucleotide sequence of cDNA encoding Fra e 9 and deduced amino acid sequence.

The putative cleavage site for the signal peptide is indicated by an arrowhead. The catalytic residues are boxed. The cysteine residues of the C-terminal domain are circled. The potential N-glycosylation sites are framed.

Fig 3. Alignment of Fra e 9 with the β-1,3-glucanases from other plant sources.

Ash (Fra e 9, KC920916), olive (Ole e 9, Q94G86), pear (B9VQ36), A. thaliana (Q06915), latex (Hev b 2, A2TJX4), banana (Mus a 5, A7U7Q7), tomato (Q01413), barley (P15737). (A) Dashes indicate gaps. Letters over black shading are conserved residues in all sequences; dark gray indicates residues conserved in at least six sequences; light gray indicates residues conserved in five sequences. % I and %S represent identity and similarity percentages of these sequences comparing to that of Fra e 9. (B) Independent comparison of the amino acid sequences of N-terminal and C-terminal domains of the β-1,3-glucanases with Fra e 9 domains.

Fig 4. Recombinant expression and molecular and functional characterization of the NtD-Fra e 9.

(A) Time-course of the expression of rNtD-Fra e 9 in E. coli. Supernatants and pellets from cultures were harvested at different times after induction and stained with Coomassie Blue (CBS) after SDS-PAGE. (B) Purified domain was analysed by CBS and by immunostaining with a pool of sera from olive pollen allergic patients (Sera) or specific antiserum (pAb). (C) Mass spectrometry analysis. (D) CD spectrum in the far-UV (190–250 nm). In the inset are included the porcentages of secondary structure. (E) Thermal unfolding measured as ellipticity at 220nm during heating from 20°C to 80°C. (F) Schematic representation of the 3D-model of NtD-Fra e 9 and NtD-Ole e 9; The main structural differences are highlighted. (G) The pH dependence for the enzymatic activity of rNtD-Fra e 9 was assayed at different pH values.

Fig 5. Recombinant expression and molecular characterization of CtD-Fra e 9.

(A) Time-course of the expression of rCtD-Fra e 9 in P. pastoris. Extracellular medium was harvested at different times after induction and stained with Coomassie Blue (CBS) after SDS-PAGE. (B) Purified domain was analysed by CBS, immunostaining with a pool of sera from olive pollen allergic patients (Sera) and with a Ole e 9-specific antiserum (pAb), and staining with ConA-rNtD was loaded in the same lane of the gel as a control for no glycosylation-. (C) Mass spectrometry analysis. (D) CD spectra in the far-UV (190–250 nm). (E) thermal unfolding assay during heating from 20°C to 80°C as in Fig 4. (F) Schematic representation of the 3D of Fra e 9 modeled against Ole e 9 3D structure determined by NMR [26]. (G) Affinity gel electrophoresis (AGE) analysis of rCtD-Fra e 9 (Fra e 9), rCtD-Ole e 9 (Ole e 9) and BSA as negative control; both proteins were electrophoresed under non-denaturing conditions in polyacrylamide gels in the presence and absence (-) of laminarin.

Fig 6. Analysis of the IgG and IgE reactivity of Fra e 9 and Ole e 9.

(A) Identification of the isoforms pattern of both β-1,3-glucanases by 2-DE with pAbs raised against rNtD and rCtD of Fra e 9 in ash and olive pollen extracts. (B) IgE-binding inhibition analysis by ELISA of Fra e 9 recombinant domains after preincubation of the pool of sera from patients allergic to olive with increasing amounts of ash or olive pollen extracts.