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

<p>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.</p

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

<p>(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.</p

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

<p>(A) Time-course of the expression of rNtD-Fra e 9 in <i>E</i>. <i>coli</i>. 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.</p

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

<p>(A) Time-course of the expression of rCtD-Fra e 9 in <i>P</i>. <i>pastoris</i>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133066#pone.0133066.g004" target="_blank">Fig 4</a>. (F) Schematic representation of the 3D of Fra e 9 modeled against Ole e 9 3D structure determined by NMR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133066#pone.0133066.ref026" target="_blank">26</a>]. (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.</p

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

<p>Ash (Fra e 9, KC920916), olive (Ole e 9, Q94G86), pear (B9VQ36), <i>A</i>. <i>thaliana</i> (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.</p

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

<p>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.</p