1. Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4 
Domen Zafred, MSc, Andreas Nandy, PhD, Linda Pump, Helga Kahlert, PhD, Walter Keller, PhD 
Journal of Allergy and Clinical Immunology 
Volume 132, Issue 3, Pages e10 (September 2013)
DOI: /j.jaci
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Comparison of IgE reactivity and allergenicity of nPhl p 4 and recombinant allergens. A, ELISA plates in the inhibition assay were coated with nPhl p 4 (first row) and rPhl p (second row), and the sera of individual patients with GPA allergy were preincubated with increasing amounts of inhibitor (x-axis). The inhibition values are shown as percentages of the maximum inhibition detected, and vertical lines represent data points (protein concentration). B, Basophil activation assays were performed to compare the allergenicity of nPhl p 4, rPhl p 4, and Phl p 1 and also, for 3 subjects, Phl p 5. Vertical lines represent data points (protein concentration). C, Plotted are concentrations needed for the specific activation of 50% of the basophils (C50 [nmol/L]) or the ratio of these concentrations needed with different allergens (Prel). Boxes present the medians and first quartiles, and whiskers present the ninth and 91st percentiles of the plotted data.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Representation and structural details of Phl p 4. A, The F-domain is in a red color scheme, with helixes in salmon and β-strands in brown. The S-domain is in a green color scheme, with helixes in olive and β-strands in dark green. The borders of the F-domain are marked with the corresponding residues (P226 and N431), and the termini of the protein are marked with N and C. The broad catalytic cavity extending on the si side of the FAD is shaped by a broad β-sheet (green). Residues Q61 and Q330, which are glycosylated in the wild-type protein rPhl p (N61 and N330, respectively), are in stick representation (a representation that shows covalent bonds as sticks between the atoms), with blue spheres placed where the glycan is attached in the wild-type protein. B, FAD (black) is in stick representation with a view along the N5-N10 axis, visualizing the bent isoalloxazine ring and the broad cavity beneath. The covalent bonds with residues H86 and C150 are seen on the right side of the ring. C, A metal ion is octahedrally coordinated by 4 water molecules and residues E89 and E338. D, The 2 cysteine bridges are in stick representation, connecting C14 and C71 in the F-domain and C281-C302 in the S-domain.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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4. Fig 3
Structure-based sequence alignment of the Phl p 4 and BBE. The alignment of the Phl p 4 (3TSH) and BBE (3D2H) yielded 37% sequence identity. The unaligned residues are shown in boldface. Annotations used were as follows: identical residues (*) and strongly (:) and weakly (.) conserved residues. These signs are underlined for completely or partially buried residues of Phl p 4 having less than 25% FSASs. Secondary structure elements (arrows for β-strands and cylinders for helixes) are in the same color scheme as in Fig 2. The F-domain of Phl p 4 (salmon) consists of 2 sequences flanking the S-domain (olive). Cysteines colored yellow form disulfide bonds. Residues binding FADs are orange: H86 and C150 in Phl p 4 and H104 and C166 in BBE. Glycosylation sites are in blue, and the glycosylation present in Phl p was eliminated by the mutations N61Q and N330Q.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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5. Fig 4
Comparison of the structures of Phl p 4 and BBE. A, The 3-dimensional alignment reveals a high structural similarity of the 2 proteins, which are shown in the same orientation as in Fig 2, A. BBE is colored green, and Phl p 4 is colored in a gradient from blue (aligned residues with low root mean SD [RMSD]) to red (unaligned residues). B, Surface representation of Phl p 4 and BBE (oriented as in Fig 4, A) shows charge distribution on the surface as a gradient from blue (positively charged residues) over white (uncharged residues) to red (negatively charged residues).
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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6. Fig E1
Purity of nPhl p 4 and detection of the conjugated carbohydrates. A, nPhl p 4 was purified from pollen extract (lane A) to homogeneity (lane B). A clear distinction in size to the nPhl p 13 can be seen (lane C). Dot blot analysis with 3 different specific mAbs proved the purity of our samples: the nPhl p 4–specific murine IgG mAbs 3C4 (lane D) and 5H1 (lane E) bound to a purified sample of nPhl p 4 but not nPhl p 13. The nPhl p 13–specific antibody 4G6 did not bind to the membrane loaded with nPhl p 4 (lane F). B, The purified allergens rPhl p (lanes G and J), nonglycosylated rPhl p (lanes H and K), and nPhl p 4 (lanes I and L) were applied to an SDS-PAGE gel and transferred to a nitrocellulose membrane. Glycosylation was detected with the DIG Glycan Detection Kit (Roche, Mannheim, Germany). No glycosylation was detected for the nonglycosylated mutant.
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7. Fig E2
SDS-PAGE and Western blot analysis. A, Nonglycosylated rPhl p (lane A), rPhl p (lane B), rPhl p (lane C), rSec c (lane D), and nPhl p 4 (lane E) were applied to an SDS-PAGE gel. The double band of the nonglycosylated protein is a consequence of partly broken bonds with the FAD. B and C, Proteins from Fig E2, A, were blotted onto a nitrocellulose membrane, where they were detected with the murine anti–nPhl p 4 mAbs 5H1 (membrane B) and 3C4 (membrane C). The latter did not bind rSec c (membrane C, lane D).
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8. Fig E3
Gel filtration analysis of the purified proteins. The column was calibrated with ferritin (F; 440 kDa), catalase (C; 232 kDa), albumin (A; 67 kDa), ovalbumin (O; 43 kDa), and ribonuclease (R; 13.7 kDa). V0, Void volume. Both recombinant proteins form a single peak corresponding to a monomer. The natural protein has a shoulder approximately at the elution volume of the nonglycosylated protein, suggesting that the plant-derived glycosylation is not homogenous. No aggregates or oligomers are visible in the elution diagrams.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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9. Fig E4
CD spectra of the nPhl p 4 and recombinant isoallergens. The CD signal is plotted as the mean residue ellipticity (y-axis) against the wavelength (x-axis), indicating that the proteins are completely folded and exhibit very similar fold. Minor differences in the CD signal might be attributed to differences in the flexible parts of the structure or to glycosylation.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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10. Fig E5
IgE Inhibition assays with immobilized nPhl p 4. ELISA plates were coated with nPhl p 4, and sera of 5 patients with GPA allergy were preincubated with increasing amounts of inhibitors: glycosylated and nonglycosylated rPhl p No significant difference in inhibition properties was observed among the recombinant allergens.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
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11. Fig E6
Topologic diagram of Phl p 4. For an easier comparison, the orientations and positions of the secondary structure elements in the diagram are based on the diagram of PMCH (1DII), the model structure from this structural clan of the FAD-binding enzymes.E7,E9 Secondary structure elements in this diagram have the same color and size as in Fig 3 and thus represent the number of amino acids in the element and not its relative size in the structure. The F-domain is in red, and the S-domain is in green. Elements in bright colors (α6, α7, α14, α16, β19, β20, and the 310 helix from the S-domain) are not present in the structure of PMCH. Elements α8, β7, and β11 are present in both structures but were not displayed in the topologic diagram of the PMCH. The sequence flow of the β-strands β3 and β4 is correct in our topologic diagram and not in the published topologic diagram of PCMH.E7
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12. Fig E7
Enzymatic activity. A, Using 4 nmol/L protein, the highest turnover of 2,6-dichlorophenolindophenol was detected with 10 mmol/L glucose. B, FAD levels were reduced on titration with glucose (gray curve). Titration was paused at a glucose/protein ratio of After 12 hours of incubation at room temperature, approximately 10% of FAD reoxidized (black curve; glucose/protein ratio, 0.65). The titration was continued (black curve), and after reaching the molar ratio of 1.5:1, the absorbance remained constant.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions