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A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity

García-Ramírez, Benjamín, Mares-Mejía, Israel, Rodríguez-Hernández, Annia, Cano-Sánchez, Patricia, Torres-Larios, Alfredo, Ortega, Enrique, Rodríguez-Romero, Adela·2022·Communications Biology
DOI 10.1038/s42003-022-03718-wPMC PMC9334453
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Abstract

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis(Hev b 8). The crystallographic models revealed that the IgE’s six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å 2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE’s elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

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Fig. 1 rHev b 8 profilin stability. a Far-UV CD spectra at different temperatures: at 25 °C before heat treatment (blue), at 90 °C (gray), and first (orange) and second (yellow) annealing processes (n = 3). The Table shows percentages of secondary structure (αH, α-helix; βS, β-sheet, Turns, and Other). b Box and whisker plot showing ELISA results of the recognition of rHev b 8 after the first and second annealing procedures, rZea m 12 was used as a negative control. Error bars represent standard deviations of the means of four replications. c Left: SDS-Page showing rHev b 8 and rZea m 12, Right: Western blot. IgE 2F5 recognized rHev b 8 but did not recognize rZea m 12.
Fig. 1 rHev b 8 profilin stability. a Far-UV CD spectra at different temperatures: at 25 °C before heat treatment (blue), at 90 °C (gray), and first (orange) and second (yellow) annealing processes (n = 3). The Table shows percentages of secondary structure (αH, α-helix; βS, β-sheet, Turns, and Other). b Box and whisker plot showing ELISA results of the recognition of rHev b 8 after the first and second annealing procedures, rZea m 12 was used as a negative control. Error bars represent standard deviations of the means of four replications. c Left: SDS-Page showing rHev b 8 and rZea m 12, Right: Western blot. IgE 2F5 recognized rHev b 8 but did not recognize rZea m 12.CC BY · PubMed Central
Fig. 2 Basis of allergen recognition through the free Fab/IgE 2F5 and the complex with DDDK-rHev b 8 structures. a MALDI-TOF spectrum for the DDDK-Hev b 8 construct. b and c Ribbon diagrams of the complex Fab/IgE 2F5-DDDK-rHev b 8 at 3.04 (gold) and 3.34 Å resolution (blue), respectively. d Fab constant domains show a displacement of 19.2 degrees due to flexibility at the elbow region. e The three free Fab/IgE 2F5 molecules in the asymmetric unit (3.75 Å resolution). f Superposition of the three free Fab/IgE 2F5 molecules in the asymmetric unit (red, blue, and purple), with the complex Fab/IgE 2F5-DDDK-rHev b 8 at 3.04 Å resolution (gold).
Fig. 2 Basis of allergen recognition through the free Fab/IgE 2F5 and the complex with DDDK-rHev b 8 structures. a MALDI-TOF spectrum for the DDDK-Hev b 8 construct. b and c Ribbon diagrams of the complex Fab/IgE 2F5-DDDK-rHev b 8 at 3.04 (gold) and 3.34 Å resolution (blue), respectively. d Fab constant domains show a displacement of 19.2 degrees due to flexibility at the elbow region. e The three free Fab/IgE 2F5 molecules in the asymmetric unit (3.75 Å resolution). f Superposition of the three free Fab/IgE 2F5 molecules in the asymmetric unit (red, blue, and purple), with the complex Fab/IgE 2F5-DDDK-rHev b 8 at 3.04 Å resolution (gold).CC BY · PubMed Central
Fig. 3 IgE 2F5 and its Fab are glycosylated. a SDS-PAGE stained with the Pierce Glycoprotein Staining Kit. Lane one: Molecular weight markers. Second lane: the Fab/IgE 2F5 heavy chain (VH and Cε1) is stained; Third lane: the complete IgE 2F5 heavy chain is stained; Fifth and Seventh lanes: Heavy chains of the complete IgG 2D10 and 1B4, which are glycosylated are stained. No stained bands were observed in the Fourth and Sixth lanes. Eighth and Tenth lanes, profilin (rHev b 8) and the soy trypsin inhibitor (STI) were used as negative controls: The Ninth lane shows horseradish peroxidase (HRP) used as a positive control. b The same gel was stained with Coomassie Blue G250. The Fab/IgE 2F5 light chain, IgG light and heavy chains, rHev b 8 profilin, and STI are observed. c Polder map contoured at 3σ (blue) and omit map 3σ (green) for the glycosylation at N189. d Composition of the oligosaccharide bound to N189 on the Fab/IgE 2F5, which comprises a core of di-mannose and fucose connected to the chitobiose core (GlcNAc2). Abbreviations: N, GlcNAc; M, mannose; F, fucose. e Mass spectra of DDDK-rHev b 8 (14,625 Da) and of the Fab/IgE 2F5 (57,397.3 Da). Lysozyme (14,100 Da) and bovine serum albumin (66,410 Da) were used to calibrate the spectrum. Interestingly, peaks of the Fab/IgE 2F5-DDDK-rHev b 8 complex (72,397.9 Da) and the dimeric Fab (115,371 Da) were also detected; however, these two peaks were not calibrated.
Fig. 3 IgE 2F5 and its Fab are glycosylated. a SDS-PAGE stained with the Pierce Glycoprotein Staining Kit. Lane one: Molecular weight markers. Second lane: the Fab/IgE 2F5 heavy chain (VH and Cε1) is stained; Third lane: the complete IgE 2F5 heavy chain is stained; Fifth and Seventh lanes: Heavy chains of the complete IgG 2D10 and 1B4, which are glycosylated are stained. No stained bands were observed in the Fourth and Sixth lanes. Eighth and Tenth lanes, profilin (rHev b 8) and the soy trypsin inhibitor (STI) were used as negative controls: The Ninth lane shows horseradish peroxidase (HRP) used as a positive control. b The same gel was stained with Coomassie Blue G250. The Fab/IgE 2F5 light chain, IgG light and heavy chains, rHev b 8 profilin, and STI are observed. c Polder map contoured at 3σ (blue) and omit map 3σ (green) for the glycosylation at N189. d Composition of the oligosaccharide bound to N189 on the Fab/IgE 2F5, which comprises a core of di-mannose and fucose connected to the chitobiose core (GlcNAc2). Abbreviations: N, GlcNAc; M, mannose; F, fucose. e Mass spectra of DDDK-rHev b 8 (14,625 Da) and of the Fab/IgE 2F5 (57,397.3 Da). Lysozyme (14,100 Da) and bovine serum albumin (66,410 Da) were used to calibrate the spectrum. Interestingly, peaks of the Fab/IgE 2F5-DDDK-rHev b 8 complex (72,397.9 Da) and the dimeric Fab (115,371 Da) were also detected; however, these two peaks were not calibrated.CC BY · PubMed Central
Fig. 4 Paratope-epitope interactions. a Epitope residues. Structural alignment of the Fab/IgE-rHev b 8 models at 3.04 Å (gold) and 3.34 Å (blue). b Paratope residues alignment of the models at 3.04 Å (gold) and 3.34 Å (blue).
Fig. 4 Paratope-epitope interactions. a Epitope residues. Structural alignment of the Fab/IgE-rHev b 8 models at 3.04 Å (gold) and 3.34 Å (blue). b Paratope residues alignment of the models at 3.04 Å (gold) and 3.34 Å (blue).CC BY · PubMed Central
Fig. 5 Stereo-view of the paratope-epitope interface. Fab/IgE 2F5 light chain (yellow), heavy chain (green) and profilin (pink), the 2Fo-Fc electron density map (1σ) is shown in light blue. The epitope-paratope interface area (926 Å2).
Fig. 5 Stereo-view of the paratope-epitope interface. Fab/IgE 2F5 light chain (yellow), heavy chain (green) and profilin (pink), the 2Fo-Fc electron density map (1σ) is shown in light blue. The epitope-paratope interface area (926 Å2).CC BY · PubMed Central
Fig. 6 Paratope-epitope interactions and affinity of the Fab/IgE 2F5. a Interaction network summary. Heavy chain (H), light chain (L), and profilin (P). Salt bridges are represented as red lines, hydrogen bonds as blue lines, and nonbonded contacts (electrostatics and van der Waals interactions) with orange lines. b BLI fitting considering nonspecific binding (NSB) using the Association-Dissociation model implemented in GraphPad Prism 8 and a 1:1 model. Six different concentrations were tested and are shown in different colors.
Fig. 6 Paratope-epitope interactions and affinity of the Fab/IgE 2F5. a Interaction network summary. Heavy chain (H), light chain (L), and profilin (P). Salt bridges are represented as red lines, hydrogen bonds as blue lines, and nonbonded contacts (electrostatics and van der Waals interactions) with orange lines. b BLI fitting considering nonspecific binding (NSB) using the Association-Dissociation model implemented in GraphPad Prism 8 and a 1:1 model. Six different concentrations were tested and are shown in different colors.CC BY · PubMed Central
Fig. 7 Generating cross-reactivity. a Ribbon and stick models of rHev b 8 (pink) and rZea m 12 (cyan) profilins (RMSD 0.307 Å). Different residues between both profilins (E14, N98, I118, and D128) are circled in dotted lines; this epitope region is part of the polyproline binding site in profilins. b Sequence alignment between rHev b 8 and rZea m 12, green arrows show conserved residues in the epitope, and yellow arrows show different residues. c Paratope residues of Fab/IgE 2F5 and the four residues in rHev b 8 that are different in rZea m 12 are displayed as sticks. rHev b 8 epitope residues are shown in pink, the Fab heavy chain residues in green, and the Fab light chain residues in yellow. Dotted lines indicate residues interactions.
Fig. 7 Generating cross-reactivity. a Ribbon and stick models of rHev b 8 (pink) and rZea m 12 (cyan) profilins (RMSD 0.307 Å). Different residues between both profilins (E14, N98, I118, and D128) are circled in dotted lines; this epitope region is part of the polyproline binding site in profilins. b Sequence alignment between rHev b 8 and rZea m 12, green arrows show conserved residues in the epitope, and yellow arrows show different residues. c Paratope residues of Fab/IgE 2F5 and the four residues in rHev b 8 that are different in rZea m 12 are displayed as sticks. rHev b 8 epitope residues are shown in pink, the Fab heavy chain residues in green, and the Fab light chain residues in yellow. Dotted lines indicate residues interactions.CC BY · PubMed Central
Fig. 8 Cross-reactivity immunoassay. Box and whisker plot showing ELISA results for Zea m 12, and its mutants compared with rHev b 8 and the Sidak’s multiple comparisons tests. IgE interaction ELISA using rHev b 8, and the DDDK-rHev b 8 construct as positive controls, rZea m 12 as a negative control, rZea m 12-E128D single mutant, rZea m 12-E128D-G98N double mutant, and PBS as background control (ns not significant, all P values less than 0.001 are summarized with three asterisks). For these experiments, n = 4.
Fig. 8 Cross-reactivity immunoassay. Box and whisker plot showing ELISA results for Zea m 12, and its mutants compared with rHev b 8 and the Sidak’s multiple comparisons tests. IgE interaction ELISA using rHev b 8, and the DDDK-rHev b 8 construct as positive controls, rZea m 12 as a negative control, rZea m 12-E128D single mutant, rZea m 12-E128D-G98N double mutant, and PBS as background control (ns not significant, all P values less than 0.001 are summarized with three asterisks). For these experiments, n = 4.CC BY · PubMed Central

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Authors
García-Ramírez, B., Mares-Mejía, I., Rodríguez-Hernández, A., Cano-Sánchez, P., Torres-Larios, A., Ortega, E., & Rodríguez-Romero, A.
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2022-07-27
Volume
5
Issue
1
ISSN
2399-3642
DOI
10.1038/s42003-022-03718-w
PMC
PMC9334453
Cited by
16
References
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Language
en
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