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Non-Specific Lipid Transfer Protein Amb a 6 Is a Source-Specific Important Allergenic Molecule in Ragweed Pollen

Grijincu, Manuela, Tănasie, Gabriela, Zbîrcea, Lauriana-Eunice, Buzan, Maria-Roxana, Tamaș, Tudor-Paul, Cotarcă, Monica-Daniela, Huțu, Ioan, Babaev, Elijahu, Stolz, Frank, Dorofeeva, Yulia, Valenta, Rudolf, Păunescu, Virgil, Panaitescu, Carmen, Chen, Kuan-Wei·2024·International Journal of Molecular Sciences
DOI 10.3390/ijms25126513PMC PMC11204090
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Abstract

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

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Figure 1 Overview of recombinant allergen production: (a) SDS-PAGE with the purified rAmb a 6 separating under non-reducing (NR) and reducing (R) conditions; (b) IgE binding towards rAmb a 6 (A6) separated under NR and R conditions using serum from five ragweed-allergic patients, one non-allergic subject (N.C.), and buffer used as control. A molecular weight marker is shown on the left for both SDS-PAGE and immunoblot.
Figure 1 Overview of recombinant allergen production: (a) SDS-PAGE with the purified rAmb a 6 separating under non-reducing (NR) and reducing (R) conditions; (b) IgE binding towards rAmb a 6 (A6) separated under NR and R conditions using serum from five ragweed-allergic patients, one non-allergic subject (N.C.), and buffer used as control. A molecular weight marker is shown on the left for both SDS-PAGE and immunoblot.CC BY · PubMed Central
Figure 2 Physicochemical characterization of the recombinant allergen Amb a 6 (rAmb a 6): (a) MALDI-ToF mass spectrum for rAmb a 6. The mass/charge ratio is shown on the x-axis; (b) Far UV spectrum of rAmb a 6. The mean residue ellipticity θ (deg × cm2/dmol) is shown on the y-axis and the wavelength (nm) is shown on the x-axis; the 3D model of Amb a 6, excluding the signal peptide is also shown [40].
Figure 2 Physicochemical characterization of the recombinant allergen Amb a 6 (rAmb a 6): (a) MALDI-ToF mass spectrum for rAmb a 6. The mass/charge ratio is shown on the x-axis; (b) Far UV spectrum of rAmb a 6. The mean residue ellipticity θ (deg × cm2/dmol) is shown on the y-axis and the wavelength (nm) is shown on the x-axis; the 3D model of Amb a 6, excluding the signal peptide is also shown [40].CC BY · PubMed Central
Figure 3 Homology of Amb a 6 with other known nsLTP allergens: (a) Sequence alignment of Amb a 6 with 41 other similar allergenic nsLTPs, with amino acids conserved among all nsLTPs highlighted in yellow and cysteine residues of Amb a 6 evidenced in bold red text; (b) Maximum parsimony phylogenetic tree of the allergenic proteins detected by NCBI Blast.
Figure 3 Homology of Amb a 6 with other known nsLTP allergens: (a) Sequence alignment of Amb a 6 with 41 other similar allergenic nsLTPs, with amino acids conserved among all nsLTPs highlighted in yellow and cysteine residues of Amb a 6 evidenced in bold red text; (b) Maximum parsimony phylogenetic tree of the allergenic proteins detected by NCBI Blast.CC BY · PubMed Central
Figure 4 IgE recognition of rAmb a 6: (a) Correlation between the optical densities (OD) obtained for rAmb a 6 in IgE ELISA and the specific IgE levels determined by ImmunoCAP for 150 ragweed pollen-allergic patients; (b) Specific IgE reactivity towards rAmb a 6, Amb a 1, and ragweed pollen extract for 150 ragweed-allergic patients. In the boxplot, the line shows the median, the box represents the 25th and 75th percentile, and the whiskers show the minimum and maximum values. Statistically significant differences are marked as *** p < 0.001; (c) Specific IgE levels towards rAmb a 6 and Amb a 1 for 45 patients reactive towards rAmb a 6 in ImmunoCAP.
Figure 4 IgE recognition of rAmb a 6: (a) Correlation between the optical densities (OD) obtained for rAmb a 6 in IgE ELISA and the specific IgE levels determined by ImmunoCAP for 150 ragweed pollen-allergic patients; (b) Specific IgE reactivity towards rAmb a 6, Amb a 1, and ragweed pollen extract for 150 ragweed-allergic patients. In the boxplot, the line shows the median, the box represents the 25th and 75th percentile, and the whiskers show the minimum and maximum values. Statistically significant differences are marked as *** p < 0.001; (c) Specific IgE levels towards rAmb a 6 and Amb a 1 for 45 patients reactive towards rAmb a 6 in ImmunoCAP.CC BY · PubMed Central
Figure 5 Mediator release assay towards rAmb a 6, nAmb a 1.01, and Par j 2 using serum from ragweed-allergic patients. The results are shown as the percentage of total release, bar charts represent the median release, and the error bars show the 95% confidence interval. The dashed line indicates the degranulation obtained from cells with serum without allergen (cut-off). Allergen concentrations (ng/mL) are displayed on the x-axes, with the y-axes showing the percentages of total release (%).
Figure 5 Mediator release assay towards rAmb a 6, nAmb a 1.01, and Par j 2 using serum from ragweed-allergic patients. The results are shown as the percentage of total release, bar charts represent the median release, and the error bars show the 95% confidence interval. The dashed line indicates the degranulation obtained from cells with serum without allergen (cut-off). Allergen concentrations (ng/mL) are displayed on the x-axes, with the y-axes showing the percentages of total release (%).CC BY · PubMed Central
Figure 6 Lack of clear association between reactivity to rAmb a 6 and the clinical features of ragweed-allergic patients: (a) Allergy-related symptoms reported by patients positive towards rAmb a 6 in ImmunoCAP compared to those which were negative in ImmunoCAP. Statistically significant results are marked as ** p < 0.01; (b) Symptom types reported by patients positive and negative towards rAmb a 6 in ImmunoCAP; (c) Boxplots showing sIgE levels towards rAmb a 6 in monosensitized, oligo-, and polysensitized ragweed-allergic patients according to SPT among rAmb a 6-positive and -negative patients. In the boxplots, the horizontal lines show the median, the box represents the 25th and 75th percentile, and the whiskers show the minimum and maximum values.
Figure 6 Lack of clear association between reactivity to rAmb a 6 and the clinical features of ragweed-allergic patients: (a) Allergy-related symptoms reported by patients positive towards rAmb a 6 in ImmunoCAP compared to those which were negative in ImmunoCAP. Statistically significant results are marked as ** p < 0.01; (b) Symptom types reported by patients positive and negative towards rAmb a 6 in ImmunoCAP; (c) Boxplots showing sIgE levels towards rAmb a 6 in monosensitized, oligo-, and polysensitized ragweed-allergic patients according to SPT among rAmb a 6-positive and -negative patients. In the boxplots, the horizontal lines show the median, the box represents the 25th and 75th percentile, and the whiskers show the minimum and maximum values.CC BY · PubMed Central

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Metadata

Authors
Grijincu, M., Tănasie, G., Zbîrcea, L.-E., Buzan, M.-R., Tamaș, T.-P., Cotarcă, M.-D., Huțu, I., Babaev, E., Stolz, F., Dorofeeva, Y., Valenta, R., Păunescu, V., Panaitescu, C., & Chen, K.-W.
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2024-06-13
Volume
25
Issue
12
Pages
6513
ISSN
1422-0067
DOI
10.3390/ijms25126513
PMC
PMC11204090
Cited by
7
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • INSPIRED (Innovative Strategies for Prevention, Diagnosis, and Therapy of Ragweed Pollen Induced Respiratory Diseases) project (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)
  • European Regional Development Fund (ERDF) (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)
  • Romanian national budget (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)
  • Danube Allergy Research Cluster of the Country of Lower Austria (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)
  • Russian Science Foundation (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)
  • Victor Babes University of Medicine and Pharmacy, Timișoara (2014–2020 92/09.09.2016, P_37_747, 23-75-30016)

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