Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.W_01213P22236_B1(VLID)404835

Nitration of the birch pollen allergen Bet v 1.0101: efficiency and site-selectivity of liquid and gaseous nitrating agents.

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O₃/NO₂). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O₃/NO₂. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O₃/NO₂ was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein

PLoS ONE / The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma.

Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. METHODS: In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with (68)Ga via a NOTA derivative. Administered activities were 53-174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. RESULTS: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. CONCLUSION: (68)Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial

Nitration of β-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice

<div><p>Background</p><p>We revealed in previous studies that nitration of food proteins reduces the risk of <i>de novo</i> sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, <i>in vitro</i> experiments suggested an increased capacity of effector cell activation by nitrated food proteins.</p><p>Objective</p><p>The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.</p><p>Design</p><p>BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.</p><p>Results</p><p>A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.</p><p>Conclusion</p><p>Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.</p></div

Cytokine profiles of primDCs stimulated with LPS-depleted allergen + LPS.

<p>Profile of cytokines released by primary dendritic cells after 24 hours of stimulation with different concentrations of LPS (0.1, 1 or 10 ng/ml) alone, or in combination with 50 µg/ml LPS-depleted Bet v 1.0101 or LPS-depleted nitro-Bet v 1.0101. Each dot represents one donor. The mean and SD for all donors are shown. <i>P</i> values were calculated by one-way ANOVA (*<i>P</i><.05; **<i>P</i><.01).</p

Structural analysis of nitro-Bet v 1.0101.

<p>Crystal structure analysis of monomeric nitro-Bet v 1.0101 revealed that monomeric nitro-Bet v 1.0101 displays the same backbone as Bet v 1.0101 with nitration of Y5, Y66, Y83 and Y150 <i>(A)</i>, the electron density corresponding to the nitration of tyrosine 66 <i>(B)</i>, the detection of two positions of the nitro group on tyrosine 83 <i>(C)</i>, the influence of nitration of Y83 on Asp 69 <i>(D)</i> and the influence of nitration of Y66 on Glu 87 <i>(E)</i>. HPLC-MS analysis of five separately nitrated Bet v 1.0101 samples for calculation of the mean percentage of nitration for each Y showed as main difference between the complete nitrated sample and the monomeric one the additional nitration of Y81 <i>(F)</i>.</p

Biochemical characterization.

<p>Oligomerization and aggregation behavior of Bet v 1.0101, mock-Bet v 1.0101 and nitro-Bet v 1.0101 analyzed by size exclusion chromatography (SEC), by gel electrophoresis and by DLS. Highly concentrated sample (15 mg/ml) was loaded onto a Superdex 75 10/300 GL column for SEC analysis. Bet v 1.0101 and mock-Bet v 1.0101 eluted as a monomeric peak, whereas nitro-Bet v 1.0101 showed several peaks corresponding to oligomers. An SDS-PAGE (15%, non-reducing) with the corresponding fractions as eluted during the SEC is shown. Bet v 1.0101 was collected starting from an elution volume of 11 ml, nitro-Bet v 1.0101 was collected starting from an elution volume of 7 ml. <i>(A)</i> Nitro-tyrosine was detected with anti-nitrotyrosine antibody on Western blot and the whole protein was visualized by silver staining on SDS-PAGE. <i>(B)</i> DLS measurements revealed different aggregation states for Bet v 1.0101, mock-Bet v 1.0101 and nitro-Bet v 1.0101. <i>(C)</i>.</p

T-cell line proliferation.

<p>Proliferative responses of Birch pollen (BP)-specific TCLs from BP-allergic donors stimulated with decreasing amounts of LPS-contaminated Bet v 1.0101, mock-Bet v 1.0101 and nitro-Bet v 1.0101 (protein) and stimulated with decreasing amounts of three different LPS-free immunodominant epitopes (peptides) of Bet v 1.0101 containing a tyrosine, in native form or nitrated. The SI was calculated as the ratio between counts per minute (cpm) in stimulated cultures and cpm of the medium control. Each symbol represents a single donor (n = 5), the mean is indicated with a horizontal bar and the SD is represented by the vertical bar. Statistical significance was calculated with One-Way Anova and the Tukey's test per concentration (untreated, mock-nitrated and nitrated protein): <i>P</i> = .2506 for 5 µg/ml protein; <i>P</i> = .1354 for 2.5 µg/ml protein; <i>P = </i>.0815 for 1.25 µg/ml protein and <i>P</i> = .0618 for 0.625 µg/ml protein. Statistical significance was calculated with the <i>t</i>-test for the untreated and nitrated peptide per concentration. No values below <i>P</i> = .0824 could be measured.</p