Novel post-translationally cleaved Ara h 2 proteoforms: Purification, characterization and IgE-binding properties.

The 2S albumins Ara h 2 and Ara h 6 have been shown to be the most important source of allergenicity in peanut. Several isoforms of these allergens have been described. Using extraction and liquid chromatography we isolated proteins with homology to Ara h 2 and characterized hitherto unknown Ara h 2 proteoforms with additional post-translational cleavage. High-resolution mass spectrometry located the cleavage site on the non-structured loop of Ara h 2 while far UV CD spectroscopy showed a comparable structure to Ara h 2. The cleaved forms of Ara h 2 were present in genotypes of peanut commonly consumed. Importantly, we revealed that newly identified Ara h 2 cleaved proteoforms showed comparable IgE-binding using sera from 28 peanut-sensitized individuals, possessed almost the same IgE binding potency and are likely similarly allergenic as intact Ara h 2. This makes these newly identified forms relevant proteoforms of peanut allergen Ara h 2.

Defining the cross-reactivity between peanut allergens Ara h 2 and Ara h 6 using monoclonal antibodies.

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

Unveiling the critical pH values triggering the unfolding of soy 7S and 11S globulins and enhancing their encapsulation efficiency.

The pH-shifting process is an effective encapsulation method, and it is typically performed at extreme alkaline pH, which severely limits the application. In this study, we found that there were critical pH for the unfolding proteins during pH-shifting from 7 to 12, and upon the critical pH, physiochemical characteristics of protein greatly changed, leading to a sharp increase of encapsulation of hydrophobic actives. Firstly, the critical pH for ß-conglycinin (7S) or Glycinin (11S) unfolding was determined by multispectral technology. The critical pH for 7S and 11S were 10.5 and 10.3, respectively. The encapsulation efficiency (EE) obtained by ß-conglycinin-curcumin nanocomposite (7S-Cur) (88.80 %) and Glycinin-curcumin nanocomposite (11S-Cur) (88.38 %) at critical pH was significantly higher than that obtained by pH 7 (7S-Cur = 16.66 % and 11S-Cur = 15.78 %), and both values were close to EE obtained by at 12 (7S-Cur = 95.16 % and 11S-Cur = 94.63 %). The large-scale application of hydrophobic functional compounds will be enhanced by the experimental results.

Atmospheric cold plasma reduces Ara h 1 antigenicity in roasted peanuts by altering the protein structure and amino acid profile.

Ara h 1 is the major allergen in peanuts. To enhance the unique flavor, peanuts are usually roasted at high temperatures. However, roasting can increase the allergenic potential, owing to glycation of allergens. Atmospheric cold plasma (ACP) is a non-thermal processing technology that generates reactive species, enabling protein structural changes. Herein, glucose was also added to the ACP-treated peanut protein before roasting. The content and antigenicity of the advanced glycation end products were measured. The antigenicity was evaluated by ELISA and in vitro digestion assays. The amino acid profile and secondary and tertiary protein structures were also assessed. The antigenicity of Ara h 1 decreased by 91 % and 76 % after 30 min of air and nitrogen plasma treatment, respectively. The glycation degree and thermal and digestive stabilities were also reduced. These results correlated with the structural changes, denaturation, and aggregation. Therefore, cold plasma may reduce the allergic effects of peanuts.

Investigation of peanut allergen-procyanidin non-covalent interactions: Impact on protein structure and in vitro allergenicity.

Interactions between plant polyphenols and food allergens may be a new way to alleviate food allergies. The non-covalent interactions between the major allergen from peanut (Ara h 2) with procyanidin dimer (PA2) were therefore characterized using spectroscopic, thermodynamic, and molecular simulation analyses. The main interaction between the Ara h 2 and PA2 was hydrogen bonding. PA2 statically quenched the intrinsic fluorescence intensity and altered the conformation of the Ara h 2, leading to a more disordered polypeptide structure with a lower surface hydrophobicity. In addition, the in vitro allergenicity of the Ara h 2-PA2 complex was investigated using enzyme-linked immunosorbent assay (ELISA) kits. The immunoglobulin E (IgE) binding capacity of Ara h 2, as well as the release of allergenic cytokines, decreased after interacting with PA2. When the ratio of Ara h 2-to-PA2 was 1:50, the IgE binding capacity was reduced by around 43 %. This study provides valuable insights into the non-covalent interactions between Ara h 2 and PA2, as well as the potential mechanism of action of the anti-allergic reaction caused by binding of the polyphenols to the allergens.

Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. METHODS: The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. RESULTS: A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. CONCLUSIONS: Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.

Film-forming properties and mechanisms of soy protein: Insights from ß-conglycinin and glycinin.

Extensive research has been conducted on soy protein films; however, limited information is available regarding the influence of the major components, ß-conglycinin (7S) and glycinin (11S), on the film-forming properties of soy protein. This study aimed to isolate the 7S and 11S fractions in order to prepare films and investigate the impact of varying 7S/11S ratios on the film-forming solutions (FFS) and film properties. The findings revealed that higher 11S ratios led to increased protein aggregation, consequently elevating the storage modulus (G') of the FFS. Notably, an optimal 7S/11S ratio of 7S1:11S2 (CF3) significantly enhanced the film's water resistance. Specifically, it enhanced the water contact angle by an impressive 17.44 % and reduced the water vapor transmission rate by 27.56 %. These improvements were attributed to intermolecular interactions, involving hydrogen bonds and salt bridges, between the amino acid residues of 7S and 11S. As a result, a more uniform and dense microstructure was achieved. Interestingly, the mechanical and optical properties of the film were maintained by the different protein fractions examined. In summary, this study contributes to the understanding of the film-forming properties of soy protein, particularly the role of 7S and 11S.

Influence of pH and ionic strength on the physicochemical and structural properties of soybean ß-conglycinin subunits in aqueous dispersions.

Understanding the impact of pH and ionic strength on the physicochemical and structural properties of soy proteins at subunit level is essential for design and fabrication of many plant-based foods. In this study, soybean ß-conglycinin and its subunit fractions αα' and ß were dispersed in solutions with different pH values (3.7, 7.6, and 9.0) at low (5 mM NaCl) and high (400 mM NaCl) ionic strengths, respectively. The solubility, rheology, particle size, zeta potential, microstructure, secondary structure, and tertiary structure of the different dispersions were analyzed using a range of analytical methods. The ß-conglycinin, αα'- and ß-subunits aggregated near the isoelectric point (pH 3.7). Increasing the ionic strength led to the assembly of more homogeneous units. An increase in ionic strength at pH 7.6 and pH 9.0 led to electrostatic screening, which promoted dissociation of the aggregates. The ß-subunit showed a greater sensitivity to pH and ionic strength than the αα'-subunits. Based on the evidence from a range of analytical methods, the highly hydrophilic extension region of the αα'-subunits played an important role in determining the stability of the ß-conglycinin dispersions under different environmental conditions. Moreover, the N-linked glycans appeared to impact the conformation and aggregation state of the ß-conglycinin.

Alanine Scanning of the Unstructured Region of Ara h 2 and of a Related Mimotope Reveals Critical Amino Acids for IgE Binding.

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.

Insights from ß-Conglycinin and Glycinin into the Mechanism of Nanoliposome-Soybean Protein Isolate Interactions.

The interaction mechanism between nanoliposomes (NL) and a soybean protein isolate (SPI) was investigated via the complexation between NL and two major components of SPI, i.e., ß-conglycinin (7S) and glycinin (11S). The endogenous fluorescence emissions of 7S and 11S were statically quenched after complexation with NL, and the polarity of the SPI fluorophore increased. The interaction between NL and SPI was exothermic and spontaneous, 7S/11S secondary structures were altered, and more hydrophobic groups were exposed on protein surfaces. Moreover, the NL-SPI complex had a large zeta potential to attain system stability. Hydrophobic forces and hydrogen bonds played vital roles in the interaction between NL and 7S/11S, and a salt bridge was also involved in the NL-11S interaction. The binding characteristics between NL and 7S/11S were chiefly governed by the protein characteristics, such as amino acid composition, surface hydrophobicity, and advanced structure. These findings could deepen the understanding of the interaction mechanism between NL and SPI.

Expression and purification of 15N-labeled Fra a 1, a strawberry allergen, to prepare samples for NMR measurements.

Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (∼20 kDa) and a truncated (∼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 15N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1.

IgE Recognition and Structural Analysis of Disulfide Bond Rearrangement and Chemical Modifications in Allergen Aggregations in Roasted Peanuts.

Given that roasting changes the structure and allergenicity of peanut allergens, the structural information of peanut allergens must be expounded to explain the alteration in their allergenicity. This work focused on allergen aggregations (AAs) in roasted peanuts. IgE recognition capability was assessed via western blot analysis. The disulfide bond (DB) rearrangement and chemical modification in AAs were identified by combining mass spectroscopy and software tools, and structural changes induced by cross-links were displayed by molecular dynamics and PyMOL software. Results showed that AAs were strongly recognized by IgE and cross-linked mainly by DBs. The types of DB rearrangement in AAs included interprotein (98 peptide pairs), intraprotein (22 peptide pairs), and loop-linked (6 peptides) DBs. Among allergens, Ara h 2 and Ara h 6 presented the most cysteine residues to cross-linkf with others or themselves. DB rearrangement involved IgE epitopes and induced structural changes. Ara h 1 and Ara h 3 were predominantly chemically modified. Moreover, chemical modification altered the local structures of proteins, which may change the allergenic potential of allergens.

Effect of ultrahigh-pressure treatment on the structure and allergenicity of peach allergenic proteins.

Peach is a common plant-derived allergenic food and ultrahigh-pressure treatment is often used in peach products. In our study, an in-depth analysis of the structural and allergenicity changes of peach allergenic proteins after UHP treatment was performed by spectroscopy, mass spectrometry combined with serology and cytology. The results indicated that UHP treatment could reduce the content of peach soluble proteins and cause changes in secondary and tertiary structures. In addition, more hydrophobic residues were exposed and proteins tended to polymerize after UHP-treatment. The results of immunological assays showed that UHP treatment could reduce the IgE binding capacity of peach proteins and affect the ability of basophil degranulation, the upregulation of some cytokines may contribute to the reduction of peach protein allergenicity. Notably, UHP treatment may lead to the masking of some digestion sites in Pru p 3 epitopes, thus impeding human digestion and increasing the potential risk of allergenicity.

Bile Acid Profile Influences Digestion Resistance and Antigenicity of Soybean 7S Protein.

Soybean 7S storage protein (ß-conglycinin) is the most important allergen, exhibits resistance in gastrointestinal (GI) digestion, and causes allergies in humans and animals. A previous study has demonstrated that 7S proteins contained innate amyloid aggregates, but the fate of these specific protein aggregates in intestinal digestion and correlation to allergenicity are unclear. In this study, via a modified INFOGEST static in vitro digestion and IgE binding test, we illustrate that the survived amyloid aggregates of soybean 7S protein in GI digestion might be dominant IgE epitopes of soybean protein in humans. The impact of conjugated primary bile acid salt (BS) profile on digestion resistance and immunogenicity of soybean protein is assessed, regarding the binding affinity of BS to protein aggregates with consideration of the BS composition and the physiologically relevant colloidal structure. The results show that chenodeoxycholate-containing colloidal structures exhibit high affinity and unfolding capacity to protein amyloid aggregates, promoting proteolysis by pancreatic enzymes and thus mitigating the antigenicity of soybean protein. This study presents a novel understanding of bile acid profile and colloidal structure influence on the digestibility and antigenicity of dietary proteins. It should be helpful to design in vitro digestion protocol and accurately replicate physiologically relevant digestion conditions.

Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1.

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.

Mechanistic insights into silica nanoparticle-allergen interactions on antigen presenting cell function in the context of allergic reactions.

The incorporation of nanomaterials into consumer products has substantially increased in recent years, raising concerns about their safety. The inherent physicochemical properties of nanoparticles allow them to cross epithelial barriers and gain access to immunocompetent cells. Nanoparticles in cosmetic products can potentially interact with environmental allergens, forming a protein corona, and together penetrate through damaged skin. Allergen-nanoparticle interactions may influence the immune response, eventually resulting in an adverse or beneficial outcome in terms of allergic reactivity. This study determines the impact of silica nanoparticle-allergen interactions on allergic sensitization by studying the major molecular mechanisms affecting allergic responses. The major birch pollen allergen Bet v 1 was chosen as a model allergen and the birch pollen extract as a comparator. Key events in immunotoxicity including allergen uptake, processing, presentation, expression of costimulatory molecules and cytokine release were studied in human monocyte-derived dendritic cells. Using an in vivo sensitization model, murine Bet v 1-specific IgG and IgE levels were monitored. Upon the interaction of allergens with silica nanoparticles, we observed an enhanced uptake of the allergen by macropinocytosis, improved proteolytic processing, and presentation concomitant with a propensity to increase allergen-specific IgG2a and decrease IgE antibody levels. Together, these events suggest that upon nanoparticle interactions the immune response is biased towards a type 1 inflammatory profile, characterized by the upregulation of T helper 1 (Th1) cells. In conclusion, the interaction of the birch pollen allergen with silica nanoparticles will not worsen allergic sensitization, a state of type 2-inflammation, but rather seems to decrease it by skewing towards a Th1-dominated immune response.

Mitigating the allergenicity of peanut allergen Ara h 1 by cold atmospheric pressure argon plasma jet.

BACKGROUND: Peanut allergy is recognized as a major food allergy that triggers severe and even fatal symptoms. Avoidance of peanuts in the diet is the main option for current safety management. Processing techniques reducing peanut allergenicity are required to develop other options. Cold plasma is currently considered as a novel non-thermal approach to alter protein structure and has the potential to alleviate immunoreactivity of protein allergen. RESULTS: The application of a cold argon plasma jet to peanut protein extract could reduce the amount of a 64 kDa protein band corresponding to a major peanut allergen Ara h 1 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but the overall protein size distribution did not change significantly. A decrease in peanut protein solubility was a possible cause that led to the loss of protein content in the soluble fraction. Immunoblotting and enzyme-linked immunosorbent assay elucidated that the immunoreactivity of Ara h 1 was significantly decreased with the time treated with plasma. Ara h 1 antigenicity reduced by 38% after five scans (approximately 3 min) of cold argon plasma jet treatment, and the reduction was up to 66% after approximately 15 min of treatment. CONCLUSION: The results indicate that cold argon plasma jet treatment could be a suitable platform for alleviating the immunoreactivity of peanut protein. This work demonstrates an efficient, compact, and rapid platform for mitigating the allergenicity of peanuts, and shows great potential for the plasma platform as a non-thermal technique in the food industry. © 2023 Society of Chemical Industry.

Soy protein-phlorizin conjugate prepared by tyrosinase catalysis: Identification of covalent binding sites and alterations in protein structure and functionality.

Tyrosinase-catalyzed synthesis of soy 7S/11S-phlorizin conjugates was performed, and the reaction sites, conformation alterations and functional properties of complexes were evaluated using proteomic, in combination with multispectral technologies. Phlorizin was conjugated to 7S/11S primarily via residues of Lys, Cys, His and Arg residues. The phlorizin binding equivalents and decreased contents of free and total sulfhydryl groups and free amino groups confirmed the covalent interaction in the 7S/11S-phlorizin complexes. Conjugation with phlorizin promoted the conversion of α-helix to ß-sheet and ß-turn, with simultaneous transformation of the microenvironments around Trp and Tyr residues to hydrophilic and hydrophobic microenvironments, respectively, and lowering of the surface hydrophobicity of 7S/11S. The DPPH and ABTS radical scavenging abilities and α-glucosidase inhibitory activities of 7S/11S were increased by three-, two- and three-fold after the covalent binding of phlorizin. The study provided an ideal tyrosinase-catalyzed approach to fabricate custom-tailored nutritional soy protein-polyphenol products.

Complexation of ß-conglycinin or glycinin with sodium alginate blocks: Complexation mechanism and structural and functional properties.

Sodium alginate (SA), α1 â†’ 4 linked copolymer of ß-d-mannuronic acid (M) and α-L guluronic acid (G) forms two homopolymeric fractions (MM and GG) and a heteropolymeric fraction (MG). The main components of soybean protein isolate are ß-conglycinin (7S) and glycinin (11S). However, accurate structural analyses of the 7S/11S and MM/MG/GG complexes are lacking. The complexation mechanism, structure, and functional properties of the complexes of 7S/11S with SA blocks was investigated at pH 4. The number of intermolecular hydrogen bonds exceeded that of the intramolecular hydrogen bonds. Secondary and tertiary structures and molecular weights of the complexes were significantly different from those of 7S/11S. The crystalline structure transformed to an amorphous structure, and the complexes underwent fluorescence quenching. Complexes 11S-MM and 11S-MG exhibited good emulsifying properties of 37.88 % and 38.13 %, respectively; 7S-GG and 7S-MM exhibited excellent surface hydrophobicity and emulsifying properties; and 11S-MM, 11S-GG, and 11S-MG exhibited excellent thermal stability.

Formation, characterization, and antigenicity of lecithin-ß-conglycinin complexes.

Lipid binding has been proposed to represent a functional property of many allergenic proteins. This study investigated the formation, characterization, and antigenicity of lecithin-ß-conglycinin complexes. The results indicate that lecithin was combined with ß-conglycinin via static quenching and primarily driven by hydrogen bonds and van der Waals forces. In addition, heat treatment reduced the antigenicity of complexes, as evidenced by changes in molecular weight and secondary and tertiary structures. It revealed that large aggregates developed and more hydrophobic regions were exposed for complexes after heat treatment, as well as a decrease in the ß-sheet contents and an increase in the ß-turn and random coil contents. Furthermore, the average particle size of the complexes increased with increased temperature treatment, and the morphology of the complexes exhibited an amorphous polymer. These findings shedlight on the interaction between lecithin and ß-conglycinin and help us understand the role of lecithin in allergic reactions.