Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE.

Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products.

Quantitative In Silico Evaluation of Allergenic Proteins from Anacardium occidentale, Carya illinoinensis, Juglans regia and Pistacia vera and Their Epitopes as Precursors of Bioactive Peptides.

The aim of the study presented here was to determine if there is a correlation between the presence of specific protein domains within tree nut allergens or tree nut allergen epitopes and the frequency of bioactive fragments and the predicted susceptibility to enzymatic digestion in allergenic proteins from tree nuts of cashew (Anacardium occidentale), pecan (Carya illinoinensis), English walnut (Juglans regia) and pistachio (Pistacia vera) plants. These bioactive peptides are distributed along the length of the protein and are not enriched in IgE epitope sequences. Classification of proteins as bioactive peptide precursors based on the presence of specific protein domains may be a promising approach. Proteins possessing a vicilin, N-terminal family domain, or napin domain contain a relatively low occurrence of bioactive fragments. In contrast, proteins possessing the cupin 1 domain without the vicilin N-terminal family domain contain a relatively high total frequency of bioactive fragments and predicted release of bioactive fragments by the joint action of pepsin, trypsin, and chymotrypsin. This approach could be utilized in food science to simplify the selection of protein domains enriched for bioactive peptides.

Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt.

Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers. Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1-3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ∼2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcεRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.

Polyphenol-rich pomegranate juice reduces IgE binding to cashew nut allergens.

BACKGROUND: Food allergy negatively impacts quality of life and can be life-threatening. Cashew nuts can cause severe reactions in very small amounts, and they are included in a group of foods most commonly responsible for causing food allergy. Polyphenols and polyphenol-rich juices have been demonstrated to complex with peanut allergens. Here, the interaction between cashew nut allergens and polyphenol-rich juices is evaluated biochemically and immunologically. RESULTS: Various juices, including pomegranate (POM), blueberry (BB), and concord grape (CG) juices, were evaluated for polyphenol content and formation of polyphenol-cashew allergen complexes. Among the various juices studied, POM juice showed a greater capacity to form complexes with cashew proteins. Dynamic light scattering (DLS) demonstrated a sharp increase in cashew protein extract particle size to around 3580 nm, and fewer cashew proteins were resolved by electrophoresis after treatment with POM juice. Immunoassays demonstrated reduced IgG and IgE binding to cashew allergens due to allergen precipitation by POM juice. These observations support the formation of complexes between polyphenol and cashew proteins that can prevent antibody recognition of cashew allergens through allergen precipitation. CONCLUSION: POM juice treatment of cashew extract effectively reduces antibody binding through allergen precipitation, and these findings could be applied to the development of less allergenic cashew nut products and oral immunotherapy. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility.

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear ß-2,6-fructofuranosyl polysaccharide with occasional (5.7%) ß-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

Proteomic characterization of peanut flour fermented by Rhizopus oryzae.

Fermentation alters the protein content and composition of foods. To characterize fungal catabolism of peanut proteins, defatted peanut flour was fermented by Rhizopus oryzae (R. oryzae) for up to 48 h and evaluated by SDS-PAGE, mass spectrometry, and antibody binding. A clear change in peanut protein migration was observed by SDS-PAGE after 16 h of fermentation. Mass spectrometric analysis indicated changes in allergen peptides and R. oryzae proteins. Several low molecular weight allergen fragments produced during fermentation were identified by mass spectrometry. Immunoassays using anti-peanut allergen antibodies demonstrated reduced allergen content as early as 16 h of fermentation. However, ELISA with peanut allergic IgE indicated only slightly reduced allergen binding even after 48 h. These results indicate that while R. oryzae fermentation efficiently metabolizes peanut allergens, significant IgE binding remains in lower molecular mass peptides, and therefore R. oryzae fermented peanut products would not be safe for peanut allergic individuals.

Proteomic Analysis of Pecan (Carya illinoinensis) Nut Development.

Pecan (Carya illinoinensis) nuts are an economically valuable crop native to the United States and Mexico. A proteomic summary from two pecan cultivars at multiple time points was used to compare protein accumulation during pecan kernel development. Patterns of soluble protein accumulation were elucidated using qualitative gel-free and label-free mass-spectrometric proteomic analyses and quantitative (label-free) 2-D gel electrophoresis. Two-dimensional (2-D) gel electrophoresis distinguished a total of 1267 protein spots and shotgun proteomics identified 556 proteins. Rapid overall protein accumulation occurred in mid-September during the transition to the dough stage as the cotyledons enlarge within the kernel. Pecan allergens Car i 1 and Car i 2 were first observed to accumulate during the dough stage in late September. While overall protein accumulation increased, the presence of histones diminished during development. Twelve protein spots accumulated differentially based on 2-D gel analysis in the weeklong interval between the dough stage and the transition into a mature kernel, while eleven protein spots were differentially accumulated between the two cultivars. These results provide a foundation for more focused proteomic analyses of pecans that may be used in the future to identify proteins that are important for desirable traits, such as reduced allergen content, improved polyphenol or lipid content, increased tolerance to salinity, biotic stress, seed hardiness, and seed viability.

Structural and immunologic characterization of Ara h 1, a major peanut allergen.

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170-586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.

Sensitive and selective detection of peanut allergen Ara h 1 by ELISA and lateral flow immunoassay.

The Ara h1 protein is a peanut allergen and it provides a useful biomarker for the detection of peanut protein. In this manuscript, we describe the generation of monoclonal antibodies (MAbs) against the Ara h1 protein and their development into sensitive and selective immunoassays for peanut detection. Our enzyme-linked immunosorbent assay (sELISA) detects a peanut meal standard with a sensitivity of 10 ng/mL and 500 ng/mL by lateral flow immunoassay (LFIA). MAb Ara h1 binding epitopes were identified, and immunoassay detection was limited to peanut meal varieties irrespective of thermal treatment. No binding was observed from tree nut meals (100-0.4 µg/mL). Peanut allergen detection during food manufacturing can limit the incidence of product recall resulting from cross-contact contamination or improper labeling of finished food products. Detection of Ara h1 by immunoassay can provide a cost-effective method for rapid surveillance of peanut during food production and prior to consumption.

Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Centrosomes and tumour suppressors.

Centrosomes are microtubule organising centres that act as spindle poles during mitosis. Recent work implicates centrosomes in many other processes, and shows that centrosome defects can cause genetic instability. Many regulators of mammalian centrosome function were predicted from studies of model systems. Surprisingly, some well-known tumour suppressors have recently been found at centrosomes, where they influence centrosome duplication and function, suggesting that control of centrosome function is central to genetic stability.

Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed.

In this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110-10 kDa), respectively. Liquid chromatography-electrospray ionization-tandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications.

Characterization of Anti-Ana o 3 Monoclonal Antibodies and Their Application in Comparing Brazilian Cashew Cultivars.

Ana o 3 is an immuno-dominant cashew nut allergen. Four monoclonal antibodies to Ana o 3 (2H5, 6B9C1, 19C9A2, and 5B7F8) were characterized by ELISA and in silico modeling. The 2H5 antibody was the only antibody specific for cashew nut extract. In addition to cashew nut extract, the 6B9C1 and 19C9A2 antibodies recognized pistachio extract, and the 5B7F8 recognized pecan extract. All four antibodies recognized both recombinant Ana o 3.0101 and native Ana o 3. ELISA assays following treatment of purified Ana o 3 with a reducing agent indicated that the 6B9C1 and 19C9A2 antibodies likely recognize conformational epitopes, while the 2H5 and 5B7F8 antibodies likely recognize linear epitopes. In silico modeling predicted distinct epitopes for each of the anti-Ana o 3 antibodies. Screening extracts from 11 Brazilian cashew nut cultivars using all four antibodies showed slight differences in Ana o 3 bindings, demonstrating that these antibodies could identify cultivars with varying allergen content.

Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding.

Genome-enabled biotechnologies have the potential to accelerate breeding efforts in long-lived perennial crop species. Despite the transformative potential of molecular tools in pecan and other outcrossing tree species, highly heterozygous genomes, significant presence-absence gene content variation, and histories of interspecific hybridization have constrained breeding efforts. To overcome these challenges, here, we present diploid genome assemblies and annotations of four outbred pecan genotypes, including a PacBio HiFi chromosome-scale assembly of both haplotypes of the 'Pawnee' cultivar. Comparative analysis and pan-genome integration reveal substantial and likely adaptive interspecific genomic introgressions, including an over-retained haplotype introgressed from bitternut hickory into pecan breeding pedigrees. Further, by leveraging our pan-genome presence-absence and functional annotation database among genomes and within the two outbred haplotypes of the 'Lakota' genome, we identify candidate genes for pest and pathogen resistance. Combined, these analyses and resources highlight significant progress towards functional and quantitative genomics in highly diverse and outbred crops.

A cashew specific monoclonal antibody recognizing the small subunit of Ana o 3.

Food allergies represent a substantial medical liability and preventing accidental exposure to food allergens requires constant attention. Allergic reaction to cashew nuts is frequently serious, and the small 2S albumin, Ana o 3, is an immuno-dominant cashew allergen. Ana o 3 is composed of five alpha helices, contains 2 subunits linked by cysteine disulfide bonds, and remains soluble even after extensive heating of cashew nuts. The stability and solubility properties of Ana o 3 make it an excellent target for diagnostic and detection methods and tools. In this work, a monoclonal antibody, designated 2H5, aimed at amino acids 39-54 within helices I and II of the small subunit of Ana o 3 was developed that recognizes both recombinant and native Ana o 3 and is cashew specific in ELISA experiments. The KD against the targeted amino-acid sequence was found to be approximately 7.0 × 10-6 mg/ml (3.3 nM), while the KD against the native protein was found to be approximately 1.2 × 10-3 mg/ml (92 nM). The 2H5 monoclonal anti-Ana o 3 antibody can distinguish between native and recombinant proteins and represents a useful reagent for the study of antibody cashew-allergen interactions and may enable the development of cashew-specific diagnostic tools that can be used to prevent accidental cashew allergen exposures.