Christopher P. Mattison

In vitro digestion of soluble cashew proteins and characterization of surviving IgE-reactive peptides

SCOPE The stability of food allergens to digestion varies. We characterized the stability of cashew allergens to digestion by pepsin and trypsin and identified IgE-binding epitopes that survive digestion. METHODS AND RESULTS The ability of pepsin and trypsin to digest cashew allergens was assessed with an in vitro digestion model. Samples were evaluated by SDS-PAGE, MS, ELISA, and immunoblotting to compare IgE binding. Increasing amount of protease resulted in greater degradation of higher molecular weight cashew proteins. Among cashew proteins, the 2S albumin, Ana o 3, was most resistant to digestion by both pepsin and trypsin. MS identified digestion resistant Ana o 3 protein fragments that retained reported IgE-binding epitopes. Pretreatment of extracts or purified Ana o 3 with reducing agent increased the sensitivity of Ana o 3 to protease digestion. Circular dichroism revealed the structure of purified Ana o 3 was largely alphahelical and was disrupted following reduction. Ana o 3 reduction followed by protease digestion decreased binding of serum IgE from cashew allergic patients. Our results indicate that the Ana o 3 disulfide bond dependent structure protects the protein from proteolysis. CONCLUSION Ana o 3 is the cashew allergen most likely to survive gastrointestinal digestion intact.

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.

Decreased immunoglobulin E (IgE) binding to cashew allergens following sodium sulfite treatment and heating.

Cashew nut and other nut allergies can result in serious and sometimes life-threatening reactions. Linear and conformational epitopes within food allergens are important for immunoglobulin E (IgE) binding. Methods that disrupt allergen structure can lower IgE binding and lessen the likelihood of food allergy reactions. Previous structural and biochemical data have indicated that 2S albumins from tree nuts and peanuts are potent allergens, and that their structures are sensitive to strong reducing agents such as dithiothreitol. This study demonstrates that the generally regarded as safe (GRAS) compound sodium sulfite effectively disrupted the structure of the cashew 2S albumin, Ana o 3, in a temperature-dependent manner. This study also showed that sulfite is effective at disrupting the disulfide bond within the cashew legumin, Ana o 2. Immunoblotting and ELISA demonstrated that the binding of cashew proteins by rabbit IgG or IgE from cashew-allergic patients was markedly lowered following treatment with sodium sulfite and heating. The results indicate that incorporation of sodium sulfite, or other food grade reagents with similar redox potential, may be useful processing methods to lower or eliminate IgE binding to food allergens.

Mip1 associates with both the Mps1 kinase and actin, and is required for cell cortex stability and anaphase spindle positioning.

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.

In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides.

Ara h 1 is a major peanut allergen. Processing-induced modifications may modulate the allergenic potency of Ara h 1. Carboxymethyl lysine (CML) modifications are a commonly described nonenzymatic modification on food proteins. In the current study, we tested the ability of digestive and endolysosomal proteases to cleave CML-modified and unmodified Ara h 1 peptides. Mass spectrometric analyses of the digested peptides demonstrate that carboxymethylation of lysine residues renders these peptides refractory to trypsin digestion. We did not detect observable differences in the simulated gastric fluid or endolysosomal digestion between the parental and CML-modified peptides. One of the tested peptides contains a lysine residue previously shown to be CML modified laying in a previously mapped linear IgE epitope, but we did not observe a difference in IgE binding between the modified and parental peptides. Our findings suggest a molecular mechanism for the increased resistance of peanut allergens modified by thermal processing, such as Ara h 1, to digestion in intestinal fluid after heating and could help explain how food processing-induced modifications may lead to more potent food allergens by acting to protect intact IgE epitopes from digestion by proteases targeting lysine residues.

Myosin Gene Expression and Protein Abundance in Different Castes of the Formosan Subterranean Termite (Coptotermes formosanus).

The Formosan subterranean termite (Coptotermes formosanus) is an important worldwide pest, each year causing millions of dollars in structural damage and control costs. Termite colonies are composed of several phenotypically distinct castes. Termites utilize these multiple castes to efficiently perform unique roles within the colony. During the molting/caste differentiation process, multiple genes are believed to be involved in the massive reorganization of the body plan. The objective of this research was to analyze the muscle gene, myosin, to further understand the role it plays in C. formosanus development. We find that comparing worker vs. solider caste myosin gene expression is up-regulated in the soldier and a myosin antibody-reactive protein suggests changes in splicing. Comparison of body regions of mature soldier and worker castes indicates a greater level of myosin transcript in the heads. The differential expression of this important muscle-related gene is anticipated considering the large amount of body plan reorganization and muscle found in the soldier caste. These results have a direct impact on our understanding of the downstream genes in the caste differentiation process and may lead to new targets for termite control.

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies.

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.

RNA-Seq Analysis of Developing Pecan (Carya illinoinensis) Embryos Reveals Parallel Expression Patterns among Allergen and Lipid Metabolism Genes

The pecan nut is a nutrient-rich part of a healthy diet full of beneficial fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. The transcriptome of a developing pecan nut was characterized to identify the gene expression occurring during the process of nut development and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages. Library preparation and sequencing were performed using Illumina-based mRNA HiSeq with RNA from four time points during the growing season during August and September 2012. Sequence analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with 52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by GO annotation. Gene expression clustering analysis identified 12 different gene expression profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, Car i 2, and Car i 4, were significantly up-regulated during the time course. Up-regulated fatty acid metabolism genes that were identified included acyl-[ACP] desaturase and omega-6 desaturase genes involved in oleic and linoleic acid metabolism. Notably, a few of the up-regulated acyl-[ACP] desaturase and omega-6 desaturase genes that were identified have expression patterns similar to the allergen genes based upon gene expression clustering and qPCR analysis. These findings suggest the possibility of coordinated accumulation of lipids and allergens during pecan nut embryogenesis.

Effects of industrial cashew nut processing on anacardic acid content and allergen recognition by IgE

Cashew nuts are important both nutritionally and industrially, but can also cause food allergies in some individuals. The present study aimed to assess the effect(s) of industrial processing on anacardic acids and allergens present in cashew nuts. Sample analyses were performed using liquid chromatography coupled with mass spectrometry, SDS-PAGE and immunoassay. The anacardic acid concentration ranged from 6.2 to 82.6mg/g during processing, and this variation was attributed to cashew nut shell liquid incorporation during storage and humidification. Dehydrated and selected samples did not significantly differ in anacardic acid content, having values similar to the raw sample. SDS-PAGE and immunoassay analysis with rabbit polyclonal sera and human IgE indicated only minor differences in protein solubility and antibody binding following processing steps. The findings indicate that appreciable amounts of anacardic acid remain in processed nuts, and that changes to cashew allergens during industrial processing may only mildly affect antibody recognition.