Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterialcodAgene

Genetically modified (GM) mustard line (V4) with increased carotenoid content was compared with native mustard to find the difference in allergenic potential, if any. Simulated gastric fluid (SGF) digestibility of crude protein extract from GM as well as its native counterpart mustard crop was envisaged to understand the intended or unintended changes in GM crop along with IgE immunoblotting. BALB/c mice were used as model for allergenicity studies for monitoring total and specific IgE, specific IgG1, histamine level, histopathology, and systemic anaphylaxis score. Allergenicity of mustard was checked in humans by clinical history, skin prick test and IgE levels. Similar results were evident by significant increase in total IgE, specific IgE, IgG1, histamine levels, in GM and native mustard in comparison to control group. Prominent anaphylactic symptoms (score 2: 60%; score 3: 20%; score 4: 20% in native mustard and score 2: 40%; score 3: 40%; score 4: 20% in GM mustard) and eruptive histopathological changes were observed in both GM and native mustard when compared with controls. One protein of approximately 16 kDa was found stable up to 1 h in both GM as well as non GM mustard. IgE immunoblotting detected three protein components of approximately 29, 24 and 16 kDa in both GM and non GM varieties. Collectively, our data demonstrate substantially equivalent allergic responses against GM as well as its native counterpart. Therefore, the GM mustard may be as safe as its native counterpart with reference to allergenic responses.

Genetically modified crops have resistance to abiotic stress by introduction of choline oxidase protein. In the present study, the safety of choline oxidase protein derived from Arthrobacter globiformis was assessed for toxicity and allergenicity. The protein was stable at 90 degrees C for 1 h. Toxicity studies of choline oxidase in mice showed no significant difference (p > 0.05) from control in terms of growth, body weight, food consumption, and blood biochemical indices. Histology of gut tissue of mice fed protein showed normal gastric mucosal lining and villi in jejunum and ileum sections. Specific IgE in serum and IL-4 release in splenic culture supernatant were low in choline oxidase treated mice, comparable to control. Intravenous challenge with choline oxidase did not induce any adverse reaction, unlike ovalbumin group mice. Histology of lung tissues from choline oxidase sensitized mice showed normal airways, whereas ovalbumin-sensitized mice showed inflamed airways with eosinophilic infiltration and bronchoconstriction. ELISA carried out with food allergic patients' sera revealed no significant IgE affinity with choline oxidase. Also, choline oxidase did not show any symptoms of toxicity and allergenicity in mice.

Genetically modified (GM) soybean (carrying the EPSPS transgene) is the most common GM food in Korea. In order to assess whether genetic modification increases the allergenic risk of soybeans, the allergenicity and IgE-reactive components of wild-type and GM soybean extracts were compared in allergic adults who had been sensitized to soybeans. We enrolled 1,716 adult allergy patients and 40 healthy, non-atopic controls. Skin prick tests and IgE enzyme linked immunosorbent assays (ELISAs) were performed using wild-type and GM soybean extracts, along with other common inhaled allergens. The specificities of serum IgE antibodies from allergic patients and the identities of the IgE-reactive components of the soybean extracts were compared using ELISA inhibition testing, 2-dimensional gel electrophoresis, and IgE immunoblotting. To evaluate the effects of digestive enzymes and heat treatment, the soybean extracts were heated or pre- incubated with or without simulated gastric and intestinal fluids. The IgE sensitization rates to wild-type and GM soybeans were identical (3.8% of allergic adults), and circulating IgE antibodies specific for the two extracts were comparable. The results of the ELISA inhibition test, SDS-PAGE, and IgE immunoblotting showed a similar composition of IgE-binding components within the wild-type and GM extracts, which was confirmed using two-dimensional gel electrophoresis, IgE immunoblotting, and amino acid sequencing. None of the subjects had a positive response to purified EPSPS protein in the skin prick test, ELISA, or IgE immunoblot analysis. These findings suggest that the IgE sensitization rate to GM soybean extracts is identical to that of wild-type soybean extracts in adult allergy patients. In addition, based on both in vivo and in vitro methods, the allergenicity of wild type and GM soybean extracts was identical.

Myiasis Due to Hypoderma lineatum Infection Mimicking the Hypereosinophilic Syndrome

Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials

Leguminous crops are the main source of protein in Asian subcontinent including India and their proteins may induce allergic reactions in sensitized individuals. Pepsin resistance of proteins is a characteristic feature of most of the allergens. Simulated gastric fluid (SGF) assay as validated by digestion of purified known allergenic and non-allergenic proteins was the basis of this study. Purified allergenic proteins were stable to SGF digestion contrary to rapidly digested non-allergenic proteins. Crude proteins extracts (CPE) of soybean, peanut, chickpea, black gram, kidney bean and Bengal gram were digested in vitro to detect their non-digestible proteins. Six proteins from soybean and seven from peanut remained undigested after SGF digestion. Likewise, seven proteins from chickpea (70, 64, 55, 45, 35, 20 and 18 kDa), ten from black gram (47, 30, 29, 28, 26, 24, 22, 16, 14 and 12 kDa), five from kidney bean (45, 29, 24, 20 and 6.5 kDa) and one from Bengal gram (20 kDa) remained undigested in SGF. Most of the proteins stable in SGF for more than 2 min showed similarity with characterized allergens on the basis of their molecular weights as in case of soybean, peanut, chickpea and black gram. Also, soybean and chickpea stable proteins showed IgE binding property with respective allergic patient’s sera. The non-digestible proteins from the chickpea, black gram, kidney bean and Bengal gram are being reported for the first time by our group. IgE binding of SGF resistant soybean and chickpea proteins is being reported first time as well.

Targeting prokaryotic choline oxidase into chloroplasts enhance the potential of photosynthetic machinery of plants to withstand oxidative damage

Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation

The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carrying amino acid residue was released by acid hydrolysis. The fluorescence excitation maxima of the isolated aminoacylriboflavin, showing a hypsochromic shift of the near-ultraviolet band relative to riboflavin, and the pH-dependent flavin fluorescence confirmed the presence of an 8alpha-substituted flavin linked to histidine. Similarly, MALDI-TOF mass spectrometry showed a molecular mass corresponding to histidylriboflavin. Classical experiments used to distinguish between the N(1) and N(3) isomers all indicated that the flavin was linked to the N(1) position of the histidine residue. The position of the FAD-carrying histidine residue in the choline oxidase polypeptide was identified by tryptic cleavage of the denatured enzyme, HPLC separation of the proteolytic peptide fragments, and characterization of the purified flavin-carrying peptide by mass spectrometry and spectroscopy. The FAD moiety was assigned to the tryptic peptide, His-Ala-Arg, corresponding to residues 87-89 in the open reading frame of the previously published cDNA sequence. Further analysis of the flavopeptide by collision-induced dissociation mass spectrometry confirmed that the flavin cofactor was attached to His(87). We conclude that this variant of choline oxidase contains 8alpha-[N(1)-histidyl]FAD at position 87 in the polypeptide chain.

The ability of certain proteins to induce an allergic response in susceptible individuals is well established. Symptoms can range from mild erythema or rhinitis, to acute, and possibly fatal, anaphylactic shock. Because such allergic responses require complex interactions between the protein and the immune system, they are notoriously difficult to predict. Nevertheless, it is clear that some proteins are intrinsically more allergenic than others. The challenge for toxicologists is to identify those characteristics that confer on proteins the potential to induce allergic sensitization and allergic disease. Here, we first consider the potential contribution that individual epitopes may make to the allergenicity of a protein. These are the minimal peptide units within proteins that can be recognized by the immune system and are a fundamental requirement for all immune responses, including those resulting in allergic sensitization. It appears that allergens must necessarily contain B-cell epitopes to which immunoglobulin E (IgE) can bind, and T-cell epitopes capable of inducing a type 2 T-lymphocyte response. Nevertheless, it appears doubtful that the presence of appropriate epitopes alone is sufficient to endow a protein with allergenic potential. We therefore consider also the contribution that other features and characteristics of proteins may make to their overall allergenicity. In particular, we consider the effects that resistance to proteolysis, post-translational glycosylation, and enzymatic activity may have. It appears that relative stability in simulated gastric fluid (SGF) sometimes correlates with allergenic activity. However, this is not universally true, and it is known that there are protein allergens, such as some of those associated with oral allergy syndrome, that are unstable. Nevertheless, if stability in SGF is associated with the intrinsic allergenicity of many proteins irrespective of the route of exposure, then this may reflect some more fundamental property of proteins, and possibly their stability in other biologic matrices and/or to intracellular proteases. Post-translational modification appears generally to enhance allergenicity, perhaps by increasing uptake and detection of the protein by the immune system. Some enzymatic activities also enhance allergenicity through what appear to be several different mechanisms, including nonspecific activation of cells participating in the immunologic response. Overall, it appears likely that many factors can contribute to the overall allergenicity of any given protein. Some, such as the presence of epitopes with allergenic potential, may be essential. Others, such as the glycosylation status, resistance to proteolysis, and enzymatic activity, may play a subsidiary but nevertheless critically important role. By better defining the limits within which these factors operate, we can hope to gain a better understanding of the fundamental origins of protein allergenicity, and therefore be in a position to identify and characterize the hazards and risks of allergic disease associated with novel proteins.

Mechanisms of allergen-specific immunotherapy.

BackgroundSafety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.ResultsThe model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile.ConclusionThe results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food.

An integral part of the safety assessment of genetically modified plants is consideration of possible human health effects, especially food allergy. Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult because of the absence of valid methods and models. Food allergens may share physicochemical properties that distinguish them from nonallergens, properties that may be used as a tool to predict the inherent allergenicity of proteins newly introduced into the food supply by genetic engineering. One candidate property is stability to digestion. We have systematically evaluated the stability of food allergens that are active via the gastrointestinal tract in a simple model of gastric digestion, emphasizing the major allergens of plant-derived foods such as legumes (peanuts and soybean). Important food allergens were stable to digestion in the gastric model (simulated gastric fluid). For example, soybean beta-conglycinin was stable for 60 min. In contrast, nonallergenic food proteins, such as spinach ribulose bis-phosphate carboxylase/oxygenase, were digested in simulated gastric fluid within 15 sec. The data are consistent with the hypothesis that food allergens must exhibit sufficient gastric stability to reach the intestinal mucosa where absorption and sensitization (development of atopy) can occur. Thus, the stability to digestion is a significant and valid parameter that distinguishes food allergens from nonallergens.

The quantitation of parasite-specific human IgG and IgE in sera: Evaluation of solid-phase RIA and ELISA methodology

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions. This is relevant for the genetic engineering of crops with economic interest that do not naturally possess efficient pathways for the synthesis of glycine betaine and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, the best characterized choline-oxidizing enzyme is the flavin-dependent choline oxidase from Arthrobacter globiformis, for which structural, mechanistic, and biochemical data are available. Here, we have replaced a hydrophobic residue (Val464) lining the active site cavity close to the N(5) atom of the flavin with threonine or alanine to investigate its role in the reaction of choline oxidation catalyzed by choline oxidase. The reductive half-reactions of the enzyme variants containing Thr464 or Ala464 were investigated using substrate and solvent kinetic isotope effects, solvent viscosity effects, and proton inventories. Replacement of Val464 with threonine or alanine uncovered a kinetically slow equilibrium between a catalytically incompetent form of enzyme and an active species that can efficiently oxidize choline. In both variants, the active form of enzyme shows a decreased rate of hydroxyl proton abstraction from the alcohol substrate, with minimal changes in the subsequent rate of hydride ion transfer to the flavin. This study therefore establishes that a hydrophobic residue not directly participating in catalysis plays important roles in the reaction of choline oxidation catalyzed by choline oxidase.