Potential Of Novel Proteins Research Articles

Allergenicity prediction of novel and modified proteins: Not a mission impossible! Development of a Random Forest allergenicity prediction model

Alternative and sustainable protein sources (e.g., algae, duckweed, insects) are required to produce (future) foods. However, introduction of new food sources to the market requires a thorough risk assessment of nutritional, microbial and toxicological risks and potential allergic responses. Yet, the risk assessment of allergenic potential of novel proteins is challenging. Currently, guidance for genetically modified proteins relies on a weight-of-evidence approach. Current Codex (2009) and EFSA (2010; 2017) guidance indicates that sequence identity to known allergens is acceptable for predicting the cross-reactive potential of novel proteins and resistance to pepsin digestion and glycosylation status is used for evaluating de novo allergenicity potential. Other physicochemical and biochemical protein properties, however, are not used in the current weight-of-evidence approach. In this study, we have used the Random Forest algorithm for developing an in silico model that yields a prediction of the allergenic potential of a protein based on its physicochemical and biochemical properties. The final model contains twenty-nine variables, which were all calculated using the protein sequence by means of the ProtParam software and the PSIPred Protein Sequence Analysis program. Proteins were assigned as allergenic when present in the COMPARE database.Results show a robust model performance with a sensitivity, specificity and accuracy each greater than ≥85%. As the model only requires the protein sequence for calculations, it can be easily incorporated into the existing risk assessment approach. In conclusion, the model developed in this study improves the predictability of the allergenicity of new or modified food proteins, as demonstrated for insect proteins.

Allergenic potential of novel proteins – What can we learn from animal production?

Currently, risk assessment of the allergenic potential of novel proteins relies heavily on evaluating protein digestibility under normal conditions based on the theory that allergens are more resistant to gastrointestinal digestion than non-allergens. There is also proposed guidance for expanded in vitro digestibility assay conditions to include vulnerable sub-populations. One of the underlying rationales for the expanded guidance is that current in vitro assays do not accurately replicate the range of physiological conditions. Animal scientists have long sought to predict protein and amino acid digestibility for precision nutrition. Monogastric production animals, especially swine, have gastrointestinal systems similar to humans, and evaluating potential allergen digestibility in this context may be beneficial. Currently, there is no compelling evidence that the mechanisms sometimes postulated to be associated with allergenic sensitization, e.g. antacid modification of stomach pH, are valid among production animals. Furthermore, examples are provided where non-biologically representative assays are better at predicting protein and amino acid digestibility compared with those designed to mimic in vivo conditions. Greater emphasis should be made to align in vitro assessments with in vivo data.

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.

Proteins from an unevolved library of de novo designed sequences bind a range of small molecules.

The availability of large collections of de novo designed proteins presents new opportunities to harness novel macromolecules for synthetic biological functions. Many of these new functions will require binding to small molecules. Is the ability to bind small molecules a property that arises only in response to biological selection or computational design? Or alternatively, is small molecule binding a property of folded proteins that occurs readily amidst collections of unevolved sequences? These questions can be addressed by assessing the binding potential of de novo proteins that are designed to fold into stable structures, but are "naïve" in the sense that they (i) share no significant sequence similarity with natural proteins and (ii) were neither selected nor designed to bind small molecules. We chose three naïve proteins from a library of sequences designed to fold into 4-helix bundles and screened for binding to 10,000 compounds displayed on small molecule microarrays. Several binders were identified, and binding was characterized by a series of biophysical assays. Surprisingly, despite the similarity of the three de novo proteins to one another, they exhibit selective ligand binding. These findings demonstrate the potential of novel proteins for molecular recognition and have significant implications for a range of applications in synthetic biology.

Evaluation of biotechnology-derived novel proteins for the risk of food-allergic potential: advances in the development of animal models and future challenges

Increasing concern from the public about the safety of genetically modified food has made critical to have suitable methods for recognizing associated potential hazards. Hierarchical approaches to allergenicity determination were proposed, and these include evaluation of the structural and sequence homology and serological identity of novel proteins with existing allergens, measuring the resistance to proteolytic digestion and assessment of sensitizing potential using animal models. Allergic individuals have a predisposed (i.e. atopic) genetic background, and a close resemblance to this setup is therefore desirable in animal models, which is possible by using a strain of an animal species that is prone for allergic disorders. So far, none of the animal model has been validated for the purpose of hazard identification in the context of safety assessment. However, the available knowledge suggests that the judicious use of an appropriate animal model could provide important information about the allergic potential of novel proteins. This paper provides an up-to-date review of the progress made in the field of development of in vivo models in this direction and the further goals that have to be achieved.

In Vivo Digestion of a Thaumatin-Like Kiwifruit Protein in Rats

Food allergens must exhibit sufficient gastrointestinal stability to reach the intestinal mucosa where absorption and sensitization can occur. Therefore, investigation of protein stability within the gastrointestinal tract may provide a prospective test for the allergenic potential of novel proteins. The aim of this work was to examine the effect of the fruit matrix and purified pectin on the digestion in vivo of kiwifruit allergens in the rat gastrointestinal system. The major kiwi allergen, Act d 2, was quantified in several compartments of the gastrointestinal tract by a monoclonal antibody-based ELISA. Protein intactness was demonstrated by immunoblot. Under conditions of complex food digestion in vivo, a pepsin-labile protein survived passage from the stomach to the caecum during a 3-h period. Decay of Act d 2 in the rat gut exhibited an exponential pattern. Ingestion of kiwifruit was followed by a decrease in both total and specific pepsin activity. When purified, Act d 2 allergen was consumed together with pure apple pectin; both gastric acidity as well as specific and total pepsin activity declined and thus protected 23% of the ingested allergen from digestion for 90 min. In conclusion, ingestion of pectin-rich fruits and particularly pectin supplements may have a protective action on pepsin-labile allergens and prolong their survival in both gastric and duodenal juices, enabling efficient uptake and presentation to the immune system.

Approaches to Assess the Allergenic Potential of Novel Proteins: Are We on the Right Track?

Approaches to Assess the Allergenic Potential of Novel Proteins: Are We on the Right Track?

Review of the development of methodology for evaluating the human allergenic potential of novel proteins

Safety assessment of novel proteins in genetic-engineered foods is a key component of the overall safety evaluation for these products. Since allergens are typically proteins, assessment of the potential allergenicity of the novel proteins in genetically engineered foods is critical. This article reviews methods available to assess the potential allergenicity of novel proteins, as well as problems and deficiencies in the existing methods. The role of bioinformatics and knowledge of allergenic epitopes in developing new approaches to this problem is discussed.

Pollen allergens are restricted to few protein families and show distinct patterns of species distribution

Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing. To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability. Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments. Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man. This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.

Characterisation of immune responses to food allergens in mice

There is considerable interest in the development and evaluation of approaches for the safety assessment of novel foods, and in particular in methods for characterisation of allergenic potential. One strategy that has found favour is a tiered approach in which the potential of novel proteins to induce allergic sensitisation is assessed based on considerations of stability of the protein in a simulated gastric juice and homology with, or structural similarity to, known allergens. Linked to such an approach may be evaluation of serological identity with proteins known to cause allergic disease. With the aim of supplementing such approaches with a more direct measurement of potential allergenic activity, attempts have been made to characterise the quality of immune responses elicited in BALB/c strain mice. Such evaluations comprise measurement of IgG and IgE antibody production and (to a lesser extent) of induced cytokine expression patterns. Investigations to date suggest that in mice proteins provoke variable immune responses, those with the potential to cause allergic sensitisation stimulating IgE (and IgG) antibody production. In contrast, non-allergenic, but nevertheless immunogenic, proteins are associated with IgG antibody responses in the absence of marked IgE production. Consistent with the selective activation of selective type 2 T lymphocyte responses, exposure of mice to allergenic protein is associated with preferential expression of IL-4, -5, -10 and -13. Collectively these data suggest that characterisation of the nature of immune response induced in mice by proteins may provide a useful adjunct or alternative to current strategies for the assessment of allergenic potential.

In Silico Methods for Evaluating Human Allergenicity to Novel Proteins: International Bioinformatics Workshop Meeting Report, 23–24 February 2005

The ILSI Health and Environmental Sciences Institute (HESI) hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-science for conducting a sequence homology/bioinformatics evaluation in the context of a comprehensive allergenicity assessment for novel proteins, to obtain consensus on the value and role of bioinformatics in evaluating novel proteins, and to discuss the utility and methods of allergen-specific IgE testing in the diagnosis of food allergy. The workshop participants included over forty international experts from academia, industry, and government. The workshop was hosted by the HESI Protein Allergenicity Technical committee, which has established a long-term program whose mission is to advance the scientific understanding of the relevant parameters for characterizing the allergenic potential of novel proteins.

Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons

There is a growing interest in the development of methods for the evaluation of the allergenic potential of novel proteins. One approach is the measurement of specific IgE antibody production stimulated by systemic (intraperitoneal; ip) exposure of BALB/c strain mice. In the current investigations, inter-laboratory comparisons have been performed of IgE antibody production induced in mice by food proteins of differing sensitizing potential. Female BALB/c strain mice ( n=5) were exposed to 0.1% peanut agglutinin, an allergenic constituent of peanuts, to 2% ovalbumin (OVA), a major allergenic constituent of hens' egg, or to a protein considered to lack significant allergenicity, potato agglutinin (5%). Specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay and IgG and IgG1 antibody production was analysed by enzyme-linked immunosorbent assay (ELISA). Two independent experiments were conducted in each laboratory, but with all serological analyses conducted in one of the laboratories. Each of the proteins induced vigorous IgG and IgG1 antibody responses, with no statistically significant differences in titres recorded between laboratories. Furthermore, OVA and potato agglutinin induced responses of equivalent immunogenicity with respect to both IgG and IgG1 antibody titres. Administration of peanut agglutinin and OVA each stimulated marked IgE antibody responses in every experiment. In the two laboratories, titres ranged from 1:32 and 1:64 for peanut agglutinin, and from 1:8 and 1:32 for OVA. In contrast, exposure to potato agglutinin failed to induce vigorous IgE production, with no detectable IgE (negative with neat serum), or titres of 1 (positive with neat serum only) recorded. These data demonstrate that the induction of IgE antibody by food proteins of differing allergenic potential is a relatively robust phenomenon and transferable between laboratories. Furthermore, these results provide additional evidence that the measurement of antibody (IgE) responses in BALB/c mice may allow discrimination between allergens and those materials that apparently lack allergenicity.

Protein digestibility and relevance to allergenicity.

In January 2001 a Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Consultation Committee on Allergenicity of Foods Derived from Biotechnology published a report outlining in detail an approach for assessing the allergenic potential of novel proteins. One component of this decision tree is a determination of whether the protein of interest is resistant to proteolytic digestion. Although these (Italic)in vitro(/Italic) methodologies have been useful, the correlation between resistance to proteolysis and allergenic activity is not absolute. Two views and highlights of supporting research regarding the relationship of resistance to digestion and allergenicity are presented in this article.

Approaches to assessment of the allergenic potential of novel proteins in food from genetically modified crops.

The safety assessment of food derived from genetically modified plants continues to attract considerable attention. Among the important issues that need to be considered is whether the products of novel genes introduced into crop plants will have the potential to induce allergic sensitization or to elicit allergic disease. Hierarchical approaches to allergenicity testing have been proposed, and these incorporate evaluation of the structural and sequence homology and serological identity of novel proteins with known allergens, measurement of resistance to proteolytic digestion, and assessment of allergenic potential using animal models. Accounts of these approaches are available elsewhere, and it is not the purpose of this article to provide a detailed critique of specific methods. Our intention is rather to look more broadly at the strategy for assessment of allergenic potential, the challenges such assessments pose for the practicing toxicologist, and how some of these might best be addressed.

Digestion stability as a criterion for protein allergenicity assessment.

Assessing the allergenic potential of transgenic proteins introduced into genetically engineered food remains a critical part of the overall safety assessment of these foods. Stability to digestion has been proposed as one of the steps in the decision tree approach to assess the allergenic potential of transgenic proteins. The validity of digestion stability as a criterion for protein allergenicity assessment, however, has encountered some criticism in recent years. This chapter gives an overview of the rationale behind the use of digestion stability as a criterion for protein allergenicity assessment and reviews the available data that may or may not support its use. The application of in vitro digestion assays for the assessment of allergenic potential of novel proteins and the factors that affect the assay results are also discussed. There is a need to establish standardized assay conditions so that direct comparison of results from different laboratories can be made. Consensus also needs to be reached on relating the measured digestibility to the allergenic potential of proteins.

Sequence analysis and resistance to pepsin hydrolysis as part of an assessment of the potential allergenicity of ice structuring protein type III HPLC 12

The recently published WHO/FAO guidelines on the assessment of allergenicity of novel food proteins provide a strategy with which to approach the determination of the potential of novel proteins in foods to be allergens. Key to this strategy are the assessment of sequence similarity to known allergens and the assessment of the resistance to pepsin hydrolysis. Ice structuring proteins (also commonly referred to as anti-freeze or thermal hysteresis proteins) are a group of naturally occurring proteins that bind to ice and structure ice crystal formation. The amino acid sequence of the ice structuring protein (ISP) type III HPLC 12 (ISP type III) was compared in silico with the sequences of known allergens. Secondly, the resistance to pepsin hydrolysis of ISP type III and its glycoconjugates (produced in recombinant baker's yeast) was assessed. The results indicate that ISP type III has no sequence similarity with known allergenic proteins. Both ISP type III and ISP type III glycoconjugates contained within the fermentation product were hydrolysed readily by pepsin (50% loss in <10 min at pH 1.5) to give peptide fragments that were too small to be allergenic or to trigger cross-linking to IgE. In an accompanying study, we demonstrated that IgE from fish-allergic individuals did not bind ISP Type III. Therefore, in accordance with the WHO/FAO strategy, the assessment of ISP type III and ISP type III glycoconjugates by sequence analysis together with lack of resistance to pepsin hydrolysis and the absence of IgE binding supports the conclusion that both are unlikely to present a potential sensitisation hazard.

Can animal models predict food allergenicity?

Summary The development of genetically modified crops requires that appropriate safety assessment procedures are in place. One important consideration is food allergy, the need being to determine whether, and to what extent, the products of novel genes introduced into crop plants have the potential to cause allergic sensitisation. Although there are available approaches to allergenicity testing, there is a need for appropriate animal models that are able to supplement and support existing methods and provide a more direct assessment of the allergenic potential of novel proteins. Although doubts have been expressed regarding the feasibility of developing animal models suitable for this purpose, progress has been made. The purpose of this article is to review recent developments and to consider the potential value of animal models in food allergy safety assessment.

Determination of protein allergenicity: studies in mice

There is a need to identify and characterize the allergenic potential of novel proteins introduced into genetically-modified crop plants. Although several approaches have already been described, none of these measures directly the ability of proteins to cause allergic sensitization. For this reason there has been a growing interest in the development of suitable animal models. This article describes experience to date with a method based upon assessment of serological (IgG and IgE antibody) responses induced in BALB/c strain mice by proteins. Comparisons have been made between intraperitoneal (i.p.) administration and exposure by gavage using both allergenic and non-allergenic proteins. The available data indicate that responses provoked by i.p. exposure permit the identification of proteins that have the inherent potential to induce IgE antibody production and allergic sensitization. Moreover, this approach also provides a rank order of proteins with respect to allergenic potency that apparently reflects what is known of their relative sensitizing activity in humans. By comparison, oral exposure of mice by gavage is somewhat less sensitive. On this basis it is proposed that the inherent sensitizing potential of novel proteins can be evaluated as a function of IgE antibody responses stimulated by parenteral (i.p.) exposure of BALB/c mice.