Safety Assessment of Biotechnology Products for Potential Risk of Food Allergy: Implications of New Research

Food allergy is a potential risk associated with use of transgenic proteins in crops. Currently, safety assessment involves consideration of the source of the introduced protein, in silico amino acid sequence homology comparisons to known allergens, physicochemical properties, protein abundance in the crop, and, when appropriate, specific immunoglobulin E binding studies. Recently conducted research presented at an International Life Sciences Institute/Health and Environmental Sciences Institute–hosted workshop adds to the scientific foundation for safety assessment of transgenic proteins in five areas: structure/activity, serum screening, animal models, quantitative proteomics, and basic mechanisms. A web-based tool is now available that integrates a database of allergenic proteins with a variety of computational tools which could be used to improve our ability to predict allergenicity based on structural analysis. A comprehensive strategy and model protocols have been developed for conducting meaningful serum screening, an extremely challenging process. Several animal models using oral sensitization with adjuvant and one dermal sensitization model have been developed and appear to distinguish allergenic from nonallergenic food extracts. Data presented using a mouse model suggest that pepsin resistance is indicative of allergenicity. Certain questions remain to be addressed before considering animal model validation. Gel-free mass spectrometry is a viable alternative to more labor-intensive approaches to quantitative proteomics. Proteomic data presented on four nontransgenic varieties of soy suggested that if known allergen expression in genetically modified crops falls within the range of natural variability among commercial varieties, there appears to be no need to test further. Finally, basic research continues to elucidate the etiology of food allergy

The Utility of an International Sera Bank for Use in Evaluating the Potential Human Allergenicity of Novel Proteins

In the safety assessment of novel foods produced through biotechnology, careful consideration is given to determining the allergenic potential of newly introduced proteins. IgE serum screening is one tool for evaluating whether the protein in question has sequence identity to a known allergen or if the source of the gene encoding the protein is a known allergenic food. A "specific” serum screen involves testing a gene product with sera from patients with documented clinical allergy to a specific allergen to confirm that the gene product of interest is not the same protein to which the patient produces IgE antibodies. A "targeted” serum screen involves testing the gene product of interest with sera from patients sensitive to food or aeroallergens from the same broad group. The concept of a global sera bank with accessible, well-characterized sera for use in such assays is an appealing option. This paper summarizes the consensus elements from a workshop to evaluate the potential utility of an international sera bank for evaluating the allergenicity of novel proteins. Areas of agreement following the workshop included the following: (1) specific sera screens are appropriate for exploring potentially cross-reactive proteins that have been identified through bioinformatics analyses; however, additional validation is needed, particularly for targeted sera screens, (2) practical and ethical considerations may preclude the formation of a global sera bank, and therefore, (3) a regional network of clinicians who could serve as sources of patient sera or be approached to conduct sera studies would be the most practical alternativ

Genetic basis and detection of unintended effects in genetically modified crop plants

In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging “omics” technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that “unintended” does not necessarily mean “harmful”. This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics

Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

The steroidogenic acute regulatory (StAR) protein, a novel mitochondrial protein, is involved in the regulation of steroid hormone biosynthesis through its mediation of the intramitochondrial transport of the steroid substrate, cholesterol, to the cytochrome P450 cholesterol side chain cleavage (P450scc) enzyme. The expression of StAR protein is regulated by cAMP-dependent signaling in steroidogenic cells. During the course of our studies in mouse Leydig cells, we employ several methods for studying the regulation of StAR protein expression by human chorionic gonadotropin (hCG). A sensitive quantitative reverse transcription and polymerase chain reaction (RT-PCR) was utilized for determining StAR mRNA expression. Stimulation of mLTC-1 mouse Leydig tumor cells with hCG resulted in the coordinate regulation of StAR mRNA expression and progesterone accumulation in a time-response manner. The validity and accuracy of quantitative RT-PCR results in mLTC-1 cells were verified by a competitive PCR approach and were further confirmed in primary cultures of isolated mouse Leydig cells. Immunoblotting studies demonstrated an increase in the levels of the StAR protein in a concentration dependent manner following hCG stimulation in mLTC-1 cells. Northern hybridization analysis revealed three StAR transcripts, all of which were of sufficient size to encode functional StAR protein, and which were coordinately expressed in response to hCG. Collectively, the experimental approaches utilized in the present investigation allow for the demonstration and characterization of hCG mediated regulation of StAR mRNA and StAR protein expression in mouse Leydig cells

A Mitochondrial Kinase Complex Is Essential to Mediate an ERK1/2-Dependent Phosphorylation of a Key Regulatory Protein in Steroid Biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393