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Background: Oral immunotherapy (OIT) with peanut ( Arachis hypogaea ) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants., Objective: Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants ( n  = 20) enrolled in phase 3 PTAH OIT clinical trials., Methods: Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides., Results: Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered., Conclusion: At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut., Competing Interests: SAT is an employee of Aimmune Nestle Health Science. This study was funded by The United States Department of Agriculture (USDA), Agricultural Research Service and by Aimmune Nestlé Health Science, via a Cooperative Research and Development Agreement (CRADA agreement #58-6054-1-0011). Aimmune Nestlé was involved in performing the OIT study with Palforzia, which is published information, but was not involved in the design and interpretation of this study. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2023 Rambo, Kronfel, Rivers, Swientoniewski, McBride, Cheng, Simon, Ryan, Tilles, Nesbit, Kulis, Hurlburt and Maleki.)

Peanut and tree-nut allergies are frequently comorbid for reasons not completely understood. Vicilin-buried peptides (VBPs) are an emerging family of food allergens whose conserved structural fold could mediate peanut/tree-nut co-allergy. Peptide microarrays were used to identify immunoglobulin E (IgE) epitopes from the N-terminus of the vicilin allergens Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from three patient diagnosis groups: monoallergic to either peanuts or cashew/pistachio, or dual allergic. IgE binding peptides were highly prevalent in the VBP domains AH1.1, AO1.1, JR2.1, and PV3.1, but not in AO1.2, JR2.2, JR2.3, and PV3.2 nor the unstructured regions. The IgE profiles did not correlate with diagnosis group. The structure of the VBPs from cashew and pistachio was solved using solution-NMR. Comparisons of structural features suggest that the VBP scaffold from peanuts and tree-nuts can support cross-reactivity. This may help understand comorbidity and cross-reactivity despite a distant evolutionary origin.

Non-specific lipid transfer proteins (LTPs) are well studied allergens that can lead to severe reactions, but often cause oral allergy syndrome in the Mediterranean area and other European countries. However, studies focused on LTP reactivity in allergic individuals from the United States are lacking because they are not considered major allergens. The goal of this study is to determine if differences in immunoglobulin (Ig) E binding patterns to the peanut allergen Ara h 9 and two homologous LTPs (walnut Jug r 3 and peach Pru p 3) between the US and Spain contribute to differences observed in allergic reactivity. Synthetic overlapping 15-amino acid-long peptides offset by five amino acids from Ara h 9, Jug r 3, and Pru p 3 were synthesized, and the intact proteins were attached to microarray slides. Sera from 55 peanut-allergic individuals from the US were tested for IgE binding to the linear peptides and IgE binding to intact proteins using immunofluorescence. For comparison, sera from 17 peanut-allergic individuals from Spain were also tested. Similar IgE binding profiles for Ara h 9, Jug r 3, and Pru p 3 were identified between the US and Spain, with slight differences. Certain regions of the proteins, specifically helices 1 and 2 and the C-terminal coil, were recognized by the majority of the sera more often than other regions of the proteins. While serum IgE from peanut-allergic individuals in the US binds to peptides of Ara h 9 and its homologs, only IgE from the Spanish subjects bound to the intact LTPs. This study identifies Ara h 9, Jug r 3, and Pru p 3 linear epitopes that were previously unidentified using sera from peanut-allergic individuals from the US and Spain. Certain regions of the LTPs are recognized more often in US subjects, indicating that they represent conserved and possible cross-reactive regions. The location of the epitopes in 3D structure models of the LTPs may predict the location of potential conformational epitopes bound by a majority of the Spanish patient sera. These findings are potentially important for development of peptide or protein-targeting diagnostic and therapeutic tools for food allergy., Competing Interests: Author RK and PB were employed by the company Rho Federal Systems Division. Authors RR and RS were employed by the company Aimmune Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Kronfel, Cheng, McBride, Nesbit, Krouse, Burns, Cabanillas, Crespo, Ryan, Simon, Maleki and Hurlburt.)

Vicilin-buried peptides (VBPs) from edible plants are derived from the N-terminal leader sequences (LSs) of seed storage proteins. VBPs are defined by a common α-hairpin fold mediated by conserved CxxxCx (10-14) CxxxC motifs. Here, peanut and walnut VBPs were characterized as potential mediators of both peanut/walnut allergenicity and cross-reactivity despite their low (∼17%) sequence identity. The structures of one peanut (AH1.1) and 3 walnut (JR2.1, JR2.2, JR2.3) VBPs were solved using solution NMR, revealing similar α-hairpin structures stabilized by disulfide bonds with high levels of surface similarity. Peptide microarrays identified several peptide sequences primarily on AH1.1 and JR2.1, which were recognized by peanut-, walnut-, and dual-allergic patient IgE, establishing these peanut and walnut VBPs as potential mediators of allergenicity and cross-reactivity. JR2.2 and JR2.3 displayed extreme resilience against endosomal digestion, potentially hindering epitope generation and likely contributing to their reduced allergic potential.

Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small-molecule ligands. Ligand-binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds. Pollen and plant food allergens of the family 10 of pathogenesis-related proteins bind a variety of small molecules such as glycosylated flavonoid derivatives, flavonoids, cytokinins, and steroids in vitro. However, their natural ligand binding was reported to be highly specific. Insect and mammalian lipocalins transport odorants, pheromones, catecholamines, and fatty acids with a similar level of specificity, while the food allergen β-lactoglobulin from cow's milk is notably more promiscuous. Non-specific lipid transfer proteins from pollen and plant foods bind a wide variety of lipids, from phospholipids to fatty acids, as well as sterols and prostaglandin B2, aided by the high plasticity and flexibility displayed by their lipid-binding cavities. Ligands increase the stability of allergens to thermal and/or proteolytic degradation. They can also act as immunomodulatory agents that favor a Th2 polarization. In summary, ligand-binding allergens expose the immune system to a variety of biologically active compounds whose impact on the sensitization process has not been well studied thus far., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Peanut is a potent inducer of proallergenic T H 2 responses in susceptible individuals. Antigen-presenting cells (APCs) including dendritic cells and monocytes instruct naive T cells to differentiate into various effector cells, determining immune responses such as allergy and tolerance., Objective: We sought to detect peanut protein (PN)-induced changes in gene expression in human myeloid dendritic cells (mDCs) and monocytes, identify signaling receptors that mediate these changes, and assess how PN-induced genes in mDCs impact their ability to promote T-cell differentiation., Methods: mDCs, monocytes, and naive CD4 + T cells were isolated from blood bank donors and peanut-allergic patients. APCs were incubated with PN and other stimulants, and gene expression was measured using microarray and RT quantitative PCR. To assess T-cell differentiation, mDCs were cocultured with naive T H cells., Results: PN induced a unique gene expression profile in mDCs, including the gene that encodes retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in the retinoic acid (RA)-producing pathway. Stimulation of mDCs with PN also induced a 7-fold increase in the enzymatic activity of RALDH2. Blocking antibodies against Toll-like receptor (TLR)1/TLR2, as well as small interfering RNA targeting TLR1/TLR2, reduced the expression of RALDH2 in PN-stimulated APCs by 70%. Naive T H cells cocultured with PN-stimulated mDCs showed an RA-dependent 4-fold increase in production of IL-5 and expression of integrin α 4 β 7 ., Conclusions: PN induces RALDH2 in human APCs by signaling through the TLR1/TLR2 heterodimer. This leads to production of RA, which acts on T H cells to induce IL-5 and gut-homing integrin. RALDH2 induction by PN in APCs and RA-promoted T H 2 differentiation could be an important factor determining allergic responses to peanut., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Understanding the discrepancy between IgE sensitization and allergic reactions to peanut could facilitate diagnosis and lead to novel means of treating peanut allergy., Objective: To identify differences in IgE and IgG4 binding to peanut peptides between peanut-allergic (PA) and peanut-sensitized but tolerant (PS) children., Methods: PA (n = 56), PS (n = 42) and nonsensitized nonallergic (NA, n = 10) patients were studied. Synthetic overlapping 15-mer peptides of peanut allergens (Ara h 1-11) were spotted onto microarray slides, and patients' samples were tested for IgE and IgG4 binding using immunofluorescence. IgE and IgG4 levels to selected peptides were quantified using ImmunoCAP. Diagnostic model comparisons were performed using likelihood-ratio tests between each specified nominal logistic regression models., Results: Seven peptides on Ara h 1, Ara h 2, and Ara h 3 were bound more by IgE of PA compared to PS patients on the microarray. IgE binding to one peptide on Ara h 5 and IgG4 binding to one Ara h 9 peptide were greater in PS than in PA patients. Using ImmunoCAP, IgE to the Ara h 2 peptides enhanced the diagnostic accuracy of Ara h 2-specific IgE. Ratios of IgG4/IgE to 4 out of the 7 peptides were higher in PS than in PA subjects., Conclusions: Ara h 2 peptide-specific IgE added diagnostic value to Ara h 2-specific IgE. Ability of peptide-specific IgG4 to surmount their IgE counterpart seems to be important in established peanut tolerance., (© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Food allergies are a major clinical problem and are driven by IgE antibodies (Abs) specific for food antigens (Ags). T follicular regulatory (Tfr) cells are a specialized subset of FOXP3+ T cells that modulate Ab responses. Here, we analyzed the role of Tfr cells in regulating Ag-specific IgE using a peanut-based food allergy model in mice. Peanut-specific IgE titers and anaphylaxis responses were significantly blunted in Tfr cell-deficient Foxp3-Cre Bcl6fl/fl mice. Loss of Tfr cells led to greatly increased nonspecific IgE levels, showing that Tfr cells have both helper and suppressor functions in IgE production in the germinal center (GC) that work together to facilitate the production of Ag-specific IgE. Foxp3-Cre Ptenfl/fl mice with augmented Tfr cell responses had markedly higher levels of peanut-specific IgE, revealing an active helper function by Tfr cells on Ag-specific IgE. The helper function of Tfr cells for IgE production involves IL-10, and the loss of IL-10 signaling by B cells led to a severely curtailed peanut-specific IgE response, decreased GCB cell survival, and loss of GC dark zone B cells after peanut sensitization. We thus reveal that Tfr cells have an unexpected helper role in promoting food allergy and may represent a target for drug development.

Many individuals with peanut (PN) allergy have severe reactions to tree nuts (TN) such as walnuts or cashews. Although allergenic proteins in TN and PN have overall low identity, they share discrete sequences similar in physicochemical properties (PCP) to known IgE epitopes. Here, PCP-consensus peptides (cp, 13 aa and 31 aa) were identified from an alignment of epitope rich regions of walnut vicilin, Jug r 2, leader sequence (J2LS) and cross-reactive epitopes in the 2S albumins of peanut and synthesized. A peptide similarity search in the Structural Database of Allergenic Proteins (SDAP) revealed a network of peptides similar (low property distance, PD) to the 13 aa cp (13cp) in many different plant allergens. Peptides similar to the 13cp in PN and TN allergens bound IgE from sera of patients allergic to PN and TN in peptide microarray analysis. The 13cp was used to produce a rabbit consensus peptide antibody (cpAB) that detected proteins containing repeats similar to the 13cp in western blots of various nut extracts, in which reactive proteins were identified by mass spectrometry. The cpAB bound more specifically to allergens and nut extracts containing multiple repeats similar to the 13 cp, such as almond (Pru du 6), peanut (Ara h 2) and walnut (Jug r 2). IgE binding to various nut extracts is inhibited by recombinant J2LS sequence and synthetic 31cp. Thus, several repeated sequences similar to the 13cp are bound by IgE. Multiple similar repeats in several allergens could account for reaction severity and clinically relevant cross-reactivity to PN and TN. These findings may help improve detection, diagnostic, and therapeutic tools., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier Ltd.)

Oral allergy syndrome (OAS) describes an allergic reaction where an individual sensitized by pollen allergens develops symptoms after eating certain foods. OAS is caused by cross-reactivity among a class of proteins ubiquitous in plants called pathogenesis related class 10 (PR-10) proteins. The best characterized PR-10 protein is Bet v 1 from birch pollen and its putative function is binding hydrophobic ligands. We cloned a subset of seven recombinant PR-10 proteins from pollens, peanuts, and hazelnuts and developed a standard purification method for them. Immunoglobulin E (IgE) binding of purified PR-10 proteins was analyzed by ImmunoCAP ISAC microarray and enzyme-linked immunosorbent assays (ELISAs) with sera from allergic patients. We investigated the binding activities of PR10s by testing 16 different ligands with each protein and compared their secondary structures using circular dichroism (CD). The PR-10s in this study had very similar CD spectra, but bound IgE with very different affinities. All seven proteins showed a similar pattern of binding to the polyphenol ligands (resveratrol, flavonoids, and isoflavones) and variable binding to other potential ligands (fatty acids, sterols, and plant hormones). We suggest our protocol has the potential to be a near-universal method for PR-10 purification that will facilitate further research into this important class of panallergens., Competing Interests: The authors declare no conflict of interest.

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

Quantitative guidelines to distinguish allergenic proteins from related, but non-allergenic ones are urgently needed for regulatory agencies, biotech companies and physicians. In a previous study, we found that allergenic proteins populate a relatively small number of protein families, as characterized by the Pfam database. However, these families also contain non-allergenic proteins, meaning that allergenic determinants must lie within more discrete regions of the sequence. Thus, new methods are needed to discriminate allergenic proteins within those families. Physical-Chemical Properties (PCP)-motifs specific for allergens within a Pfam class were determined for 17 highly populated protein domains. A novel scoring method based on PCP-motifs that characterize known allergenic proteins within these families was developed, and validated for those domains. The motif scores distinguished sequences of allergens from a large selection of 80,000 randomly selected non-allergenic sequences. The motif scores for the birch pollen allergen (Bet v 1) family, which also contains related fruit and nut allergens, correlated better than global sequence similarities with clinically observed cross-reactivities among those allergens. Further, we demonstrated that the average scores of allergen specific motifs for allergenic profilins are significantly different from the scores of non-allergenic profilins. Several of the selective motifs coincide with experimentally determined IgE epitopes of allergenic profilins. The motifs also discriminated allergenic pectate lyases, including Jun a 1 from mountain cedar pollen, from similar proteins in the human microbiome, which can be assumed to be non-allergens. The latter lacked key motifs characteristic of the known allergens, some of which correlate with known IgE binding sites., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

For years, the use of polyhistidine tags (His-tags) has been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a consequence of His-tag addition is that it can affect protein function and stability. Functional proteins are essential in the elucidation of their biological, kinetic, structural, and thermodynamic properties. In this study, we determine the effect of N-terminal His-tags on the thermal stability of select proteins using differential scanning fluorimetry and identify that the removal of the His-tag can have both beneficial and deleterious effects on their stability., Competing Interests: The authors declare no competing financial interest.

Aspartate β-semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45 Å resolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and have extensive interchain contacts, which are thought to create a half-sites reactivity enzyme. ASADH from higher organisms shows a tetrameric oligomerization, which also has implications for both reactivity and regulation. This work analyzes the apo form of F. tularensis ASADH, as well as the binding of the enzyme to its cofactor NADP + .

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A 71 G 74 S 69 D 75 T 73 F 72 V 67 ) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The dry roasting of peanuts is suggested to influence allergic sensitization as a result of the formation of advanced glycation end products (AGEs) on peanut proteins. Identifying AGEs is technically challenging. The AGEs of a peanut allergen were probed with nano-scale liquid chromatography-electrospray ionization-mass spectrometry (nanoLC-ESI-MS) and tandem mass spectrometry (MS/MS) analyses. Amadori product ions matched to expected peptides and yielded fragments that included a loss of three waters and HCHO. As a result of the paucity of b and y ions in the MS/MS spectrum, standard search algorithms do not perform well. Reactions with isotopically labeled sugars confirmed that the peptides contained Amadori products. An algorithm was developed on the basis of information content (Shannon entropy) and the loss of water and HCHO. Results with test data show that the algorithm finds the correct spectra with high precision, reducing the time needed to manually inspect data. Computational and technical improvements allowed for better identification of the chemical differences between modified and unmodified proteins.

Allergic reactions to food are on the rise worldwide and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut allergies are the most problematic because the reaction often persists into adulthood and can be as severe as anaphylaxis and death . The purpose of the work presented here was to develop a reproducible method to produce large quantities of pure recombinant Ara h 1(rAra h 1) that will enable standardization of immunological tests for patients and allow structural and immunological studies on the wild type and mutagenized forms of the protein. Ara h 1 is initially a pre-pro-protein which, following two endoproteolytic cleavages, becomes the mature form found in peanut. The mature form however has flexible regions that make it refractory to some structural studies including crystallography. Therefore, independent purification of the mature and core regions was desirable. Expression constructs were synthesized cDNA clones for each in a pET plasmid vector without tags. Codons were optimized for expression in E. coli . High-level expression was achieved in BL21 strains. Purification to near homogeneity was achieved by a combination of ammonium sulfate precipitation and ion exchange chromatography. The purified rAra h 1 was then compared with natural Ara h 1 for IgE binding. All patients recognized both the folded natural and rAra h 1, but the IgE binding to the rArah1 was significantly reduced in comparison to the natural allergen, which could potentially make it useful for immunotherapeutic purposes.

It has been suggested that the boiling or frying of peanuts leads to less allergenic products than roasting. Here, we have compared the digestibility of the major peanut allergens in the context of peanuts subjected to boiling, frying or roasting and in purified form. The soluble peanut extracts and the purified allergens were digested with either trypsin or pepsin and analyzed by gel electrophoresis and western blot. T-cell proliferation was measured for the purified allergens. In most cases, boiled and raw peanut proteins were similarly digestible, but the Ara h 1 protein in the boiled extracts was more resistant to digestion. Most proteins from fried and roasted peanuts were more resistant to digestion than in raw and boiled samples, and more IgE binding fragments survived digestion. High-molecular-weight fragments of Ara h1 were resistant to digestion in fried and roasted samples. Ara h 1 and Ara h 2 purified from roasted peanuts were the most resistant to digestion, but differed in their ability to stimulate T-cells. The differences in digestibility and IgE binding properties of the major allergens in roasted, fried and boiled peanuts may not explain the difference between the prevalence of peanut allergy in different countries that consume peanut following these varied processing methods.

The incidence of peanut allergy continues to rise in the United States and Europe. Whereas exposure to the major allergens Ara h 1, 2, 3, and 6 can cause fatal anaphylaxis, exposure to the minor allergens usually does not. Ara h 8 is a minor allergen. Importantly, it is the minor food allergens that are thought to be responsible for oral allergy syndrome (OAS), in which sensitization to airborne allergens causes a Type 2 allergic reaction to ingested foods. Furthermore, it is believed that similar protein structure rather than a similar linear sequence is the cause of OAS. Bet v 1 from birch pollen is a common sensitizing agent, and OAS results when patients consume certain fruits, vegetables, tree nuts, and peanuts. Here, we report the three-dimensional structure of Ara h 8, a Bet v 1 homolog. The overall fold is very similar to that of Bet v 1, Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Ara h 8 binds the isoflavones quercetin and apigenin as well as resveratrol avidly.

Background: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy., Methods: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods., Results: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE., Conclusions: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Scope: The aims of this study were to evaluate IgE-mediated hypersensitivity to pine nut with details of clinical reactions and to characterize major pine nut allergens., Methods and Results: The study included ten consecutive teenagers and adults diagnosed with IgE-mediated clinical allergy to pine nut. Two major pine nut allergens were purified and identified and the secondary structures and susceptibility to digestion were characterized. Severe reactions represent 80% of allergic reactions to pine nut in this study. Moreover, 70% of the patients were monosensitized to this nut. Two major allergens with molecular weights of 6 and 50 kDa were purified and identified as albumin and vicilin, respectively. The 6 kDa protein (albumin), rich in α-helix content, was far more stable to peptic and tryptic digestion as compared with 50 kDa protein (vicilin), which was quickly broken down. The secondary structure of the purified 50 kDa protein showed 41% β-sheet, 5% α-helix, and 54% random coil and/or loops., Conclusion: Eighty percent of allergic reactions to pine nut in the ten patients included in this study were severe. Most patients (70%) were monosensitized to this nut. Two major allergens with molecular weights of 6 and 50 kDa were purified and identified as albumin and vicilin, respectively., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Ara h 1 from roasted peanut binds higher levels of serum immunoglobulin E than raw peanuts and this is likely due to the Maillard reaction. While Ara h 1 linear IgE epitopes have been mapped, the presence and importance of structural epitopes is not clear., Methods and Results: Mass spectrometry, immunoblot, ELISA, circular dichroism (CD), and structural analysis were used to compare structural and subsequent IgE-binding differences in Ara h 1 purified from raw (N) and roasted peanuts (R) and denatured Ara h 1 (D). Although N and R had similar CD spectra, the latter bound significantly more IgE. Decreased IgE binding was seen with the loss of secondary structure. This same IgE-binding pattern [R > N > D] was seen for the sera of ten peanut allergic patients. While the majority of linear epitopes are located on surface and structured regions of Ara h 1, our study shows that conformational epitopes of Ara h 1 bind better to IgE than linear epitopes., Conclusion: Enhanced IgE binding to roasted Ara h 1 could be due to alterations such as chemical modifications to individual amino acids or increased epitope exposure. IgE binding is significantly reduced with loss of structure., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

As peanut allergy is an increasing public health risk, affecting over 1% of the United States and United Kingdom school children, it is important that methods and reagents for accurate diagnosis of food allergy and detection of allergenic foods are reliable and consistent. Given that most current experimental, diagnostic, and detection tests rely on the presence of soluble allergens in food extracts, we investigated the effects of thermal processing on the solubility and IgE binding of the major peanut allergens, Ara h 1 and Ara h 2. The soluble and insoluble fractions of peanuts that were boiled, fried, and roasted were subjected to electrophoresis and Western blot analysis using anti-Ara h 1 and anti-Ara h 2 antibodies and serum IgE from peanut allergic individuals. Overall protein solubility is reduced with processing and IgE binding increases in the insoluble fractions, due mostly to the increase in the amount of insoluble proteins, with increased time of heating in all processes tested. Therefore, it can be concluded that thermal processing of peanuts alters solubility, and the differences in protein solubility within various extract preparations may contribute to inconsistent skin prick test and immunoassay results, particularly when nonstandardized reagents are used.

Staphylococcus aureus produces a wide array of virulence factors and causes a correspondingly diverse array of infections. Production of these virulence factors is under the control of a complex network of global regulatory elements, one of which is sarA. sarA encodes a DNA binding protein that is considered to function as a transcription factor capable of acting as either a repressor or an activator. Using competitive ELISA assays, we demonstrate that SarA is present at approximately 50 000 copies per cell, which is not characteristic of classical transcription factors. We also demonstrate that SarA is present at all stages of growth in vitro and is capable of binding DNA with high affinity but that its binding affinity and pattern of shifted complexes in electrophoretic mobility shift assays is responsive to the redox state. We also show that SarA binds to the bacteriophage lambda (lambda) attachment site, attL, producing SarA-DNA complexes similar to intasomes, which consist of bacteriophage lambda integrase, Escherichia coli integration host factor and attL DNA. In addition, SarA stimulates intramolecular excision recombination in the absence of lambda excisionase, a DNA binding accessory protein. Taken together, these data suggest that SarA may function as an architectural accessory protein.

The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

The majority of foods that we eat are subjected to some type of processing either at home or by the manufacturer. The biochemical reactions that occur in foods as a result of thermal processing can be both beneficial and harmful. Here, we briefly review the effects of thermal processing and some of the effects of the Maillard reaction on the allergenicity of food proteins. Specifically, we focus on the known effects of roasting on the allergenic properties of peanut proteins and the contribution of Maillard reaction products or advanced glycation end products to these observed effects. The most thorough understanding of the effects of thermal processing on allergenicity involves the peanut proteins. Thermal processing alters specific biophysical and immunological properties of peanut proteins, such as structure, function, solubility, digestibility, immunoglobulin E (IgE) binding, and T-cell responses. A better understanding of the effects of thermal processing-induced biochemical and immunological alterations is of utmost importance for proper risk assessment of existing and newly introduced proteins in the food source, as well as development of effective diagnostic tools and therapeutic treatments for food allergy.

The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

MyoD and Myogenin are dominant myogenic regulatory factors (MRFs), which are involved in control of muscle-specific gene expression. The ubiquitously expressed E12 dimerizes with MyoD and Myogenin and has been shown to enhance their DNA-binding and transcriptional activities. In this study, fluorescence anisotropy assays have been used to determine the Gibb's free energy of dissociation (DeltaG) for MyoD, Myogenin, and E12 as homo- and heterodimers to the well-characterized myosin light chain enhancer (MLC), muscle creatine kinase (MCK) enhancer, and mutant thereof. The heterodimers of MyoD or Myogenin with E12 bound the MCK enhancer equally well (DeltaG = 21 kcal/mol). The homodimers varied dramatically in both MLC and MCK enhancer binding affinity. MyoD homodimer bound the MCK enhancer with the highest affinity (DeltaG = 19.6 kcal/mol) in comparison with the Myogenin homodimer-MCK interaction (DeltaG = 16.6 kcal/mol) and E12 homodimer-MCK interaction (DeltaG = 18.0 kcal/mol). The slope and shape of the binding isotherms revealed that with the exception of the E12 homodimer-MCK enhancer interaction, the other proteins bound with high levels of positive cooperativity. In contrast, the E12 homodimer-MCK enhancer interaction actually occurs with significant negative cooperativity. The binding of these proteins to MLC enhancer mimicked binding to the MCK enhancer, but with much lower affinities. These data support the hypothesis that DNA acts as an allosteric ligand facilitating the dimerization of these proteins. The combination of differential affinity and cooperativity explains why the heterodimers are the active species in transcriptional regulation.

The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814-4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

Staphylococcus aureus is a major human pathogen, the potency of which can be attributed to the regulated expression of an impressive array of virulence determinants. A key pleiotropic transcriptional regulator of these virulence factors is SarA, which is encoded by the sar (staphylococcal accessory regulator) locus. SarA was characterized initially as an activator of a second virulence regulatory locus, agr, through its interaction with a series of heptad repeats (AGTTAAG) within the agr promoter. Subsequent DNA-binding studies have revealed that SarA binds readily to multiple AT-rich sequences of variable lengths. Here we describe the crystal structure of SarA and a SarA-DNA complex at resolutions of 2.50 A and 2.95 A, respectively. SarA has a fold consisting of a four-helix core region and 'inducible regions' comprising a beta-hairpin and a carboxy-terminal loop. On binding DNA, the inducible regions undergo marked conformational changes, becoming part of extended and distorted alpha-helices, which encase the DNA. SarA recognizes an AT-rich site in which the DNA is highly overwound and adopts a D-DNA-like conformation by indirect readout. These structures thus provide insight into SarA-mediated transcription regulation.

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.

Comparison of Staphylococcus aureus strains carrying mutations inactivating the staphylococcal accessory regulator (sar ) and/or the accessory gene regulator (agr ) suggests that sar is the primary regulatory element controlling transcription of the collagen adhesin gene (cna ) and that the regulatory effect of sar is independent of the interaction between SarA and agr. To test this hypothesis, we cloned the regions encoding each of the overlapping sar transcripts, all of which include the sarA open reading frame (ORF), and introduced each clone into cna-positive sar and agr mutants. The introduction of each clone restored the expected sar transcripts and the temporal pattern of sar transcription. The introduction of each clone also complemented the defect in cna transcription and restored collagen binding to wild-type levels. This was true even when the clones were introduced into a sar/agr double mutant. These results confirm the hypothesis that the sar-mediated regulation of cna transcription occurs via an agr-independent pathway. Direct evidence supporting this hypothesis comes from electrophoretic mobility shift assays demonstrating that SarA exhibits high-affinity binding to cis elements upstream of the cna structural gene. We also examined the correlation between sar transcription and the production of SarA. Western blot analysis of two wild-type strains indicated that SarA was produced in indistinguishable amounts during both the exponential and the post-exponential growth phases.

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-affinity binding sites composed of two half-sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half-sites and may involve other sites located downstream of the promoters.

The NH2-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor-operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor. A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH2-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.

Muscle development is controlled by the MyoD family of basic helix-loop-helix (bHLH) DNA-binding proteins. These proteins dimerize with ubiquitous products of the E2A gene (E12 and E47) and bind in a sequence-specific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been utilized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociation (deltaG) and the equilibrium dissociation constants (K(D)) for the protein-protein interactions are reported. The deltaG for the MyoD homodimers in 100 mM KCl was 8.7 kcal/mol (K(D) = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigorous confidence limit testing. The titrations of E12-dansyl with MyoD yielded a free energy of 8.3 kcal/mol with tighter confidence limits, +0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers and MyoD-E12 heterodimers are relatively weak complexes of approximately the same stability. E12 does not form stable homo-oligomeric complexes; remaining monomeric at concentrations as high as 20 microM. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterodimers versus MyoD homodimers with DNA binding sites is not due to preferential heterodimerization.

The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF4, function as transcriptional activators of muscle-specific gene expression by forming heterodimers with the ubiquitously expressed products of the E2A gene, E12 and E47. To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants designed to reveal structure-function relationships, we developed protocols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical techniques. T7 expression systems were used to direct expression of cDNA encoded proteins in Escherichia coli. Whereas MyoD and E12 were expressed well without alteration, high-level expression of myogenin required changing several rare arginine codons by in vitro mutagenesis to a commonly used E. coli arginine codon. Presumably, inefficient translation of the rare arginine codons inhibited high-level expression of myogenin in the original expression plasmid. Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulfate precipitation, and ion-exchange chromatography. Using this approach, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% homogeneity from induced cell pellets in 1 day's time.

An investigation of DNA-protein interactions by capillary electrophoresis (CE) with laser fluorometric detection is performed that combines the rapid and minimal sample consumption methods of CE with the selective separation influence of mobility shift assays. An inspection of the well characterized interaction between the trp repressor of Escherichia coli and the trp operator (DNA) is the basis of the assay. The use of fluorescently tagged operator not only lends itself to laser-induced fluorescence detection but also precludes the use of radiolabeled detection. It is demonstrated that composition and pH of the running buffer are critical for maximized efficiency and resolution of operator from the repressor-operator complex. Quantitative studies showed reaction of repressor with operator resulted in the diminishing of free operator signal and the simultaneous creation of the repressor-operator peak that is well resolved from the free operator. Also examined was the ability to perform qualitative studies involving non-specific interactions between the operator and a complex protein sample. It is shown that the specificity of operator for repressor can be used to selectively separate the repressor from a complex sample that includes non-specific proteins.

The MyoD family of transcription factors regulates muscle-specific gene expression in vertebrates. In the adult rat, MyoD mRNA accumulates predominately in fast-twitch muscle, in particular type IIb and/or IIx fibers, whereas Myogenin mRNA is restricted to slow-twitch type I muscle fibers. Transgenic mice expressing the avian v-ski oncogene from the murine sarcoma virus (MSV) promoter-enhancer display preferential hypertrophy of type IIb fast-twitch muscle apparently because of the restricted expression of the transgene. We tested the hypothesis that preferential interactions of MyoD, as a heterodimer with E12, with the MSV enhancer, which has six E-box targets for MyoD family proteins, could contribute to v-ski gene expression in IIb muscle fibers. A series of quantitative binding studies was performed using an electrophoretic mobility shift assay to test MyoD-E12 versus Myogenin-E12 binding to the MSV enhancer. Our results indicate that MyoD-E12 binds the MSV enhancer with higher affinity and higher cooperativity than Myogenin-E12. Interestingly, MyoD-E12 bound all of the individual E-boxes tested with positive cooperativity indicating DNA-mediated dimerization of the protein subunits.

trp repressor of Escherichia coli controls transcription initiation in operons involved in tryptophan biosynthesis by binding to operator sequences within the regulated promoters. Naturally occurring operators are homologous over an 18-base pair region and display dyad symmetry. We have examined the sequence determinants of a repressor binding site using a functional selection/polymerase chain reaction (PCR) amplification strategy. A trp repressor affinity column was generated and used to select binding-competent DNAs from a randomized pool of synthetic double-stranded DNA. DNAs that showed tryptophan-dependent high-affinity binding were eluted by addition of the tryptophan analog beta-indole acrylic acid and amplified by PCR. Following iterative cycles of affinity chromatography and PCR, the selected DNAs were cloned and sequenced. The CTAG tetranucleotide, present in the consensus sequence of all natural operators, was found in all selected DNAs. Mapping experiments utilizing the repressor affinity column showed the CTAG motif to be a critical determinant for repressor binding. Quantitative electrophoretic mobility shift assays with purified trp repressor revealed that although some of the DNAs were bound by one repressor dimer, others were bound by two repressor dimers with cooperativity. Measured binding constants ranged from 0.035 to 0.5 nM for the selected DNAs, compared with 0.1 nM for the trp operator.

The trp repressor of Escherichia coli is a dimeric DNA-binding protein that regulates transcription of several operons concerned with tryptophan metabolism. Although heterodimer formation between mutant and wild type subunits occurs readily in vivo, comparable heterodimers could be formed in vitro only under extreme conditions. To explain this difference we analyzed trp repressor dimer formation and dissociation using an in vitro transcription/translation system. Nascent wild type or mutant repressor polypeptides, synthesized in the presence of an excess of a second repressor, were invariably incorporated into heterodimers. In contrast, previously synthesized and assembled wild type dimers appeared to be refractory to dissociation, since they did not form heterodimers. However, previously synthesized mutant dimeric repressors that were defective in tryptophan binding readily dissociated and formed heterodimers. We noted that the ability of a dimeric repressor to dissociate under our conditions correlated inversely with its affinity for tryptophan. Consistent with this conclusion, we found that dissociation of the wild type aporepressor (no added tryptophan) was appreciably more rapid than dissociation of the tryptophan-saturated wild type repressor.

The trp repressor of Escherichia coli regulates transcription initiation in the trp operon by binding at an operator located within the trp promoter region. We have used a filter binding assay to analyze the interaction between purified trp repressor and a synthetic 43-base pair DNA fragment containing the natural trp promoter-operator region. In equilibrium binding experiments, the KD of high affinity binding of trp repressor to this DNA fragment was determined to be 2 x 10(-10) M. Low affinity binding was observed at repressor concentrations above 10 nM. In kinetic experiments with various input ratios of repressor to operator, trp repressor-operator complexes dissociated with equivalent, first-order kinetics. Instantaneous reduction of the tryptophan concentration resulted in increased rates of complex dissociation, indicating that loss of one or both tryptophan molecules from the repressor-operator complex destabilizes the complex. A heterodimeric repressor with a single tryptophan binding site was constructed and its affinity for operator was compared with that of ligand free aporepressor and tryptophan saturated repressor. The heterodimeric repressor had a 20-25-fold higher affinity for operator than did the aporepressor, and it had a 20-25-fold lower affinity for operator than did the tryptophan-saturated repressor.

The 3-dimensional structure of the trp repressor, aporepressor, and repressor/operator complex have been described. The NH2-terminal arms of the protein, comprising approximately 12-14 residues, were not well resolved in any of these structures. Previous studies by Carey showed that the arms are required for full in vitro repressor activity. To examine the roles of the arms more fully we have removed codons 2-5 and 2-8 of the trpR gene and analyzed the resulting truncated repressors in vivo and in vitro. The delta 2-5 trp repressor was found to be approximately 25% as active as the wild type repressor in vivo. In in vitro equilibrium binding experiments, the delta 2-5 trp repressor was shown to be five-fold less active in operator binding. The rate of dissociation of the complex formed between the delta 2-5 trp repressor and operator was essentially the same as the rate of dissociation of the wild type trp repressor/operator complex. However association of the delta 2-5 trp repressor with operator was clearly defective. Since the NH2-terminal arms of the trp repressor appear to affect association predominantly they may play a role in facilitating non-specific association of repressor with DNA as repressor seeks its cognate operators. The delta 2-8 trp repressor was unstable in vivo and in vitro, suggesting that some portion of the NH2-terminal arm is required for proper folding of the remainder of the molecule.

The trp repressor of Escherichia coli binds to the operators of three operons concerned with tryptophan biosynthesis and regulates their expression. trp superrepressors can repress expression of the trp operon in vivo at lower tryptophan concentrations than those required by the wild-type repressor. The five known superrepressors have been purified and characterized using a modified filter binding assay. In four of the five superrepressors, EK13, EK18, DN46 and EK49, negatively charged wild-type residues located on the surface of the repressor that faces the operator are replaced by positively charged or neutral residues. Each of these proteins has higher affinity for the trp operator than wild-type repressor. Decreased rates of dissociation of the repressor-operator complex were found to be responsible for the higher affinities. The fifth superrepressor, AV77, has an amino acid substitution in the turn of the helix-turn-helix DNA-binding motif. This superrepressor was indistinguishable from wild-type repressor in our filter binding assay. We conclude that rapid dissociation of repressor from operator is important for trp repressor function in vivo. The negatively charged wild-type residues that are replaced in superrepressors are probably responsible for the characteristic rapid dissociation of the trp repressor from the trp operator.

The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are alpha 2 type homodimers. Enzyme-bound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. One, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. Three modes of regulation are imposed on the expression of nitrate assimilation, namely: nitrogen metabolite repression, nitrate induction and autogenous regulation by nitrate reductase. In this study, nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved proteins from crude extracts of the wild type and specific nitrate-nonutilizing (nit) mutants were examined for material cross-reactive with antibodies against nitrate reductase and nitrite reductase. The polyclonal antibody preparations used were rendered monospecific by reverse affinity chromatography. Growth conditions which alter the regulatory response of the organism were selected such that new insight could be made into the complex nature of the regulation imposed on this pathway. The results indicate that although nitrate reductase and nitrite reductase are coordinately expressed under specific nutritional conditions, the enzymes are differentially responsive to the regulatory signals.