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Introduction: Molecular studies of hen’s egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (ses-)IgE and ses-IgG4 among baked-egg reactive or tolerant children. Methods: A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. Ses-IgE and ses-IgG4 repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared. Results: 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher ses-IgE and lower ses-IgG4 to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar ses-IgG4 but greater ses-IgE than tolerant group. A combination of OVA-sIgE with ses-IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype. Conclusion: We have created a comprehensive epitope library and showed that ses-IgE is a potential biomarker of baked-egg reactivity.

ImmunoglobulinA (IgA) is the predominant antibody isotype in the gut, where it regulates commensal flora and neutralizes toxins and pathogens. The function of food-specific IgA in the gut is unknown but is presumed to protect from food allergy. Specifically, it has been hypothesized that food-specific IgA binds ingested allergens and promotes tolerance by immune exclusion; however, the evidence to support this hypothesis is indirect and mixed. Although it is known that healthy adults have peanut-specific IgA in the gut, it is unclear whether children also have gut peanut-specific IgA. We found in a cohort of non–food-allergic infants ( n = 112) that there is detectable stool peanut-specific IgA that is similar to adult quantities of gut peanut-specific IgA. To investigate whether this peanut-specific IgA is associated with peanut tolerance, we examined a separate cohort of atopic children ( n = 441) and found that gut peanut-specific IgA does not predict protection from development of future peanut allergy in infants nor does it correlate with concurrent oral tolerance of peanut in older children. We observed higher plasma peanut-specific IgA in those with peanut allergy. Similarly, egg white–specific IgA was detectable in infant stools and did not predict egg tolerance or outgrowth of egg allergy. Bead-based epitope assay analysis of gut peanut-specific IgA revealed similar epitope specificity between children with peanut allergy and those without; however, gut peanut-specific IgA and plasma peanut-specific IgE had different epitope specificities. These findings call into question the presumed protective role of food-specific IgA in food allergy.

Background Accurate diagnosis of peanut allergy is a significant clinical challenge. Here, a novel diagnostic blood test using the peanut bead-based epitope assay ("peanut BBEA") was developed utilizing the LEAP cohort and then validated using two independent cohorts. Methods The development of the peanut BBEA diagnostic test followed the National Academy of Medicine's established guidelines with discovery performed on 133 subjects from the non-interventional arm of the LEAP trial and an independent validation performed on 82 subjects from the CoFAR2 and 84 subjects from the POISED study. All samples were analyzed using the peanut BBEA methodology, which measures levels of IgE to two Ara h 2 sequential (linear) epitopes and compares their combination to a threshold pre-specified in the model development phase. When a patient has an inconclusive outcome by skin prick testing (or sIgE), IgE antibody levels to this combination of two epitopes can distinguish whether the patient is "Allergic" or "Not Allergic." Diagnoses of peanut allergy in all subjects were confirmed by double-blind placebo-controlled food challenge and subjects' ages were 7-55 years. Results In the validation using CoFAR2 and POISED cohorts, the peanut BBEA diagnostic test correctly diagnosed 93% of the subjects, with a sensitivity of 92%, specificity of 94%, a positive predictive value of 91%, and negative predictive value of 95%. Conclusions In validation of the peanut BBEA diagnostic test, the overall accuracy was found to be superior to existing diagnostic tests for peanut allergy including skin prick testing, peanut sIgE, and peanut component sIgE testing.

Background Nasal transcriptomics can provide an accessible window into asthma pathobiology. Objective Our goal was to move beyond gene signatures of asthma to identify master regulator genes that causally regulate genes associated with asthma phenotypes. Methods We recruited 156 children with severe persistent asthma and controls for nasal transcriptome profiling and applied network-based and probabilistic causal methods to identify severe asthma genes and their master regulators. We then took the same approach in an independent cohort of 190 adults with mild/moderate asthma and controls to identify mild/moderate asthma genes and their master regulators. Comparative analysis of the master regulator genes followed by validation testing in independent children with severe asthma (n = 21) and mild/moderate asthma (n = 154) was then performed. Results Nasal gene signatures for severe persistent asthma and for mild/moderate persistent asthma were identified; both were found to be enriched in coexpression network modules for ciliary function and inflammatory response. By applying probabilistic causal methods to these gene signatures and validation testing in independent cohorts, we identified (1) a master regulator gene common to asthma across severity and ages (FOXJ1); (2) master regulator genes of severe persistent asthma in children (LRRC23, TMEM231, CAPS, PTPRC, and FYB); and (3) master regulator genes of mild/moderate persistent asthma in children and adults (C1orf38 and FMNL1). The identified master regulators were statistically inferred to causally regulate the expression of downstream genes that modulate ciliary function and inflammatory response to influence asthma. Conclusion The identified master regulator genes of asthma provide a novel path forward to further uncovering asthma mechanisms and therapy.

Genetic predisposition increases risk for asthma, and distinct nasal microbial compositions are associated with asthma. Host genetics might shape nasal microbiome composition.We examined associations between host genetics and nasal microbiome composition.Nasal samples were collected from 584 participants from the Mount Sinai Health System, New York. Seventy-seven follow-up samples were collected from a subset of 40 participants. 16S rRNA sequencing and RNA sequencing were performed on nasal samples. Beta diversity was calculated, variant calling on RNA sequencing data was performed, and genetic relatedness between individuals was determined. Using linear regression models, we tested for associations between genetic relatedness and nasal microbiome composition.The median age of the cohort was 14.6 (interquartile range 11.2-19.5) years, with participants representing diverse ancestries and 52.7% of the cohort being female. For participants who provided follow-up samples, the median time between samples was 5.1 (interquartile range 1.4-7.2) months. Nasal microbiome composition similarity as reflected by beta diversity was significantly higher within subjects over time versus between subjects (coefficient = 0.091, P = 2.84sup-/supsup7/sup). There was no significant association between genetic relatedness and beta diversity (coefficient = -0.05, P = .29). Additional analyses exploring the relationship between beta diversity and genetic variance yielded similar results.Host genetics has little influence on nasal microbiome composition.

Background Household endotoxin levels have been variably associated with risk for asthma and atopy. Methods We studied participants from the 2005-2006 National Health and Nutrition Examination Survey (NHANES, n = 6963), a large cohort representative of the US population (aged 1-84 years). We built logistic regression models to test for associations between house dust endotoxin and sensitization to specific foods (milk, egg, and peanut). To experimentally explore the detected epidemiologic associations, peripheral blood mononuclear cells (PBMCs) were collected from 21 children (aged 1-19 years) mono-food allergic (ie, sensitized and clinically reactive) to milk, egg, or peanut and nonallergic controls for stimulation with endotoxin and secreted cytokine measurement. For each food allergy, linear mixed-effects models were built to test the association between endotoxin stimulation and cytokine level. Results Among NHANES subjects, the geometric mean household endotoxin level was 15.5 EU/mg (GSE 0.5). Prevalence of food allergen sensitization (sIgE ≥ 0.35 kUA /L) varied by food: milk 5.7%, egg 4.0%, and peanut 7.9%. In models adjusted for potential confounders (age, race, country of birth, total people per household, US region, and history of wheezing in the past year), household endotoxin level was associated with sensitization to milk (OR 1.7, 95% CI 1.2-2.1) and egg (OR 1.4, 95% CI 1.01-1.9), but not peanut (OR 0.98, 95% CI 0.8-1.2). Interferon-γ levels of endotoxin-stimulated PBMCs from children allergic to milk or egg, but not peanut, were significantly lower compared to controls in linear mixed-effects models adjusted for repeated measures, experimental variables, age, and inter-individual variability (P-values .007, .018, and .058, respectively). Conclusion Higher household endotoxin is associated with increased odds of milk and egg sensitization. Altered cytokine responsiveness to endotoxin is also observed in PBMCs from individuals with milk and egg allergy.

Biological and regulatory mechanisms underlying many multi-gene expression-based disease biomarkers are often not readily evident. We describe an innovative framework, NeTFactor, that combines network analyses with gene expression data to identify transcription factors (TFs) that significantly and maximally regulate such a biomarker. NeTFactor uses a computationally-inferred context-specific gene regulatory network and applies topological, statistical, and optimization methods to identify regulator TFs. Application of NeTFactor to a multi-gene expression-based asthma biomarker identified ETS translocation variant 4 (ETV4) and peroxisome proliferator-activated receptor gamma (PPARG) as the biomarker’s most significant TF regulators. siRNA-based knock down of these TFs in an airway epithelial cell line model demonstrated significant reduction of cytokine expression relevant to asthma, validating NeTFactor’s top-scoring findings. While PPARG has been associated with airway inflammation, ETV4 has not yet been implicated in asthma, thus indicating the possibility of novel, disease-relevant discovery by NeTFactor. We also show that NeTFactor’s results are robust when the gene regulatory network and biomarker are derived from independent data. Additionally, our application of NeTFactor to a different disease biomarker identified TF regulators of interest. These results illustrate that the application of NeTFactor to multi-gene expression-based biomarkers could yield valuable insights into regulatory mechanisms and biological processes underlying disease.

BACKGROUND: In a recent trial of milk oral immunotherapy (MOIT) with or without omalizumab in 55 patients with milk allergy treated for 28 months, 44 of 55 subjects passed a 10-g desensitization milk protein challenge; 23 of 55 subjects passed the 10-g sustained unresponsiveness (SU) challenge 8 weeks after discontinuing MOIT. OBJECTIVE: We sought to determine whether IgE and IgG(4) antibody binding to allergenic milk protein epitopes changes with MOIT and whether this could predict the development of SU. METHODS: By using a novel high-throughput Luminex-based assay to quantitate IgE and IgG(4) antibody binding to 66 sequential epitopes on 5 milk proteins, serum samples from 47 subjects were evaluated before and after MOIT. Machine learning strategies were used to predict whether a subject would have SU after 8 weeks of MOIT discontinuation. RESULTS: MOIT profoundly altered IgE and IgG(4) binding to epitopes, regardless of treatment outcome. At the initiation of MOIT, subjects achieving SU exhibited significantly less antibody binding to 40 allergenic epitopes than subjects who were desensitized only (false discovery rate ≤ 0.05 and fold change > 1.5). Based on baseline epitope-specific antibody binding, we developed predictive models of SU. Using simulations, we show that, on average, IgE-binding epitopes alone perform significantly better than models using standard serum component proteins (average area under the curve, >97% vs 80%). The optimum model using 6 IgE-binding epitopes achieved a 95% area under the curve and 87% accuracy. CONCLUSION: Despite the relatively small sample size, we have shown that by measuring the epitope repertoire, we can build reliable models to predict the probability of SU after MOIT. Baseline epitope profiles appear more predictive of MOIT response than those based on serum component proteins.

BACKGROUND: Pet allergies are common in children with asthma. Microbiota and host responses may mediate allergen sensitization. OBJECTIVE: To uncover host-microbe relationships in pet allergen sensitization via joint examination of the nasal microbiome and nasal transcriptome. METHODS: We collected nasal samples from 132 children with asthma for parallel 16S rRNA and RNA sequencing. Specific IgE levels for cat and dog dander were measured. Analyses of the nasal microbiome, nasal transcriptome, and their correlations were performed with respect to pet sensitization status. RESULTS: Among the 132 children, 91 (68.9%) were cat sensitized and 96 (72.7%) were dog sensitized. Cat sensitization was associated with lower nasal microbial diversity by Shannon Index (P=0.021) and differential nasal bacterial composition by weighted UniFrac distance (PERMANOVA P=0.035). Corynebacterium sp. and Staphylococcus epidermidis were significantly less abundant, and the metabolic process “fatty acid elongation in mitochondria” was lower in pet sensitized vs. unsensitized children. Correlation networks revealed that the nasal expression levels of 47 genes representing inflammatory processes were negatively correlated with the relative abundances of Corynebacterium sp. and Staphylococcus epidermidis. Thus, these species were not only directly associated with the absence of pet sensitization, but also with the under-expression of host gene expression of inflammatory processes that contribute to allergen sensitization. Causal mediation analyses revealed that the associations between these nasal species and pet sensitization were mediated by nasal gene expression. CONCLUSION: Higher abundances of nasal Corynebacterium sp. and Staphylococcus epidermidis are associated with absence of pet sensitization and correlate with lower expression of inflammatory genes. CAPSULE SUMMARY: Nasal microbiota and nasal gene expression vary by pet sensitization status. Higher abundances of nasal Corynebacterium sp. and Staphylococcus epidermidis are associated with absence of pet sensitization and correlate with lower host expression of inflammatory genes.

BackgroundMisdiagnosis of peanut allergy is a significant clinical challenge. Here, a novel diagnostic blood-based test using a Bead-Based Epitope Assay (“peanut BBEA”) has been developed on the LEAP cohort and then independently validated on the CoFAR2 and POISED cohorts.MethodsDevelopment of the peanut BBEA followed the National Academy of Medicine’s established guidelines with discovery performed on 133 subjects from the non-interventional arm of the LEAP trial and an independent validation performed on 81 subjects from the CoFAR2 study and 84 subjects from the POISED study. All subject samples were analyzed using the BBEA methodology. The peanut BBEA test measures levels of two Ara h 2 epitopes and compares their combination to a pre=specified threshold. If the combination of the two epitope levels is at or below the threshold, then the subject is ruled “Not Allergic”, otherwise the subject is ruled “Allergic”.All allergic diagnoses were OFC confirmed and subjects’ ages were 7-55 years.ResultsIn validation on the CoFAR2 and POISED cohorts, the peanut BBEA test had a combined sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy of 91%, 95%, 95%, 91%, 18.2, 0.09 and 93%, respectively.ConclusionThe peanut BBEA test performance in validation demonstrated overall high accuracy and compared very favorably with existing diagnostic tests for peanut allergy including skin prick testing, peanut sIgE and peanut component testing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen specific IgE-sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless specificity and sensitivity as well as a simultaneous detection of allergen specific IgG4, as a potential parameter for tolerance development, remain to be optimized. Objective We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen specific IgE and IgG4, and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intra- slide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. Methods Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4. A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4-specific fluorescence on silicon microarrays. Results Allergen specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg specific IgE was comparable to the range of 0.1 to >100 kUA/l of the ImmunoCAP system, whereas for total cow's milk the silicon microarray was less sensitive. Detectable allergen specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. Conclusions We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research. This article is protected by copyright. All rights reserved.

Background The consumption of lentil is common in the Mediterranean area and is one of the causes of IgE-mediated food allergy in many countries. Len c 1 is a well-defined allergen of lentil and approximately 80% of the patients with lentil allergy recognize the purified Len c 1 protein. We sought to identify IgE and IgG4 sequential epitopes of Len c 1 in patients with red and/or green lentil allergy. We also aimed to determine IgE and IgG4 binding differences between those patients who had outgrown or remained reactive to lentil. Methods Children with IgE-mediated lentil allergy were included in the study. We applied a microarray immunoassay to determine the characterization of positive IgE and IgG4 binding to Len c 1 epitopes in the patients' sera. Results The peptides specifically recognized by IgE and IgG4 antibodies were mainly detected between peptides 107 and 135 of Len c 1. The signal intensities of positive epitopes were significantly greater in reactive patients than tolerant ones (P = .008 for IgE and P = .002 for IgG4). Moreover, IgE and IgG4 antibodies bound largely the same sequential epitopes in patients who remained reactive or outgrew their allergy. Conclusion IgG4-binding epitopes in lentil allergy were identified and IgE and IgG4 binding to epitopes in both red and green lentils was compared. Our data regarding signal intensity differences between reactive and outgrown patients and overlap binding of IgE and IgG4 antibodies may be important for the development of more accurate diagnostic tests and understanding of natural tolerance development. David H. and Julia Koch Research Program in Food Allergy Therapeutics This study was supported in part by the David H. and Julia Koch Research Program in Food Allergy Therapeutics. WOS:000493798700001 31598994 Q1

SUMMARY Although clinical and laboratory data have long been used to guide medical practice, this information is rarely integrated with multi-omic data to identify endotypes. We present Merged Affinity Network Association Clustering (MANAclust), a coding-free, automated pipeline enabling integration of categorical and numeric data spanning clinical and multi-omic profiles for unsupervised clustering to identify disease subsets. Using simulations and real-world data from The Cancer Genome Atlas, we demonstrate that MANAclust’s feature selection algorithms are accurate and outperform competitors. We also apply MANAclust to a clinically and multi-omically phenotyped asthma cohort. MANAclust identifies clinically and molecularly distinct clusters, including heterogeneous groups of “healthy controls” and viral and allergy-driven subsets of asthmatic subjects. We also find that subjects with similar clinical presentations have disparate molecular profiles, highlighting the need for additional testing to uncover asthma endotypes. This work facilitates data-driven personalized medicine through integration of clinical parameters with multi-omics. MANAclust is freely available at https://bitbucket.org/scottyler892/manaclust/src/master/., Graphical abstract, In brief Clinical data commonly used in medical practice are underutilized in multi-omic analyses to identify disease endotypes. Tyler et al. present a python package called Merged Affinity Network Association Clustering (MANAclust) that automatically processes and integrates categorical and numeric data types, facilitating the inclusion of clinical data in multi-omic endotyping efforts.

Background DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) is a C-type lectin receptor expressed on macrophages and dendritic cells. DC-SIGN has high affinity for fucosylated glycans in several plant glycoproteins and pathogens. DC-SIGN is thought to be crucial for the development of allergic sensitization. However, the precise role of DC-SIGN in food allergy pathogenesis is not yet understood. Objective We sought to characterize DC-SIGN-binding glycoproteins in a panel of allergenic and non-allergenic foods. Methods Fluorescent-labeled peanut and soy extracts were used to test protein binding to human monocyte-derived dendritic cells (DCs) by flow cytometry. DC-SIGN-blocking assays were performed by incubating DCs with food extracts followed by staining with anti-DC-SIGN antibody. Using a DC-SIGN-Fc chimera, food extracts were tested for binding by ELISA and autoradiography. IgE immunoblotting was performed with pooled sera from food-allergic subjects. DC activation and maturation were assessed by flow cytometry. Results and conclusions We demonstrate that peanut agglutinin, a minor peanut allergen, is a novel ligand for DC-SIGN. Peanut agglutinin activates DCs to induce the expression of costimulatory molecules in vitro. We present a comprehensive report on the characterization of DC-SIGN-binding proteins in common allergenic foods such as peanut, soy, tree nuts, egg, and milk. Foods that rarely induce allergy, such as pine nuts, chickpea, and corn, showed no binding to DC-SIGN. Several DC-SIGN-binding proteins show reactivity in serum IgE immunoblots. We have also identified novel non-IgE-binding proteins that interact with DC-SIGN; these proteins may be important for regulating immune responses to these foods.

Kiwifruit allergy has been described mostly in the adult population, but immunoglobulin (Ig)E-mediated allergic reactions to kiwifruit appear to be occurring more frequently in children. To date, 13 allergens from kiwifruit have been identified. Our aim was to identify kiwifruit allergens in a kiwifruit allergic-pediatric population, describing clinical manifestations and patterns of recognition. Twenty-four children were included. Diagnosis of kiwifruit allergy was based on compatible clinical manifestations and demonstration of specific IgE by skin prick test (SPT) and/or serum-specific IgE determination. SDS-PAGE and immunoblotting were performed with kiwifruit extract, and proteins of interest were further analyzed by mass spectrometry/mass spectrometry. For component-resolved in vitro diagnosis, sera of kiwifruit-allergic patients were analyzed by an allergen microarray assay. Act d 1 and Act d 2 were bound by IgE from 15 of 24 children. Two children with systemic manifestations recognized a protein of 15 kDa, homologous to Act d 5. Act d 1 was the allergen with the highest frequency of recognition on microarray chip, followed by Act d 2 and Act d 8. Kiwifruit allergic children develop systemic reactions most frequently following ingestion compared to adults. Act d 1 and Act d 2 are major allergens in the pediatric age group.

Background The majority of children with cow's milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patient's IgE and IgG4-binding pattern to milk protein epitopes could distinguish clinical reactivity. Methods Four groups of reactivity were identified by Oral food challenge: baked milk reactive, fermented milk reactive, whole milk reactive, and outgrown. sIgE and sIgG4 binding to milk protein epitopes were assessed with a novel Luminex-based peptide assay (LPA). Using machine learning techniques, a model was developed to predict different degrees of CMA. Results The baked milk reactive patients demonstrated the highest degree of IgE epitope binding, which was followed sequentially by fermented milk reactive, whole milk reactive, and outgrown. Data were randomly divided into two groups with 75% of the data utilized for model development (n = 68) and 25% for testing (n = 21). All 68 children used for training were correctly classified with models using IgE and IgG4 epitopes. The average cross-validation accuracy was much higher for models using IgE plus IgG4 epitopes by LPA (84.8%), twice the performance of the serum component proteins assayed by UniCAP (41.9%). The performance of the model on "unseen data" was tested using the 21 withheld patients, and the accuracy of IgE was 86% (AUC = 0.89) while of IgE+IgG4 model was 81% (AUC = 0.94). Conclusion Using a novel high-throughput LPA, we were able to distinguish the diversity of IgE/IgG4 binding to epitopes in the varying CMA phenotypes. LPA is a promising tool to predict correctly different degrees of CMA.

Two-dimensional polyacrylamide gel electrophoresis (2DE) and Western immunoblotting have proven to be invaluable and fundamental techniques for comprehensive characterization of food allergens. Here, we describe and discuss detailed protocols used in our studies on identification of allergenic proteins in shrimp, sesame, hazelnut, and pistachio.

This work aimed to identify the IgE epitopes of the allergenic whey protein β-lactoglobulin (β-LG) following in vitro gastrointestinal digestion using human (HF) or simulated digestive fluids (SF). The IgE-binding of the digests was evaluated by inhibition ELISA with sera from milk allergic patients and the low molecular weight digestion products were identified by RP-HPLC-MS/MS. These peptides were then chemically synthesized and analyzed by a peptide microarray immunoassay. β-LG resisted digestion during the gastric phase, while no residual β-LG was observed at the end of the duodenal phase. The immunoreactivity of the digests was consistent with the levels of intact β-LG present, showing almost negligible IgE binding after gastrointestinal digestion with both systems. There were similarities in the peptide patterns produced, showing, respectively, 21 and 18 identical peptides in the gastric and duodenal digests. Digestion products related to five high-frequency IgE-binding synthetic peptides: β-LG (43-60), β-LG (47-62), β-LG (86-99), β-LG (86-100) and β-LG (135-147), which were released upon in vitro digestion, particularly with SF. These matched two very immunoreactive areas of the protein: 43-68 and 122-146, as mapped using a library of 36 20-amino acid peptides corresponding to the primary sequence of β-LG. However, no IgE-binding peptides comprising the residues 86-100 were detected by analyzing the 36-peptide library by microarray immunoassay. © 2014., This work was supported by the project Ministerio de Economía y Competitividad de España AGL2011-24740. The serum bank from Mount Sinai Medical Center (New York, NY) is supported by the Food Allergy Research and Education organization. S.B., I.L.-E and E.M are participants of COST-Infogest FA 1005. S. B and I. L-E acknowledge the financial support of CSIC through JAE Pre (nº JAEPre094) and JAE Doc (nº JAEDOC072) grants.

Background Nasal microbiota may influence asthma pathobiology. Objective We sought to characterize the nasal microbiome of subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls to identify nasal microbiota associated with asthma activity. Methods We performed 16S ribosomal RNA sequencing on nasal swabs obtained from 72 primarily adult subjects with exacerbated asthma (n = 20), nonexacerbated asthma (n = 31), and healthy controls (n = 21). Analyses were performed using Quantitative Insights into Microbial (QIIME); linear discriminant analysis effect size (LEfSe); Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; and Statistical Analysis of Metagenomic Profiles (PICRUSt); and Statistical Analysis of Metagenomic Profiles (STAMP). Species found to be associated with asthma activity were validated using quantitative PCR. Metabolic pathways associated with differentially abundant nasal taxa were inferred through metagenomic functional prediction. Results Nasal bacterial composition significantly differed among subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls (permutational multivariate ANOVA, P = 2.2 × 10 −2 ). Relative to controls, the nasal microbiota of subjects with asthma were enriched with taxa from Bacteroidetes (Wilcoxon-Mann-Whitney, r = 0.33, P = 5.1 × 10 −3 ) and Proteobacteria ( r = 0.29, P = 1.4 × 10 −2 ). Four species were differentially abundant based on asthma status after correction for multiple comparisons: Prevotella buccalis , P adj = 1.0 × 10 −2 ; Dialister invisus , P adj = 9.1 × 10 −3 ; Gardnerella vaginalis , P adj = 2.8 × 10 −3 ; Alkanindiges hongkongensis , P adj = 2.6 × 10 −3 . These phyla and species were also differentially abundant based on asthma activity (exacerbated asthma vs nonexacerbated asthma vs controls). Quantitative PCR confirmed species overrepresentation in asthma relative to controls for Prevotella buccalis (fold change = 130, P = 2.1 × 10 −4 ) and Gardnerella vaginalis (fold change = 160, P = 6.8 × 10 −4 ). Metagenomic inference revealed differential glycerolipid metabolism (Kruskal-Wallis, P = 1.9 × 10 −4 ) based on asthma activity. Conclusions Nasal microbiome composition differs in subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls. The identified nasal taxa could be further investigated for potential mechanistic roles in asthma and as possible biomarkers of asthma activity.

Background Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. Few options are available for treatment, and avoidance is the only therapy recommended. Objective We sought to identify relevant crustacean allergens for use as diagnostic and safe immunotherapeutic agents for subjects with shellfish allergy. Methods Thirty-eight patients were selected with immediate allergic reactions to shrimp and increased shrimp-specific serum IgE levels. One-dimensional and 2-dimensional electrophoresis of shrimp extracts were followed by IgE immunoblotting. Protein identification was done with matrix-assisted laser desorption/ionization–mass spectrometry and Edman sequencing. A cDNA library was generated from white pacific shrimp (Litopenaeus vannamei) and screened with primers designed on the basis of internal sequences obtained from 2-dimensional tryptic digests. Full-length cDNA clones were isolated from the library and sequenced. Recombinant protein was expressed and tested with sera from patients with shrimp allergy. Results Immunoblotting demonstrated IgE binding to a 20-kDa shrimp protein by 21 (55%) of 38 sera. Tryptic digestion of the protein followed by matrix-assisted laser desorption/ionization–mass spectrometric analysis and Edman sequencing identified it as a myosin light chain (MLC). Screening of the shrimp cDNA library resulted in isolation of a novel protein cDNA. Open reading frame translation provided the amino acid sequence of a new allergenic shrimp protein with high similarity to Bla g 8 (cockroach MLC). Recombinant protein was recognized by 17 patients, confirming the allergenicity of shrimp MLC. Conclusions We have identified and cloned a new major shrimp allergen, Lit v 3.0101, an MLC protein.

Background: Sesame seed allergy is becoming increasingly prevalent, probably because of its use in international fast-food and bakery products. Despite this fact, few studies have focused on the identification of its major allergenic proteins. Objective: The aim of this study was to identify allergenic proteins of sesame seeds (Sesamum indicum) . Methods: Extracted sesame seed proteins were separated by means of SDS-PAGE and 2-dimensional (2-D) PAGE. Immunolabeling was performed with individual patient sera from 20 patients with sesame seed allergy. Selected proteins were further analyzed by means of Edman sequencing. Results: IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Analyzing internal sequences, the protein at 45 kd, which was recognized by 75% of the patients, was found to be a 7S vicilin-type globulin, a seed storage protein of sesame and named Ses i 3. The protein at 7 kd was found to be a 2S albumin, another seed storage protein of sesame and named Ses i 2. Seed storage proteins are known food allergens in peanut, walnut, Brazil nut, and soybean. Interestingly, one known IgE-binding epitope of the peanut allergen Ara h 1 has 80% homology with the corresponding area of Ses i 3. The different amino acids were previously shown not to be critical for IgE binding in Ara h 1. In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively. Conclusion: The identification of 4 sesame seed allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE binding epitopes in common food allergens might be a useful tool for predicting cross-reactivity to certain foods. (J Allergy Clin Immunol 2002;110:154-9.)

Background The diagnosis of shellfish allergy remains a challenge for clinicians. Several shellfish allergens have been characterized and their IgE epitopes identified. However, the clinical relevance of this sensitization is still not clear. Objective The objective of this study was to identify allergens and epitopes associated with clinical reactivity to shrimp. Methods Shrimp-sensitized subjects were recruited and grouped based on the history of shrimp-allergic reactions and challenge outcome. IgE reactivity to recombinant crustacean allergens, and IgE and IgG4 reactivity to peptides were determined. Subjects sensitized to dust mites and/or cockroach without shrimp sensitization or reported allergic reactions, as well as nonatopic individuals, were used as controls. Results A total of 86 subjects were recruited with a skin prick test to shrimp; 74 reported shrimp-allergic reactions, 58 were allergic (38 positive double-blind placebo-controlled food challenge and 20 recent anaphylaxis), and 16 were tolerant. All subjects without a history of reactions had negative challenges. The individuals with a positive challenge more frequently recognized tropomyosin and sarcoplasmic calcium-binding proteins than those found tolerant by the challenge. Especially a sarcoplasmic-calcium-binding-protein positive test is very likely to result in a positive challenge, though the frequency of recognition is low. Subjects with dust mite and/or cockroach allergy not sensitized to shrimp recognized arginine kinase and hemocyanin. Several epitopes of these allergens may be important in predicting clinical reactivity. Conclusion Tropomyosin and sarcoplasmic-calcium-binding-protein sensitization is associated with clinical reactivity to shrimp. Myosin light chain testing may help in the diagnosis of clinical reactivity. Arginine kinase and hemocyanin appear to be cross-reacting allergens between shrimp and arthropods. Detection of IgE to these allergens and some of their epitopes may be better diagnostic tools in the routine workup of shrimp allergy.

Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitisassociated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life., The work was supported by NIH grants AI044236 (to H.A. Sampson and M.C. Berin), AI093577 (to M.C. Berin), and AI091655 (to K.M. Järvinen) and Environmental Protection Agency grant R834064 (to M.C. Berin). Clinical specimens were provided by the Jaffe Food Allergy Resource Initiative, funded by Food Allergy Research and Education.

Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cow's milk allergen β-casein (β-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. β-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: β-CN (57-68) and β-CN (82-93), which matched fragments released by β-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests. © 2013 Elsevier Ltd. All rights reserved., This work was funded by the project AGL2011-24740. S.B, I.L-E and E.M are participants of COST-Infogest FA 1005 and CYTED-IBEROFUN 110AC0386 projects. S. B and I. L-E acknowledge the financial support of CSIC through JAE Pre and JAE Doc grants.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain. An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP. Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.

Russian Abstract: В учебно-методическом пособии, входящем в серию «Инновационные социальные технологии», рассматриваются научно-практические аспекты становления и развития в России социальных инновационных технологий в области социального управления, конкретные механизмы проведения общественной экспертизы государственно значимых решений (с учетом гендерного подхода) с применением информационных технологий ситуационного центра Методика проведения экспертизы адресована преподавателям, готовящем кадры для системы государственного управления, студентам ВУЗов, а также может быть использована для экспертизы законопроектов государственными гражданскими и муниципальными служащими, независимыми общественными экспертами. English Abstract: The book is part of the series "Innovative social technologies." In it, the authors consider the theoretical and practical aspects of social development in Russia of innovative technologies in the field of social management. The authors have developed mechanisms of public expertise of state and significant decisions (including gender mainstreaming) using information technology situation center. The technique of expertise is addressed to teachers of the civil servants, as well as university students. The technique of expertise may use civil servants and independent experts for the public examination of draft laws and other official documents.

Summary Background IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. Objective We attempted to identify pistachio allergens and to clone the encoding genes. Methods Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. Results Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. Conclusion Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.

Background Shellfish allergy is a long-lasting disorder usually persisting throughout life. Despite its high prevalence, there is limited information about allergenic shrimp proteins. Objective Characterization of shrimp allergens. Methods Fifty-two adults and children with a history of immediate allergic reactions to shrimp and elevated serum IgE to shrimp were selected for this study. Tryptic digests from a 20-kd IgE-binding protein were analyzed by LC-MS/MS, identifying the protein as a sarcoplasmic-calcium-binding protein. cDNA encoding sarcoplasmic calcium-binding protein (SCP) from a shrimp cDNA library (Litopenaeus vannamei) was amplified by PCR, cloned into an expression vector, and sequenced. Recombinant SCP was tested with patients' sera. ELISA inhibition experiments determined the fraction of total shrimp IgE recognizing SCP. A functional assay with a rat basophilic leukemia cell line was used to determine the capacity for mediator release induced by SCP. Results Immunoblotting demonstrated IgE binding by 31 of 52 (59.6%) of the sera to a 20-kd shrimp protein. The protein was identified as a SCP. Amplified cDNA encoding SCP was isolated and sequenced. Open reading frame translation provided the complete amino acid sequence of shrimp SCP. Recombinant SCP was recognized by serum IgE from 20 of 52 (38.4%) subjects, of whom 17 of 20 (85%) were children. ELISA inhibition of pooled sera IgE reactivity to BS extract using recombinant SCP was significant (as high as 79%). For some subjects, mediator release induced by recombinant SCP was higher than that induced by recombinant tropomyosin. Conclusion We have identified and cloned a new shrimp allergen, Lit v 4.0101, an SCP, which appears to be of particular importance in the pediatric population.

S A T U R D A Y 107 Soy Reactivity May be Better Identified By Component Testing with Gly m 8 Than Traditional Testing Methods Jacob D. Kattan, MD, Hugh A. Sampson, MD; Icahn School of Medicine at Mount Sinai, New York, NY, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, USA. RATIONALE: Skin prick testing and serum food-specific IgE (sIgE) levels are sensitive tests for identifying soy allergy, but positive test results are common even when soy is tolerated. We sought to perform soy component testing on patients undergoing an oral food challenge (OFC) to soy to determine if this modality would improve predictability. METHODS: We recruited children in our university-based, outpatient practice referred for OFC. Challenge outcomes were compared with soy sIgE levels and the sIgE levels to the soy components Gly m 4, 5, 6, and 8. IRB approval was obtained for this study. RESULTS: We performed component testing on 16 patients, ages 4 to 19 years, who underwent an OFC to soy. Six patients passed theOFC (37.5%), while 10 failed (62.5%). The median soy-sIgE in the patients who passed OFC (16.5 kUA/L) was lower, but not significantly different from those who failed (31.9 kUA/L). The median Gly m 4, 5, and 6 levels were not significantly different between those with a negative OFC (0.16, 8.95, and 15.05 kUA/L respectively) and the soy reactive patients (0.40, 18.20, and 25.85 kUA/L respectively). There were significant differences in the median Gly m 8 levels between patients who passed their soy OFC (2.47 kUA/L) and the soy reactive patients (5.03 kUA/L; P50.023). CONCLUSIONS: Soy component testing using the component Gly m 8 may improve the specificity of soy allergy testing. Further testing is planned with a larger number of soy sensitive patients to validate the utility of soy component testing.

FEBRUARY 2015 AB36 Abstracts S A T U R D A Y 110 A New Luminex-Based Peptide Assay to Identify Different Degrees of Milk Allergic Reactivity Cansin Sackesen, MD, Jing Lin, PhD, Stephanie Schmidt, BSc, Robert C. Getts, PhD, Jim Kadushin, PhD, Gustavo Gimenez, MSc, Ebru Arik Yilmaz, MD, Ozlem Cavkaytar, MD, Ozge Soyer, MD, Galina Grishina, MSc, Ludmilla Bardina, MSc, Hugh A. Sampson, MD; Icahn School of Medicine at Mount Sinai, New York, NY, Hacettepe University School of Medicine, Ankara, Turkey, Genisphere, LLC, PA, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, USA. RATIONALE: A novel Luminex-based peptide assay (LPA) was used to identify IgE binding to allergenic epitopes in milk-allergic Turkish children who tolerated different forms of milk products in oral food challenges. We sought to establish whether LPA results could distinguish patients’ clinical reactivity to different forms of milk, e.g. baked-milk (muffin), yoghurt-cheese, and whole unprocessed milk. METHODS: Four groups of milk allergic children were identified by oral food challenge to muffin, yoghurt-cheese and whole milk: 1.Reactive to baked-milk (n520), 2.Reactive to yoghurt-cheese/tolerant to muffin (n517), 3.Reactive to milk/tolerant to muffin and yoghurt-cheese (n523), and 4.Outgrown milk allergy (n532). Milk, casein, and b-lactoglobulin sIgE and sIgG4 were determined by UniCAP;IgE and IgG4 binding to milk protein epitopes were assessed by LPA. RESULTS: Children reactive to baked-milk had the highest milk, casein and b-lactoglobulin sIgE followed by children reactive to yoghurt-cheese. In analyzing children with positive milk protein epitope-specific binding, LPA revealed the most diverse binding patterns of IgE in the baked-milk reactive group: as1-casein (12-peptides), as2-casein (8-peptides), b-casein (8-peptides), b-lactoglobulin (3-peptides) and k-casein (3-peptides). Binding of IgE was observed only in as1-casein and as2-casein regions in children reactive to yoghurt-cheese and there was no IgE binding to any peptides in groups #3 and #4. IgG4 binding patterns were similar in all four groups. CONCLUSIONS: Using a novel high-through-put LPA, we were able to successfully distinguish the reactivity and diversity of IgE binding to allergenic epitopes in the most severe milk allergy phenotypes. This assay may be useful in distinguishing different degrees of milk allergy.

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg), but not deglycosylated PNAg, activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation, and by their ability to drive T cell proliferation. Furthermore, PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg, we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner, and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs, consistent with previous reports on the effect of Th2 adjuvants on DCs.

Background : Hazelnuts are a common cause of food allergy. Allergic reactions to hazelnuts range from oral allergy syndrome caused by cross-reactivity between tree pollen and hazelnut proteins to severe anaphylactic reactions. Little information is available regarding the identification of pollen-independent hazelnut allergens. Objective : The aim of the study was to identify these pollen-independent allergens in patients with hazelnut allergy with systemic reactions. Methods : Extracted hazelnut proteins were separated by means of 2-dimensional PAGE, and immunolabeling was performed with individual sera from 14 patients with hazelnut-induced systemic reactions. Edman sequencing was performed on a 40-kd protein identified as an allergen. In parallel, RNA isolated from hazelnuts was used to construct a cDNA library. By using the peptide sequence data, oligonucleotide primers were synthesized and used to screen the library. Full-length cDNA clones were isolated, sequenced, expressed, and screened with patient sera. Results : By using 2-dimensional proteomics, a protein fraction at 40 kd was recognized by serum IgE from 86% (12/14) of the patients with hazelnut allergy with systemic reactions. Two internal amino acid sequences were determined by means of Edman sequencing. Screening of the prepared hazelnut cDNA library with oligonucleotides based on these internal peptide sequences resulted in isolation of a novel protein cDNA. The new protein, named Cor a 9, belongs to the 11S globulin seed storage protein family. This family comprises known food allergens in peanut (Ara h 3) and soybean (glycine max). The pairwise homology among these 3 proteins ranges from 45% to 50%. Interestingly, one known IgE-binding epitope of Ara h 3 shares 67% of homologous amino acid residues with the corresponding area of Cor a 9. The amino acids that differ were previously shown not to be critical for IgE binding in Ara h 3. Conclusion : Cor a 9 is the first tree pollen-unrelated hazelnut allergen isolated, sequenced, and cloned. The identification of food allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE epitopes in common food allergens, such as seed storage proteins, might be a useful tool for predicting cross-reactivity to certain foods. (J Allergy Clin Immunol 2002;110:517-23.)

G protein alpha subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous alphai2 residues for four alphas residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of alphas to be activated by the beta-adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of alphas for the beta-adrenergic receptor, based on an increased amount of high affinity binding of the beta-adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the beta-adrenergic receptor to increase the apparent affinity of alphas for the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous alphai2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.