Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free ¹⁵N-labeled antigen whose ¹H-¹⁵N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.     

Identification and fine mapping of IgG and IgE epitopes in ovomucoid

Ovomucoid is a major allergen in hen egg white which causes a serious IgE-mediated food allergy reaction. This study determined eight IgG epitopes, 5-11 amino acids in length, and nine IgE epitopes, 5-16 amino acids in length, within the primary sequence in ovomucoid using arrays of overlapping peptides synthesized on cellulose membranes. Pooled sera from eight egg-allergic patients were used to probe the membrane. We also analyzed the amino acids that are critical for antibody binding by substituting a single amino acid within each epitope. Mutational analysis of the epitopes indicated that charged amino acids (aspartic acid, glutamic acid, and lysine) and some hydrophobic (leucine, phenylalanine, and glycine) and polar (serine, threonine, tyrosine, and cystein) amino acids were important for antibody binding. These results provide useful information for the molecular design necessary to reduce the allergenicity of ovomucoid, and a better understanding of structure-function relationships of allergic epitopes in food proteins.     

Monoclonal antibody mapping of structural and functional plectin epitopes

To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association.     

Fine mapping and replication of QTL in outbred chicken advanced intercross lines

Background

Linkage mapping is used to identify genomic regions affecting the expression of complex traits. However, when experimental crosses such as F₂ populations or backcrosses are used to map regions containing a Quantitative Trait Locus (QTL), the size of the regions identified remains quite large, i.e. 10 or more Mb. Thus, other experimental strategies are needed to refine the QTL locations. Advanced Intercross Lines (AIL) are produced by repeated intercrossing of F₂ animals and successive generations, which decrease linkage disequilibrium in a controlled manner. Although this approach is seen as promising, both to replicate QTL analyses and fine-map QTL, only a few AIL datasets, all originating from inbred founders, have been reported in the literature.

Methods

We have produced a nine-generation AIL pedigree (n = 1529) from two outbred chicken lines divergently selected for body weight at eight weeks of age. All animals were weighed at eight weeks of age and genotyped for SNP located in nine genomic regions where significant or suggestive QTL had previously been detected in the F₂ population. In parallel, we have developed a novel strategy to analyse the data that uses both genotype and pedigree information of all AIL individuals to replicate the detection of and fine-map QTL affecting juvenile body weight.

Results

Five of the nine QTL detected with the original F₂ population were confirmed and fine-mapped with the AIL, while for the remaining four, only suggestive evidence of their existence was obtained. All original QTL were confirmed as a single locus, except for one, which split into two linked QTL.

Conclusions

Our results indicate that many of the QTL, which are genome-wide significant or suggestive in the analyses of large intercross populations, are true effects that can be replicated and fine-mapped using AIL. Key factors for success are the use of large populations and powerful statistical tools. Moreover, we believe that the statistical methods we have developed to efficiently study outbred AIL populations will increase the number of organisms for which in-depth complex traits can be analyzed.     

In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene.

The chicken progesterone receptor (cPR) gene and the ovalbumin (OA) gene, a target of cPR regulation, have been mapped via fluorescent in situ hybridization to the two largest chromosomes of the chicken karyotype. cPR is subtelomeric on the long arm of chromosome 1 and OA is on the long arm of chromosome 2, close to the centromere. A 35-kb cosmid probe for the cPR gene and two genomic fragments of 9.2 and 15 kb for the OA gene were biotin-labeled for nonradioactive localization of the two chicken loci.     

Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus.

Sera from virtually all individuals infected with human immunodeficiency virus contain antibodies against the viral envelope glycoproteins. By using a series of synthetic peptide antigens, we identified an immunodominant domain at amino acid position 598-609 of gp41. The minimal essential epitope is a 7-amino-acid sequence (amino acids 603-609) containing two cysteine residues. Both cysteine residues are required for the antigenic conformation of the sequence, possibly due to creation of a cyclic structure via disulfide bond formation.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene.

Double-stranded DNA molecules complementary to ovalbumin chicken messenger RNA were synthesized in vitro and integrated into the E. coli plasmid pCR1 using an oligodG-dc tailing procedure. The resultant hybrid plasmids, amplified by transfection of E. coli, were shown by hybridization and gel electrophoresis to contain extensive DNA sequences of the ovalbumin structural gene.     

Fine mapping of the chicken congenital loco locus on chromosome 12

Congenital loco in chicks is characterized by an apparent lack of control of the muscles of the neck. This disorder is inherited as a simple Mendelian recessive disease, caused by an autosomal recessive gene, lo. To date, there are no reports on the localization of this gene. The objective of this study was therefore to identify the genomic region of the lo locus. The experimental congenital loco population used here were selected from a Rhode Island Red (RIR) line and consisted of six generations, resulting in 124 chickens. A total of 113 DNA samples from offspring of four generations (G3, G4, G5, and G6) were used for genotyping. At first, genome‐wide linkage mapping was performed using 122 microsatellite markers on 22 autosomal chromosomes, and the lo locus was mapped to chromosome 12. We then performed fine mapping in two steps on chromosome 12. First, the lo locus was mapped to the interval between GGA12_5 and GGA12_11 using 13 new polymorphic markers. In the second step, fine mapping was performed by adding new families and 11 additional new polymorphic markers. Linkage mapping and haplotype information enabled the localization of the lo locus to a 1.1‐Mb region between GGA12_28 and GGA12_30. Genetic markers between GGA12_28 and GGA12_30 may be used to remove the carriers of congenital loco through this RIR line.     

Proteomic analysis of the chicken egg vitelline membrane

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.     

Fine structure analysis of interaction of FcepsilonRI with IgE

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.     

Fine structural observations of the pecten oculi capillaries of the chicken

Summary The pecten oculi of the domestic chicken was examined with light, scanning and transmission electron microscopy and with freeze-etching techniques. Particular attention has been given to the capillary structure. The capillaries form an extensive anastomotic network. Their endothelial cells have apical (luminal), as well as basal, longitudinally oriented microfolds. It is assumed that the formation of apical differentiations of the endothelial surface is due to haemodynamic influences. Thus, sufficient surface area for membrane bound enzymes is achieved. These enzymes are necessary for active transcellular transport processes that require energy. In freeze-etched material, two different structures of the membrane surface of microfolds can be recognized. These results are discussed in relation to transport functions through capillary endothelial cells of the pecten. It is assumed that the pecten plays an important role in the nourishment of the retina and vitreous body. This paper was presented in part at the inaugural session of the European Club for Ophthalmic Fine Structure in Essen on January 20 and 21, 1973.     

Conformational analysis of the immunodominant epitopes of the circumsporozoite protein of Plasmodium falciparum and knowlesi

All of the coat proteins of the sporozoite and merozoite stages of Plasmodium, determined to date, contain tandem repeats and most of these contain at least one proline residue. These tandemly repeated segments of the circumsporozite (CS) proteins of P. falciparum and P. knowlesi have been shown to constitute an immunodominant epitope. Antibodies to these peptide segments have been shown to be protective and cause the shedding of the CS protein, known as the CSP reaction. In this study, four synthetic peptides were prepared by solid-phase peptide synthesis. The first peptide corresponds to the tetrapeptide tandem repeat in the CS protein of P. falciparum, repeated eight times, (NANP)₈. The second peptide is an analogue of the first in which glycine is substituted for proline, (NANG)₈. The third peptide corresponds to the tandem repeat of P. knowlesi, PK(1–24), which is repeated twice (QAQGDGANAGQP)₂. The fourth peptide is a tetrapeptide repeat, corresponding to the C-terminal tetrapeptide of PK(1–24) and is repeated eight times, (AGQP)₈. It is shown by CD measurements that the presence of proline in these repeats induces an increase in β-sheet (β-turn) content in the (NANP)₈ peptide relative to the repeat of (NANG)₈ and PK(1–24) peptide in aqueous media. The (AGQP)₈ peptide has the highest β-sheet (β-turn) content in the synthetic peptides. It is concluded that this increase in defined structure correlates well with and hence may contribute to the increased antigenicity in these repeats.     

Fine mapping of the chicken salmonellosis resistance locus (SAL1)

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium that has a significant impact on both human and animal health. It is one of the most common food-borne pathogens responsible for a self-limiting gastroenteritis in humans and a similar disease in pigs, cattle and chickens. In contrast, intravenous challenge with S. Typhimurium provides a valuable model for systemic infection, often causing a typhoid-like infection, with bacterial replication resulting in the destruction of the spleen and liver of infected animals. Resistance to systemic salmonellosis in chickens is partly genetically determined, with bacterial numbers at systemic sites in resistant lines being up to 1000-fold fewer than in susceptible lines. Identification of genes contributing to disease resistance will enable genetic selection of resistant lines that will reduce Salmonella levels in poultry flocks. We previously identified a novel resistance locus on Chromosome 5, designated SAL1 . Through the availability of high-density SNP panels in the chicken, combined with advanced back-crossing of the resistant and susceptible lines, we sought to refine the SAL1 locus and identify potential positional candidate genes. Using a 6^th generation backcross mapping population, we have confirmed and refined the SAL1 locus as lying between 54.0 and 54.8 Mb on the long arm of Chromosome 5 ( F  = 8.72, P  = 0.00475). This region spans 14 genes, including two very striking functional candidates; CD27-binding protein ( Siva ) and the RAC -alpha serine/threonine protein kinase homolog , AKT1 ( protein kinase B , PKB ).     

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.     

Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.     

Assessment of allergenic activity of a heat-coagulated ovalbumin after in vivo digestion

We often eat heat-coagulated (H-C) food proteins, but there have been few studies on the allergenic activity of H-C proteins after digestion and absorption in vivo. To show that H-C protein is not an allergen after digestion, mice were used to investigate the digestion and absorption of the protein through the intestinal epithelium into portal blood employing immunoblotting and competitive inhibition ELISA. Ovalbumin (OVA) was used as the model protein, and H-C OVA was prepared by heating a 5% OVA solution for 15 min in boiling water. Antigenic OVA was not detected in the soluble fraction of gastrointestinal contents or the portal blood of mice administered H-C OVA. Also, voluntary physical activities, as an assessment of anaphylaxis, were monitored for 15 h using OVA sensitized mice. Compared to the voluntary physical activities of sensitized mice without any load, no decrease in activity was observed in the group administered H-C OVA, but a significant decrease in activity was found in the mice administered unheated OVA. These results strongly indicate that H-C OVA does not retain allergenic properties.     

Fine structural analysis of the effect of trypan blue on the visceral endoderm of the mouse egg cylinder

The effects of a maternal injection of trypan blue on primitive streak mouse embryos were studied with electron microscopy. Commercial trypan blue was purified by descending paper chromatography, and pregnant females received an intraperitoneal injection of the collected blue fraction on the evening of the 7th day of gestation. Ultrastructurally, the changes in the visceral endoderm were apparent 10 min after the injection and included an increase in the number of fuzzy-coated vesicles in the apical cytoplasm. By 20 min the apical cytoplasm of the extraembryonic visceral endodermal cells was filled with many fuzzy-coated vesicles and numerous vacuoles of various size and electron density. 30 min after the injection, the extraembryonic visceral endodermal cells were relatively smooth lacking a microvillous border and evidence of endocytic activity was rare. Many embryonic visceral endodermal cells were observed in various stages of degeneration although the underlying embryonic ectoderm appeared unaffected. Morphologically, it appears that trypan blue exerts its first effect by altering the endocytic activity of the visceral endoderm.     

Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Computational issues in mapping variation affecting susceptibility to complex disorders: the chicken and the egg

Linkage mapping strategies for complex disorders have evolved under a variety of constraints. Some of these constraints reflect the nature of complex disorders and are manifest in limitations on the kinds of data that can be collected, while others were (at least historically) strictly computational. This paper focuses on how computational issues have impacted the design of studies on complex disorders and, conversely, how our study designs have influenced the computational issues that have been addressed. We now have unprecedented computational resources, but also face unprecedented computational and methodological challenges as we move from the linkage mapping of genes influencing susceptibility to complex disorders toward the identification of the actual variation affecting susceptibility to these disorders. The near-term computational and methodological issues we must address will be profoundly influenced by the study designs of the recent past. But future study designs, as well as our investments in computational and methodological research, ought to be developed considering the computational and informatics resources we now have at hand.