A novel and comprehensive synthetic approach for the elucidation of protein antigenic structures. Determination of the full antigenic profile of the alpha-chain of human haemoglobin.
Ontology highlight
ABSTRACT:
SUBMITTER: Kazim AL
PROVIDER: S-EPMC1162207 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:This paper reports the localization of the regions on the beta-chain that are recognized by T cells from mice immunized with haemoglobin. The 14 overlapping peptides encompassing the entire beta-chain were examined in vitro for their ability to stimulate lymph-node cells from haemoglobin-primed B10.D2 (H-2d) and SJL (H-2s) mice. Several regions of the molecule (T sites) were found to stimulate haemoglobin-primed lymph-node cells. This strategy has enabled the localization of the full profile of T-cell recognition of the beta-chain by these mouse strains. Some of the regions that stimulated T cells appeared to coincide with those recognized by antibodies (i.e. B cells). It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites that are recognized exclusively by T cells and to which no detectable antibody response is directed.
| S-EPMC1146585 | biostudies-other
Project description:The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.
| S-EPMC1158211 | biostudies-other
Project description:A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.
| S-EPMC1146584 | biostudies-other
Project description:Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.
| S-EPMC3641798 | biostudies-other
Project description:A synthetic approach is introduced for localization of subunit interacting surfaces in oligomeric proteins. It consists of studying the binding activity of consecutive uniform overlapping peptides encompassing an entire subunit to the other, radiolabelled, subunit. This permits the establishment of the full profile of peptides that bind the other intact subunit. This approach has been demonstrated with haemoglobin, and its application here with the beta-chain peptides has enabled the localization on the beta-chain of the submolecular regions responsible for its binding to alpha-chain in solution. There was good agreement between the binding surfaces found here in solution and those expected from the crystal structure. There were also, however, some significant differences in the levels of binding found in solution and those expected from the crystal. Peptide 21-35 possessed much higher binding activity than would be expected from its contribution to subunit association in the crystal. Conversely, other regions expected to possess considerable binding capacity for alpha-chain either showed low (peptides 111-125 and 121-135) or almost no binding (peptides 91-105 and 101-115) capacity. On the other hand, two interacting surfaces (within peptides 11-25 and 71-85) that make a contribution in solution do not appear to play a role in the crystal. It is concluded that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should serve as an effective method for localization of subunit interacting surfaces of unknown proteins, even those that can be isolated only in traces.
| S-EPMC1146587 | biostudies-other
Project description:A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.
| S-EPMC1146586 | biostudies-other
Project description:Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.
| S-EPMC7759213 | biostudies-literature
Project description:BACKGROUND: It is widely known that castration has a significant effect on the accumulation of adipose tissue. microRNAs (miRNAs) are known to be involved in fat deposition and to be regulated by the androgen-induced androgen receptor (AR). However, there is little understanding of the relationship between miRNAs and fat deposition after castration. In this study, the high-throughput SOLiD sequencing approach was used to identify and characterize miRNA expression in backfat from intact and castrated full-sib male 23-week-old pigs. The patterns of adipogenesis and fat deposition were compared between castrated and intact male pigs. RESULTS: A total of 366 unique miRNA genes were identified, comprising 174 known pre-miRNAs and 192 novel pre-miRNAs. One hundred and sixty-seven pre-miRNAs were common to both castrated (F3) and intact (F4) male pig small RNA libraries. The novel pre-miRNAs encoded 153 miRNAs/miRNA*s and 141 miRNAs/miRNA*s in the F3 and F4 libraries, respectively. One hundred and seventy-seven miRNAs, including 45 up- and 132 down-regulated, had more than 2-fold differential expression between the castrated and intact male pigs (p-value < 0.001). Thirty-five miRNAs were further selected, based on the expression abundance and differentiation between the two libraries, to predict their targets in KEGG pathways. KEGG pathway analyses suggested that miRNAs differentially expressed between the castrated and intact male pigs are involved in proliferation, apoptosis, differentiation, migration, adipose tissue development and other important biological processes. The expression patterns of eight arbitrarily selected miRNAs were validated by stem-loop reverse-transcription quantitative polymerase chain reaction. These data confirmed the expression tendency observed with SOLiD sequencing. miRNA isomiRs and mirtrons were also investigated in this study. Mirtrons are a recently described category of miRNA relying on splicing rather than processing by the microprocessor complex to generate the RNAi pathway. The functions of miRNAs important for regulating fat deposition were also investigated in this study. CONCLUSIONS: This study expands the number of fat-deposition-related miRNAs in pig. The results also indicate that castration can significantly affect the expression patterns of fat-related miRNAs. The differentially expressed miRNAs may play important roles in fat deposition after castration.
| S-EPMC3901342 | biostudies-other
Project description:Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of Plasmodium falciparum rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor's mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.
| S-EPMC7553781 | biostudies-literature
Project description:The number of blood group systems, currently 35, has increased in the recent years as genetic variations defining red cell antigens continue to be discovered. At present, 44 genes and 1568 alleles have been defined as encoding antigens within the 35 blood group systems. This paper provides a brief overview of two genetic technologies: single nucleotide variant (SNV) mapping by DNA microarray and massively parallel sequencing, with respect to blood group genotyping. The most frequent genetic change associated with blood group antigens are SNVs. To predict blood group antigen phenotypes, SNV mapping which involves highly multiplexed genotyping, can be performed on commercial microarray platforms. Microarrays detect only known SNVs, therefore, to type rare or novel alleles not represented in the array, further Sanger sequencing of the region is often required to resolve genotype. An example discussed in this article is the identification of rare and novel RHD alleles in the Australian population. Massively parallel sequencing, also known as next generation sequencing, has a high-throughput capacity and maps all points of variation from a reference sequence, allowing for identification of novel SNVs. Examples of the application of this technology to resolve the genetic basis of orphan blood group antigens are presented here. Overall, the determination of a full profile of blood group SNVs, in addition to serological phenotyping, provides a basis for provision of compatible blood thus offering improved transfusion safety.
| S-EPMC4232566 | biostudies-literature