Dataset Information

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery.

ABSTRACT: The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide-MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle.

SUBMITTER: Hansen AM

PROVIDER: S-EPMC4225172 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery.

Hansen Andreas M AM Rasmussen Michael M Svitek Nicholas N Harndahl Mikkel M Golde William T WT Barlow John J Nene Vishvanath V Buus Søren S Nielsen Morten M

Immunogenetics 20140904 12

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be ...[more]

PMID: 25186069

Similar Datasets

Project description:Swine leukocyte antigen (SLA) class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs). Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and ?2-microglobulin (?2m) using splicing overlap extension polymerase chain reaction (SOE-PCR). The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV) and four porcine reproductive and respiratory syndrome virus (PRRSV) epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA)-based method. The SLA-1?13:01, SLA-1?11:10, and SLA-1?11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1?13:01 and SLA-1?04:01:01 proteins. The more flexible pocket E in the SLA-1?13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1?13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an important basis for epitope studies of infectious diseases in swine, and for the rational development of novel porcine vaccines, as well as for detailed studies of CTL responses in pigs used as animal models.

Project description:BackgroundSchistosomiasis is a chronic debilitating parasitic disease that afflicts more than 200 million individuals worldwide. Long-term administration of chemotherapy with the single available drug, praziquantel, has led to growing concerns about drug resistance. The PSD-95/Dlg/ZO-1 (PDZ) domain is an important module found in many scaffolding proteins, which has been recognized as promising targets for the development of novel drugs. However, the parasite-derived PDZ domains and their associated functions are still largely unknown.Methodology/principal findingsThe gene encoding the Schistosoma japonicum Scribble protein (SjScrib) was identified by homologous search with the S. mansoni Scrib sequence. By screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays, we identified and confirmed the ligand binding specificity for each of the four PDZ domains of SjScrib. Both SjScrib-PDZ1 and SjScrib-PDZ3 recognize type I C-terminal PDZ-domain binding motifs (PBMs), which can be deduced as consensus sequences of -[Φ][x][E][TS][x][ILF] and -[x][RKx][ETS][T][WΦ][ILV], respectively. SjScrib-PDZ2 prefers stringent type II C-terminal PBMs, which significantly differs from that of its human ortholog. SjScrib-PDZ4 binds to typical II C-terminal PBMs with a consensus sequence -[x][FW][x][LI][x][LIV], in which the aromatic residue Phe is predominantly selected at position -4. The irregular and unconventional internal ligand binding specificities for the PDZ domains of SjScrib were confirmed by point mutations of the key amino acids within the ligand binding motifs. We also compared the differences in ligand specificities between SjScrib-PDZs and hScrib-PDZs, and explored the structural basis for the ligand binding properties of SjScrib-PDZs.Conclusions/significanceIn this study, we characterized and confirmed the ligand binding specificities of all four PDZ domains of SjScrib for the first time. We denoted the differential ligand binding specificities between SjScrib-PDZs and hScrib-PDZs as well as the structural basis for these properties. This work may provide a fundamental basis for the rational design of novel anti-schistosomal drugs.

Dataset Information

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery.

Publications

Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets