Purification of correctly oxidized MHC class I heavy‐chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas; Sylvester-Hvid, Christina; Nielsen, Line; Hobley, Timothy John; Thomas, Owen R.T.; Buus, Søren

doi:10.1110/ps.0233003

Cited by 36 publications

(55 citation statements)

References 26 publications

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“…We have previously generated recombinant MHC proteins from different species such as human, swine and others (Ferre et al 2003; Ostergaard Pedersen et al 2001; Pedersen et al 2011). Here, we transfer the underlying approaches to the BoLA system.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2014

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2014

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“…Synthetic genes encoding MHC-I heavy chain were cloned as previously described (Ostergaard Pedersen et al, 2001; Ferre et al, 2003; Leisner et al, 2008). Briefly, genes encoding MHC-I heavy chains truncated at position 275 (i.e.…”

Section: Methodsmentioning

confidence: 99%

Real-time, high-throughput measurements of peptide–MHC-I dissociation using a scintillation proximity assay

Harndahl

Rasmussen

Røder

et al. 2011

Journal of Immunological Methods

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Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal requirements. To avoid labeling of the highly variable peptide, we labeled the invariant β2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually label-free assay, we showed that rates of peptide-MHC class I dissociation measured in this assay correlated well with rates of dissociation rates measured conventionally with labeled peptides. This assay can be used to measure the stability of any peptide-MHC Class I combination, it is reproducible and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed rates of dissociation that ranged from 0.1 hours to 46 hours depending on the peptide used.

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“…Briefly, a gene encoding the Factor Xa cleavage site, FXa, and the first 87 amino acids of the mature human tapasin (NP_003181) was inserted into a GrpE/pET28a vector. Expression of Tpn was done in Escherichia coli BL21(DE3) cells as described previously (35). Urea-dissolved Tpn 1-87 was purified by anion exchange and size exclusion chromatography (GE Healthcare, Ä kta FPLC).…”

Section: Methodsmentioning

confidence: 99%

“…Urea-dissolved Tpn 1-87 was purified by anion exchange and size exclusion chromatography (GE Healthcare, Ä kta FPLC). HLA-I HCs and human ␤ 2 -microglobulin (␤ 2 m) were produced as described previously (35). HLA-I HC and GrpE-FXa-Tpn 1-87 was stored individually in 8 M urea at Ϫ20°C until use.…”

Section: Methodsmentioning

confidence: 99%

Tapasin Discriminates Peptide-Human Leukocyte Antigen-A*02:01 Complexes Formed with Natural Ligands

Røder

Geironson

Rasmussen

et al. 2011

Journal of Biological Chemistry

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A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of tapasin, Tpn 1-87 , assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn 1-87 could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I-and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.Mature HLA-I 3 molecules are located on the surface of all nucleated cells where they present peptides to CD8ϩ T lymphocytes. Before the peptide-HLA-I complex is transported to the cell surface, maturation and assembly with peptides occur in the ER. During late stage maturation, HLA-I molecules are integrated in the peptide-loading complex (PLC), which at least consists of TAP1/2, tapasin, calreticulin, protein disulfide isomerase, ERp57, and HLA-I (1, 2). Integration of HLA-I into the PLC is mediated by tapasin, which structurally bridges HLA-I and TAP (3, 4). Tapasin is a multi-domain protein, which has been suggested to perform multiple functions. Tapasin has been shown to enhance peptide binding to TAP, facilitate peptide loading onto HLA-I, edit the HLA-I bound peptide repertoire, and retain and recycle suboptimally loaded peptide-HLA-I complexes (5-9). Consequently, in the absence of tapasin, cell surface-expressed HLA-I molecules are less stable and present a partly different peptide repertoire than HLA-I on wild-type cells (10 -12). The effect of tapasin depends on the HLA-I allomorph where certain allomorphs, such as HLA-A*02:01, are dependent on tapasin for efficient presentation, whereas others are less influenced (10 -12).Recently, we showed that an N-terminal fragment of tapasin, Tpn 1-87 , assi...

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Purification of correctly oxidized MHC class I heavy‐chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

Cited by 36 publications

References 26 publications

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

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

Real-time, high-throughput measurements of peptide–MHC-I dissociation using a scintillation proximity assay

Tapasin Discriminates Peptide-Human Leukocyte Antigen-A*02:01 Complexes Formed with Natural Ligands

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