Limits...
HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Davenport MP, Smith KJ, Barouch D, Reid SW, Bodnar WM, Willis AC, Hunt DF, Hill AV - J. Exp. Med. (1997)

Bottom Line: Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells.This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus.We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Headington, United Kingdom.

ABSTRACT
Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

Show MeSH

Related in: MedlinePlus

Comparison of the peptide binding motifs for HLA-A2,  HLA-B8, and HLA-B53 derived by different methods. Motifs are indicated according to the convention established by Falk et al. (2), namely,  dominant or strong anchor residues are indicated in bold type, and listed  vertically in order of descending importance. It should be noted that different criteria were used to assign anchors in some studies, particularly  when individual peptide as opposed to pool sequences were analyzed.  Hø, hydrophobic residue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196123&req=5

Figure 2: Comparison of the peptide binding motifs for HLA-A2, HLA-B8, and HLA-B53 derived by different methods. Motifs are indicated according to the convention established by Falk et al. (2), namely, dominant or strong anchor residues are indicated in bold type, and listed vertically in order of descending importance. It should be noted that different criteria were used to assign anchors in some studies, particularly when individual peptide as opposed to pool sequences were analyzed. Hø, hydrophobic residue.

Mentions: Analysis of the random peptide library by HPLC and laser desorption-mass spectrometry showed that the library was a homogeneous mix of peptides without any obvious peaks of abundant peptides and conformed to the mass range expected for nonamer peptides. Refolding of MHC class I molecules with the random peptide mixture resulted in the formation of stable complexes of heavy chain, peptide, and β2m, which migrated at the correct mass using gel filtration. A final concentration and wash step before peptide elution ensured that only peptides that formed complexes stable enough to remain associated during these procedures were analyzed. Peptides were eluted from ∼500 μg of MHC class I. The results of pool sequencing of these peptides and of the random peptide library showed that while there were no significant enrichments for any amino acids in the random library, such enrichments were found in the MHC class I–associated pool. Positions where the yield of a particular amino acid was increased to 150% or more of the previous cycle were considered significant (Fig. 1) (2). The motif identified using this method could be compared with that identified by previous studies (Fig. 2). It can be seen that although the motifs were not identical, the same anchor residues were observed by both methods with the exception of the COOH-terminal hydrophobic anchor previously observed in studies of HLA-A2 and HLA-B8. In addition, in some cases in which enrichment for anchor residues did not reach the 150% level, the same patterns of enrichment were seen for both the naturally bound peptides and the peptides bound by the recombinant class I. It is also interesting that the strength of motifs for different MHC class I alleles is conserved using both methods, so that, for example, whereas HLA-A2 shows strong enrichment for several possible minor anchors in both pool sequences, HLA-B53 shows a relatively weak proline anchor at position 2 using both methods (Fig. 2). In addition, whereas analysis of four individual self-peptides eluted from HLA-B53 and three T cell epitopes suggests a preference for hydrophobic COOH termini (Fig. 3), this was not observed in the results from pool sequencing. Thus the amino acid preferences observed from pool sequencing of HLA-B53 seem somehow less strong than those of, for example, HLA-A2.


HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Davenport MP, Smith KJ, Barouch D, Reid SW, Bodnar WM, Willis AC, Hunt DF, Hill AV - J. Exp. Med. (1997)

Comparison of the peptide binding motifs for HLA-A2,  HLA-B8, and HLA-B53 derived by different methods. Motifs are indicated according to the convention established by Falk et al. (2), namely,  dominant or strong anchor residues are indicated in bold type, and listed  vertically in order of descending importance. It should be noted that different criteria were used to assign anchors in some studies, particularly  when individual peptide as opposed to pool sequences were analyzed.  Hø, hydrophobic residue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196123&req=5

Figure 2: Comparison of the peptide binding motifs for HLA-A2, HLA-B8, and HLA-B53 derived by different methods. Motifs are indicated according to the convention established by Falk et al. (2), namely, dominant or strong anchor residues are indicated in bold type, and listed vertically in order of descending importance. It should be noted that different criteria were used to assign anchors in some studies, particularly when individual peptide as opposed to pool sequences were analyzed. Hø, hydrophobic residue.
Mentions: Analysis of the random peptide library by HPLC and laser desorption-mass spectrometry showed that the library was a homogeneous mix of peptides without any obvious peaks of abundant peptides and conformed to the mass range expected for nonamer peptides. Refolding of MHC class I molecules with the random peptide mixture resulted in the formation of stable complexes of heavy chain, peptide, and β2m, which migrated at the correct mass using gel filtration. A final concentration and wash step before peptide elution ensured that only peptides that formed complexes stable enough to remain associated during these procedures were analyzed. Peptides were eluted from ∼500 μg of MHC class I. The results of pool sequencing of these peptides and of the random peptide library showed that while there were no significant enrichments for any amino acids in the random library, such enrichments were found in the MHC class I–associated pool. Positions where the yield of a particular amino acid was increased to 150% or more of the previous cycle were considered significant (Fig. 1) (2). The motif identified using this method could be compared with that identified by previous studies (Fig. 2). It can be seen that although the motifs were not identical, the same anchor residues were observed by both methods with the exception of the COOH-terminal hydrophobic anchor previously observed in studies of HLA-A2 and HLA-B8. In addition, in some cases in which enrichment for anchor residues did not reach the 150% level, the same patterns of enrichment were seen for both the naturally bound peptides and the peptides bound by the recombinant class I. It is also interesting that the strength of motifs for different MHC class I alleles is conserved using both methods, so that, for example, whereas HLA-A2 shows strong enrichment for several possible minor anchors in both pool sequences, HLA-B53 shows a relatively weak proline anchor at position 2 using both methods (Fig. 2). In addition, whereas analysis of four individual self-peptides eluted from HLA-B53 and three T cell epitopes suggests a preference for hydrophobic COOH termini (Fig. 3), this was not observed in the results from pool sequencing. Thus the amino acid preferences observed from pool sequencing of HLA-B53 seem somehow less strong than those of, for example, HLA-A2.

Bottom Line: Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells.This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus.We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Headington, United Kingdom.

ABSTRACT
Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

Show MeSH
Related in: MedlinePlus