Masaaki Ibe

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules.

Binding of 8-mer to 11-mer peptides carrying the anchor residues to slow assembling HLA class I molecules (HLA-B*5101)

Abstract The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B*5101 cells at 26 °C for 3 h. Analysis of the binding of these peptides to HLA-B*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B*5101 binding peptides to stabilize the HLA-B*5101 molecules was markedly lower than that of HLA-B*3501 binding peptides to stabilize the HLA-B*3501 molecules. It is known that HLA-B*5101 is a slow assembling molecule, while HLA-B*3501 assembles rapidly. The results imply that the slow assembling of HLA-B*5101 molecules results from the low affinity of peptides to HLA-B*5101 molecules.

Refined peptide HLA-B^*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

Abstract HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.

PREDOMINANT ROLE OF N-TERMINAL RESIDUE OF NONAMER PEPTIDES IN THEIR BINDING TO HLA-B* 5101 MOLECULES

Previous studies on the binding of HLA class I molecules to chemically synthesized peptides carrying primary anchor residues (Ruppert et al. 1993; Parker et al. 1994; Scho ̈nbach et al. 1995, 1996) have shown that not only are primary anchor residues critical for major histocompatibility complex (MHC) class I peptide binding, but also that secondary residues play an important role in MHC class I peptide binding. Our recent studies (Scho ̈nbach et al. 1995, 1996) have demonstrated that in addition to two primary anchor residues at position 2 (P2) and the C-terminus, secondary anchor residues can be identified by statistical analysis. However, there are other methods to analyze the effect of secondary anchors on MHC class I peptide binding, as shown by Udaka and co-workers (1995). In a previous study using the HLA-B*5101 stabilization assay (Kikuchi et al. 1996), we failed to clarify the role of secondary anchor residues of HLA-B*5101 binding peptides by statistical analysis because the ability of HLAB*5101 binding peptides to stabilize HLA-B*5101 molecules is very weak. We therefore improved the stabilization assay for HLA-B*5101 binding peptides by extending the incubation time as follows: RMA-S-B*5101 cells cultured at 26°C for 18–24 h were incubated at 26 °C for 3 h with various concentrations of peptides followed by a 3 h incubation at 37°C. The cells were then stained with TP25.99 HLA class Iα3 domain specific monoclonal antibody (mAb) (D’Urso et al. 1991: Tanabe et al. 1992) and FITC-conjugated IgG of sheep mouse-specific Ig antibodies. The mean linear fluorescence intensity (MFI) of the cells was measured by using a FACScan. The relative MFI was obtained by subtracting the MFI of cells not l aded with peptide and stained with TP25.99 mAb from the MFI of peptide-loaded cells stained with TP25.99 mAb. The affinity of a peptide was represented by the halfmaximal binding level (BL50) which is the peptide concentration yielding the half-maximal mean fluorescence intensity. Binding peptides were classified according to the BL50 into three categories: high binder (BL 50 # 10–4 M), medium binder (10 –45 BL50 # 10–3 M), and low binder (10–3 M 5 BL50). High, medium, low, and nonbinders were given ranks 3, 2, 1, and 0, respectively, and the mean binding rank (MBR) was calculated. We tested 127 nonamer peptides (Sakaguchi et al. 1997) carrying the anchor residues at P2 (Pro, Ala, and Gly) and P9 (Ile, Val, Leu and Met) which were selected from the sequence of the SF2 strain of human immunodeficiency virus-1 (Sanchez-Pescador et al. 1985) and the JT strain of hepatitis C virus protein (Tanaka et al. 1992). The MBR of these peptides increased from 0.22 to 0.42 by the improved stabilization assay (Sakaguchi et al. 1997). Subsequently we conducted an analysis of these nonamer peptides to determine secondary anchor residues which contribute to the HLA-B*5101-peptide interaction. The frequency of binding peptides and the MBR was calculated for each amino acid or groups of amino acids at each non-primary anchor position (positions 1, 3, 4, 5, 6, 7, and 8) in a MannWhitney U-test (Table 1). Positive effects on the peptide binding to HLA-B*5101 molecules were found for aromatic (Tyr, Phe, Trp, and His) and aliphatic (Leu, Val, Ile, and Met) hydrophobic residues at P1 ( P50.01). Likewise, small amino acids, Ala and Gly at P6, significantly enhanced the binding to HLA-B*5101 molecules ( P50.05). In contrast, negative effects on the peptide binding to HLAB*5101 molecules were observed for Gly and Ala at P1 (p50.05). In order to confirm the effect of the secondary anchor residues, the binding of peptides mutated at P1 and P6 was tested. The substitution of hydrophobic residues Tyr and Leu for Asn at P1 of NPPIPVGEI increased the binding to HLA-B*5101 molecules. Moreover, two mutations, Tyr and Val for Leu at P1 of LPCRIKQII did not affect the binding T. Sakaguchi? M. Ibe ? C. Schönbach? M. Takiguchi ( ) Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108, Japan