Predominant role of N-terminal residue of nonamer peptides in their binding to HLA-B 5101 molecules

• Published in: Immunogenetics (New York) 1997-01, Vol.46 (3), p.245-248
• Main Authors: Sakaguchi, T, Ibe, M, Miwa, K, Yokota, S, Tanaka, K, Schönbach, C, Takiguchi, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; HLA-B Antigens - metabolism; Humans; Peptides - immunology; Peptides - metabolism; Protein Binding; Structure-Activity Relationship
• ISSN: 0093-7711; 1432-1211
• DOI: 10.1007/s002510050269

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; Schoenbach 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 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. In a previous study using the HLA-B*5101 stabilization assay, we failed to clarify the role of secondary anchor residues of HLA-B*5101 binding peptides by statistical analysis because the ability of HLA-B*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 degree C for 18-24 h were incubated at 26 degree C for 3 h with various concentrations of peptides followed by a 3 h incubation at 37 degree C. The cells were then stained with TP25.99 HLA class I alpha 3 domain specific monoclonal antibody (mAb) 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 loaded 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 half-maximal binding level (BL sub(50)) which is the peptide concentration yielding the half-maximal mean fluorescence intensity. Binding peptides were classified according to the BL sub(50) into three categories: high binder (BL sub(50) less than or equal to 10 super(-4) M), medium binder (10 super(-4) < BL sub(50) less than or equal to 10 super(-3) M), and low binder (10 super(-3) M < BL sub(50)). 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 carrying the anchor residues at P2 (Pro, Ala, and Gly) and P9 (Ile, Val, Leu and Met) which were selected from the sequence of