Identification of HLA-Cw6.02 and HLA-Cw7.01 allele-specific binding motifs by screening synthetic peptide libraries.
Unlike HLA-A and HLA-B, few peptide epitope motifs have been reported for HLA-C molecules. However, a number of cytotoxic T-lymphocyte epitopes derived from tumor antigens that bind to HLA-C molecules have been described. Here we report peptide-binding motifs for both HLA-Cw6.02 and HLA-Cw7.01 molecules. Recombinant human HLA molecules were generated and used to screen combinatorial 9mer peptide libraries. Complexes of HLA molecules properly folded and associated with beta2-microglobulin and peptides were identified using a conformation-specific HLA class I antibody conjugated to alkaline phosphatase. In the presence of substrate, peptide beads can be readily isolated and microsequenced to determine peptide identity. Of the peptides that bound to HLA-Cw6.02 and HLA-Cw7.01, 19 and 18 peptides, respectively, were sequenced, allowing motif identification for each C allele. This is the first report of an HLA-Cw7.01 peptide motif and extends the findings of Falk et al. [(1993) Proc Natl Acad Sci USA 90:12005] for an HLA-Cw6.02 motif. Anchoring amino acids for the HLA-Cw6.02 motif were phenylalanine or tyrosine in position (P)1, arginine in P2, and an aliphatic/aromatic residue at P9. Anchoring residues for HLA-Cw7.01 were positively charged amino acids in P1 and P2. Unlike most other HLA molecules, we were unable to assign P9 an anchoring residue, and we suspect that HLA-Cw7.01 binds peptides in an unconventional manner. Additionally, preferred amino acids were identified for both molecules. Identification of HLA-Cw6.02 and HLA-Cw7.01 peptide-binding motifs makes a significant contribution to the C allele peptide-binding motifs and will allow investigators to predict, design, and test HLA-Cw6.02 and HLA-Cw7.01 engineered peptides for immunotherapy.
Department of Microbiology and Immunology, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA.
Sara O
SO
Dionne
Douglas F
DF
Lake
William J
WJ
Grimes
Margaret H
MH
Smith
D000469:Alkaline Phosphatase / Q000378:metabolism; D000483:Alleles; D001665:Binding Sites; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D000939:Epitopes; D015236:HLA-C Antigens / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000378:metabolism
D000939:Epitopes; D015236:HLA-C Antigens; C528994:HLA-C*06 antigen; C040178:HLA-C*70 antigen; D018796:Immunoconjugates; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D000469:Alkaline Phosphatase