Characterization of peptide binding to the murine MHC class I H-2Kk molecule. Sequencing of the bound peptides and direct binding of synthetic peptides to isolated class I molecules.
Peptides that are bound by the murine class I MHC molecule H-2Kk have been isolated and sequenced. The initial step in the fractionation was affinity column isolation of the peptide-class I complex from either RDM-4 or x5563 tumor cell lines. Acid denaturation of the complex followed by HPLC fractionation of the peptides allowed us to sequence individual peptides, as well as pools of peptides. To date, a total of 10 sequences have been characterized, and all were 8 mers. The sequences were variable except for glutamic acid in the second position (P2) and isoleucine in the eighth (P8), which were highly conserved. To further study peptide binding to H-2Kk, a competitive binding assay consisting of the immobilized histocompatibility protein and a biotinylated self-peptide for signal generation was developed. A complete set of single-alanine variants for this one self-peptide was tested in the assay, demonstrating that substitution at P2 and P8 markedly decreased the affinity for the class I molecule; alanine at position 3 had an intermediate effect on binding. A comparison of the identified self-peptides for binding to H-2Kk showed that they differed in affinity by more than one order of magnitude. Influenza virus nucleoprotein peptide, SDY EGR LI, associated with the plate-bound class I molecule, and the resulting MHC-peptide complex could trigger TNF release by influenza-primed CTLs. This result demonstrated the functional activity of the plate-bound H-2Kk-peptide complex.
Brown
E L
EL
Genetics Institute, Inc., Cambridge, MA 02140.
Wooters
J L
JL
Ferenz
C R
CR
O'Brien
C M
CM
Hewick
R M
RM
Herrmann
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D000939:Epitopes; D006183:H-2 Antigens / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008806:Mice, Inbred AKR; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000276:immunology; D009975:Orthomyxoviridae / Q000276:immunology; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000939:Epitopes; D006183:H-2 Antigens; D008024:Ligands; D009698:Nucleoproteins; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha