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

J Immunol. 1994 Oct 1;153(7):3079-92.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding, Competitive
  • Epitopes
  • H-2 Antigens / metabolism*
  • Ligands
  • Mice
  • Mice, Inbred AKR
  • Mice, Inbred C3H
  • Molecular Sequence Data
  • Nucleoproteins / immunology
  • Orthomyxoviridae / immunology
  • Peptides / immunology*
  • Peptides / metabolism
  • Protein Binding
  • T-Lymphocytes, Cytotoxic / immunology
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Epitopes
  • H-2 Antigens
  • Ligands
  • Nucleoproteins
  • Peptides
  • Tumor Necrosis Factor-alpha