The canine MHC class I allele, DLA-88*50801, presents peptides of varying lengths with three key anchor residues (160.14)

Abstract

Abstract The binding of a peptide to a major histocompatibility (MHC) class I allele depends upon a few key residues within the peptide sequence, which is present only in ~1-2% of peptides in an average protein. Identification of this motif allows one to select candidate epitopes from a protein that may be recognized by cytotoxic T lymphocytes (CTL). While several studies have determined the binding specificities of numerous class I alleles in both humans and mice, no such study has been performed in the domestic dog. The principal aim of this study was to determine the peptide binding motif of a prevalent MHC class I allele, Dog Leukocyte Antigen (DLA) -88*50801. We hypothesized that a conserved binding motif could be identified through sequencing of endogenously presented peptides eluted from DLA-88*50801. To accomplish this goal, a plasmid encoding the DLA-88*50801 gene with an attached flag tag was stably transfected into a canine cell line (DH82). DLA-88*50801 protein was obtained through affinity purification of lysate from ~8 x 109 cells. Bound peptides were eluted from the MHC binding groove utilizing low pH and heat, isolated by size exclusion and affinity purification, and subjected to tandem mass spectrometry analysis to obtain individual peptide sequences. Sequences were manually aligned to determine the binding motif. This study provides essential information to study CTL responses in dogs.