Transport studies with the polymorphic TAP molecules in chickens

Abstract

We have proposed low recombination levels allow co-evolution in each haplotype of the chicken MHC, giving rise to the dominantly-expressed class I molecule BF2, that in turn is at least partially responsible for strong genetic associations with responses to infectious diseases. We have shown that both TAP genes are highly polymorphic and moderately diverse in sequence, and for a few haplotypes that the differences in sequence result in differences in peptide translocation that reflect the BF2 peptide motif. We wish to further understand how TAP sequence variation leads to translocation specificity. Classic transport assays with iodinated peptides and permeabilised cells were used to measure uptake and inhibition. Transformed chicken cell lines with known peptide motifs were analysed to establish the basis of specificity. TAP genes were knocked out (KO) by CRISPR-Cas9 technology in one cell line, and transfectants with wildtype and mutant genes were analysed by translocation and flow cytometry to pinpoint residues responsible for specificity. B15 cells translocate B15 peptide, but not B12 peptide. However, B12 cells translocate both B12 and B15 peptides at a lower rate, and unlabelled peptides cross-block. The historic recombinant B19 (TAP2 hybrid of B12 and B15, with some changes) transports B15 peptide better than B12 peptide, but cross-blocking was only efficient for transport of B12 peptide. B19 double KO cells do not transport peptides or express class I molecules on the cell surface. Upon transfection with TAP1 and TAP2 genes from either B15 or B19, these cells transport B15 peptide (but not B4 peptide) and restore class I levels. KO cells transfected with TAP1 and/or TAP2 genes mutated in some but not other residues allowed transport and restored class I levels. Transport assays with polymorphic TAP genes and their mutants are a powerful system for understanding specificity of peptide translocation.