Rhesus Macaque Killer Cell Ig-like Receptor Domain 0 Glycans Impact Surface Expression and Ligand Specificity

Abstract Defining the MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as a model for infectious diseases and comparative immunogenetics. Several rhesus macaque KIRs belong to a phylogenetically distinct group with a three-amino acid deletion in domain 0 (D0). This deletion results in polymorphic differences in potential N-linked glycosylation (PNG) sites adjacent to a predicted KIR-MHC class I contact site. Whereas most KIRs have two tandem PNG sites in D0 (N36FTN39FT), the KIRs containing the deletion only have a single site in this region (N36FT). To discern the contribution of glycosylation to KIR expression and ligand recognition, we constructed PNG mutants for six lineage II KIR genes that eliminate or create sites for N-glycan addition at these locations. The impact of these mutations on total and surface expression was determined by immunoblotting and flow cytometry. Ligand engagement was assessed by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion