Structural and functional studies of Ly49B, a key immune receptor in myeloid cells

Abstract

Murine Ly49 receptors are predominantly expressed on natural killer (NK) cells and play a key role in regulating immunological activity. The Ly49 family contains both inhibitory and activating receptors, which bind class I major histocompatibility complex (MHC I) or MHC-like molecules. Regulation of NK cell activity is the result of finely balanced signalling, which is delivered by the two receptor types upon ligand binding. Once activated, NK cells play an essential role in the elimination of cancerous and virus infected cells. Ly49B differs from other members of the Ly49 family in that it is expressed on the surface of myeloid, rather than NK cells. It shares only 50 % sequence identity with other Ly49s and contains an additional 20 amino acids at its carboxyl-terminus. Furthermore, there are significant variations between Ly49Bs from different mouse strains. These inter- and intra-molecular variations suggest that different allelic forms could possess distinct structural and functional properties when compared with one another and with other members of the Ly49 family. This thesis seeks to provide an in-depth characterisation of the biochemical properties of Ly49B in comparison to other Ly49 receptors. In this study a series of Ly49B mutants, created by site-directed mutagenesis, was used to identify residues critical for monoclonal anti-Ly49B antibody and ligand binding. Flow cytometric analysis of the mutants revealed that C57 Ly49B-specific residues, L166 and K167, are important for the integrity of the monoclonal 1A1 anti-Ly49B antibody epitope. The equivalent BALB/c Ly49B residues, W166 and N167, together with residue C251 were shown to play an essential role in MHC class I ligand binding. Immunoprecipitation and Western blotting were used to demonstrate that Ly49B exists in multiple molecular forms in transfected and native cells, each of which most likely represents the receptor at different stages of glycosylation. Similar results have never before been presented for any Ly49 receptor. Finally, a method for refolding and purification of the extracellular portion of Ly49B was developed, following expression in Escherichia coli.