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Ligand binding studies on Ly-49

University of British Columbia

Ly-49 is a highly polymorphic family of molecules expressed almost exclusively on murine natural killer cells ( NK cells). Ly-49 family members appear to play an important role in the recognition of tumor targets by N K cells and act as putative inhibitory receptors on NK cells for MHC class I on target cells. Ly-49 is a type-II transmembrane protein consisting of two non-covalently associated chains, a short amino terminus cytoplasmic tail, a transmembrane domain, and a carboxy terminus carbohydrate recognition domain which takes up approximately 60% of the extracellular domain. The objective of this thesis was to determine the region of Ly-49 responsible for ligand binding specificity. The approach was to generate a number of chimeric constructs in which different regions of. Ly-49 were exchanged between two members of the Ly-49 family, Ly-49 A and Ly-49C. These two members were chosen because they have distinct binding specificities and because of the availability of specific monoclonal antibodies which have been shown to inhibit binding of these two members to their H-2 ligands. The chimeric constructs were placed in a suitable expression vector and expressed on the surface of COS-1 cells using a transient expression system. Ligand binding specificity was determined using a cell adhesion assay which involved the binding of non-adherent cell lines of different haplotypes to the transfected COS-1 cells. Antibody binding was determined by staining cells with fluorescently labeled antibody followed by fluorescence activated cell scanning analysis. Although all of the Ly-49 antibodies bind completely within the carbohydrate recognition domain, this domain alone is unable to determine ligand binding specificity. Further carboxy terminal exchanges define a 19 amino acid region outside the CRD that is necessary, but by itself not sufficient to determine ligand binding. In conclusion, the region responsible for determining binding specificity is a combination of the CRD and the region defined in this study.

Thesis/Dissertation

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2009-02-02

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http://hdl.handle.net/2429/4091

Genetics

University of British Columbia

UBCV

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