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Ly49 RECEPTORS: REQUIREMENTS FOR LIGAND RECOGNITION AND STRUCTURAL ASSEMBLY Open Access

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Other title
Subject/Keyword
NK cells
Ly49 Receptors
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Craveiro Salvado, Carla M
Supervisor and department
Kane, Kevin (Medical Microbiology and Immunology)
Examining committee member and department
Mody, Christopher (University of Calgary)
Baldwin, Troy (Medical Microbiology and Immunology)
Stafford, James (Biological Sciences)
Foley, Edan (Medical Microbiology and Immunology)
Department
Department of Medical Microbiology and Immunology
Specialization
Immunology
Date accepted
2015-09-29T08:44:38Z
Graduation date
2015-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Natural killer (NK) cells in the mouse and rat rely on homodimeric Ly49 receptors to detect virally infected or transformed cells. The identification of altered cells requires the engagement of Ly49 receptors with MHC class I molecules. The engagement requires contact at three specific sites located below the floor of the peptide binding groove of MHC class I by three loop regions located within the ligand-interacting domain of Ly49s, the natural killer domain (NKD). Any alteration within either the MHC class I molecule or the Ly49 receptor could inhibit or disrupt the interaction, affecting the recognition of the ligand by the receptor, ultimately reflected in the functions of the NK cell. This relationship has not been as extensively studied in the rat as compared to the mouse. Given the sequence variability between the mouse and rat Ly49 loop regions, the significance of the loops in rat Ly49 receptors should be explored. Utilizing surface plasmon resonance (SPR), I was able to quantify the binding affinity of the rat Ly49i2 receptor for the MHC class I molecule RT1-A1c. Furthermore, I also showed that at least two of the three loop regions are required for RT1-A1c recognition by the Ly49i2 receptor. While complete binding between RT1-A1c and Ly49i2 was lost with mutated NKD L3 loop regions, only partial binding was lost with a mutated L6 loop, implying that the L6 loop may be required for MHC class I recognition, while the L3 loop may be required for MHC specificity. To assess the practical significance of the Ly49 loop regions, a functional assay is required. The RNK-16 rat leukemic cell line is heavily relied upon for NK cell functional studies. Expression of exogenous genes in RNK-16 cells, however, is difficult and extremely time consuming. I outlined a lentivirus-mediated transduction method that will allow RNK-16 cells to be transfected rapidly resulting in high transfection efficiency rate. The functional output of NK cells is a result of the signaling induced by the engagement of the MHC class I molecule with the Ly49 homodimer receptor. Utilizing biochemical assays, I demonstrate that Ly49 receptors belonging to the same group can associate as heterodimers. The ability of these receptors to form heterodimers may allow for manipulation of the receptors, altering the function of the NK cell.
Language
English
DOI
doi:10.7939/R38K75257
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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File title: CCS PhD Thesis 2015 part I
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