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Mutagenesis and expression of protein NKp80, the activation receptor of human lymphocytes
Pazderová, Kristýna ; Vaněk, Ondřej (advisor) ; Ječmen, Tomáš (referee)
The subject of this study is a receptor NKp80, also known as killer cell lectin-like subfamily F, member 1 (KLRF1). It is an activating receptor which forms homodimers on the surface of natural killer (NK) cells. Receptor NKp80 binds to a ligand, AICL, which is naturally expressed on all myeloid cells. Upon a substantial increase in AICL expression, for example in cancer cell, the cell then becomes a target for an NK cells expressing the receptor NKp80. Ultimately, the complex NKp80:AICL is therefore a potential target for the immunotherapeutic treatment of myeloid leukaemia. The aim of the study was to the produce and purify a series of mutants of an extracellular domain of NKp80 by replacing cysteins by serines in a segment of extracellular domain called the stalk region. Here, by introducing the mutations, we studied their effect on homodimer formation. The proteins were prepared in HEK293S GnTI- cells using stable transfection. Altogether, we produced seven mutants with all possible combinations of mutations of the three cysteins in the stalk region. We then analysed the proteins using size exclusion chromatography and differential scanning fluorimetry. Lastly, we deglycosylated the proteins to verify that NKp80 is present in several glycoforms. Our results show that none of the variants of...
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Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for "decoy" surface receptor - RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less...
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Production and purification of recombinant receptor Clrb
Prokopová, Tereza ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb ("C- type-lectin-related protein b")....
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