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 [1]. 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 NKRP1B and its ligand Clrb, which play a 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 [2].
During the infection of RCMV the target cell downregulates cell surface expression of Clrb, thus decreasing the inhibitory signal transmitted through NKRP1B 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 escapes the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the rat strain WAG, it has been shown that the NKRP1B homologue from rat strain SD ligates only Clrb and does not recognize RCTL. Thus the SD rat strain is less susceptible to the RCMV infection [2].
This research aims to elucidate the molecular basis of the NKRP1:Clr receptor ligand recognition, based on previous successful results with homologous human NKRP1:LLT1 receptor ligand pair[3]. For protein crystallization, it was found out that the best recombinant expression system for production of soluble extracellular domains from this family of receptors is transiently or stably transfected HEK293S GnTI- human cell line possessing homogeneous N‑glycosylation profile. To increase the yields of recombinant proteins, we have optimized transposon-based doxycycline inducible mammalian cell expression system piggyBac [4] within HEK293S GnTI- cell line using Clrb soluble expression construct as target protein.
This study was supported Czech Science Foundation (15-15181S), Charles University (UNCE 204025/2012, SVV 260079/2014), BioStruct-X and Instruct European infrastructure projects.