Natural killer cells (NK cells) play a key role in innate immunity. Their function is to recognize and kill infected, stressed or malignantly transformed cells. A range of surface receptors promotes this recognition. Cytotoxic mechanisms lead to induction of apoptosis in the target cell [1]. Activating and inhibitory NK cell receptors (NCRs) can be subdivided into immunoglobulin-like family and C‑type lectin-like family. NCR NKp80 and its myeloid-specific activating ligand AICL are both C‑type lectin-like receptors (CTLRs) with C-type lectin-like domain (CTLD) [2]. Immunocomplex NKp80-AICL promotes lysis of malignant myeloid cells, mediates crosstalk between NK cells and monocytes, is engaged in cytokine release and contributes to initiation of immune response during inflammation [3].
AICL ectodomain contains odd cysteine which is believed to be responsible for formation of dimers on the cell surface. However, this cysteine in position 87 is not conserved, compared to other CTLD receptors. AICL ectodomain was expressed in E. coli BL21 Gold (DE3) strain as inclusion bodies and we have found out that upon mutation of this cysteine to serine (C87S mutation) the yield of subsequent in vitro refolding and purification as well as the stability of prepared protein are greatly enhanced compared to wild-type construct.
We used mammalian expression system of modified human embryonic kidney cell line 293 (HEK293) to produce glycosylated NKp80 ectodomain. Unfortunately, transient transfection was not successful. Using pOPINTTGneo plasmid as well as PiggyBac cloning system we were able to create stable cell lines expressing soluble extracellular parts of NKp80 in constitutive or inducible way. Thanks to these approaches, we are able to produce both proteins in sufficient amount to initiate structural studies using analytical ultracentrifuge, dynamic light scattering and finally crystallization of this immunocomplex.
This study was supported by Czech Science Foundation (15-15181S), Charles University (UNCE 204025/2012, SVV 260079/2014), BioStruct-X and Instruct European infrastructure projects.