Immune system is able to recognize tumor cells and subsequently to eliminate them. Special sort of lymphocytes –natural killer (NK) cells are able to provide this function by causing apoptosis of tumor cells using Fas ligand binding or granzymes. These processes are activated when signals from their inhibitory receptors are decreased and on the other hand, signals from activating receptors are increased. NKp30 is an activation receptor of NK cells with one Ig-like extracellular domain. Out of its ligands, two of them are stimulating NK cells through NKp30 when bound to membrane: B7-H6, a membrane protein with two Ig‑like domains, which is present on the surface of some tumor cells; and BAG-6, a large multifunctional protein normally found in cytoplasm or nucleus which is transported onto the cell surface under stress condition by an unknown way .
The structure of the B7-H6 ligand bound to NKp30 produced in E. coli has already been solved ; however, the structure of the BAG-6 ligand is yet to be elucidated. Moreover, the glycosylation and length of C‑terminal chain of NKp30 extracellular domain as well as its oligomerization status influence its ability to bind ligands . The structural basis of these effects is not known.
For our studies the extracellular domains of NKp30 and B7-H6 have been cloned into the pTW5sec vector with C-terminal histidine tag. To study the effect of C-terminal region of extracellular domain of NKp30, shorter and longer constructs have been cloned. Both proteins have been produced in human HEK293S GnTI- cell line possessing homogeneous N‑glycosylation profile, purified by TALON affinity column and size exclusion chromatography. Glycosylation of NKp30 was confirmed by mass spectrometry and formation of its oligomers was observed by analytical ultracentrifugation and transmission electron microscopy. Impact of glycosylation and C-terminal length of NKp30 construct was measuredusing analytical ultracentrifugation. Crystallization screens of the complex with glycosylated NKp30 constructs have been set up, too.
Library of 47 plasmids of BAG-6 was generated by cloning constructs of various lengths into vectors with TriEx plasmid backbone. Production of these constructs is to be screened in E. coli, as well as in Sf9 insect and HEK293S GnTI- human cell lines.
This study was supported by BIOCEV (ERDF CZ.1.05/1.1.00/02.0109), Czech Science Foundation (15-15181S), Ministry of Education, Youth and Sports of the Czech Republic (LG14009), Charles University (UNCE 204025/2012, SVV 260079/2014), BioStruct-X and Instruct European infrastructure projects.