Recombinant expression of protein NKp46 and its ligand Epa1
and study of their interaction

J. Houserová, B. Kalousková, O. Vaněk

Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague, Czech Republic

j.houserova@email.cz

One part of innate immunity is natural killer cells that play an essential role in the immune response of an organism. In contact with infected, stressed or tumour cells, the NK cells can trigger a cytotoxic response. Initiation of this mechanism depends on the presence of activating or inhibiting ligands on the surface of the target cells. NKp46 is one of the activation receptors. This receptor belongs to the group of natural cytotoxicity receptors and has many ligands. One of them is Epa1, a lectin-like epithelial adhesin occurring on the surface of yeast Candida glabrata. That is one of the most common agents of invasive candidiasis in the world. The interaction between NKp46 and its ligands in some cases (e.g., for hemagglutinin) depends on sialic acid O‑glycosylation on the surface of NKp46. It was found that the interaction of adhesin of C. glabrata depends on glycans located on the host cell surface.
One of those glycans is very similar to glycosylation on the NKp46 surface [1, 2]. The short-term objectives of this project were recombinant expressions of both Epa1 and NKp46 proteins. Protein Epa1 was successfully produced in bacterial expression system and purified using lactose affinity chromatography. Production of protein NKp46 is now being optimised. The protein is produced via recombinant expression in HEK293S GnTI^- and purified using TALON affinity a gel permeability chromatography. Building on that, the long-term goals are (i) biophysical description of the interaction of these proteins and (ii) crystallization of the NKp46:Epa1 complex. These results may contribute to the understanding of innate immune mechanisms and could be used in the design of immunotherapeutics.

1. Zupancic, M. L., Frieman, M., Smith, D., Alvarez, R. A., Cummings, R. D. and Cormack, B. P. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Molecular Microbiology, 68, (2008),
547–559.

2. Vitenshtein, A., Charpak-Amikam, Y., Yamin, R., Bauman, Y., Isaacson, B., Stein, N., Berhani, O., Dassa, L., Gamliel, M., Gur, C., et al. NK cell recognition of Candida glabrata through binding of NKp46 and NCR1 to fungal ligands Epa1, Epa6, and Epa7. Cell Host Microbe, 20, (2016), 527–534.

This study was supported by Czech Science Foundation (18-10687S) and Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126).