NMR study of protein CD69

Monika Nálezková, Lukáš Gorčík, Lukáš Žídek and Vladimír Sklenář

National Centre for Biomolecular Research, Faculty of Science, Masaryk University in Brno, Czech Republic

Keywords: NMR, CD69, lectin-type proteins, NK cell, glycoproteins

The lectin-type proteins, like CD69, are transmembrane, disulphide-linked glycoproteins of natural killer (NK) cells. NK cells play an important role in the immune response to cell-surface displayed antigens, such as those found on virus infected cells, tumor cells, etc. Several oligosacharides were suggested to be natural ligands of CD69 receptor and NMR method was found convenient for observation of CD69 sacharide interaction.

NMR is noninvasive method, able to observe molecules in environment close to physiological conditions. Unfortunately, NMR is not very sensible and big amount of sample (300μl of 0.2mM at least), usually enriched by nuclei ¹⁵N and/or ¹³C, is needed. Preparation of well folded highly concentrated protein sample is thus crucial. At the beggining of this study, the construct of approximatelly 130 amino acid long extracelular part of human CD69 was prepared by the group of Karel Bezouška, Department of Biochemistry, Faculty of Science, Prague. NMR measurement of the construct showed that protein is totally unfolded. A construct of homologue of rat CD69 was then delivered. Basic 2D NMR experiment found partially structured protein.

In the summer 2005, the problem with folding of human CD69 was solved by our collaborators and experiments aimed at ligand binding were started. As binding of sacharides to CD69 receptor is supposed to be dependent on presence of Ca²⁺, titration experiments with CaCl₂ were performed, followed by titration with GlcNAc and pentaantenary oligosacharide. It was posible to observe several small changes of chemical shift in NMR spectra correlating chemical shifts of hydrogen and nitrogen. In order to study protein-ligand interactions in more details it was decided to prepare double (¹⁵N, ¹³C) labelled sample, and to assign side chain nuclei of the protein. The double-labelled sample has been obtained recently and assignment is currently in progress.

1. Pavlíček, J. et. al, Molecular Characterization of Binding of Calcium an Carbohydrates by an Early Activation Antigen of Lymphocytes CD69, Biochemistry 2003, 42, 9295-9306.

2. Pavlíček, J. et. al, Lymphocyte activation receptors: new structural paradigms in group V of C-type animal lectins, Biochemical Society Transactions 2004, 32, part 6.

Acknowledgements:

Karel Bezouška and his group for the sample, Ministry of Education. Youth and Sports of the Czech Republic, Grant Agency of the Czech Republic