Preparation of antennary oligosaccharide ligands from ovomucoid and their binding to CD69 receptor

M. Navrátil¹, P. Pompach², K. Bezouška^1,2

¹ Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8,

CZ-12840 Prague 2.

² Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 2.

Oligosaccharides form a very heterogeneous group of compounds. The ability of their monosaccharide units to bind to each other in many ways predicts them to be very variable in both structure and function. It is well known that saccharide structures play important role in immune system, from direct recognition of pathogenic saccharide structures to protein glycosylation, which can have also modulatory or self-protective function. One group of very interesting oligosaccharide compounds are branched antennary oligosaccharides which are present at the surface of tumour cells and are recognized by C-type lectin-like receptors of NK cells. Immune recognition is highly specific and only small difference in epitope structure may lead to opposite response, exact knowledge of this structure is therefore a necessity. Chemical synthesis¹ of antennary oligosaccharide structures is quite difficult. In contrast, there are glycoproteins that provide defined oligosaccharides structures in sufficient yields. Thus, the isolation of native structures from natural sources seems to be a more efficient and direct way. In order to research for physiological ligands^2,3 for C-type lectin-like receptors of NK cells we developed a relatively fast method for preparation of pure and well defined antennary oligosaccharides from hen egg white protein ovomucoid. This procedure involves isolation of ovomucoid by fractional precipitation, followed by its purification on HPLC, enzymatic deglycosylation of ovomucoid and finally purification and separation of cleaved oligosaccharides by HPLC. The purity and identity was verified by MALDI-TOF mass spectrometry. Biochemical binding and inhibiton experiments with CD69 receptor revealed the highest affinity for the pentaantennary oligosaccharide. Oligosaccharides were used for structural studies by means of cocrystalisation with CD69, and examination of CD69-saccharide complexes by MS and NMR titration.

1. Y. Kajihara, N. Yamamoto, T. Miyazaki, H. Sato, Curr. Med. Chem., 12(5), (2005), 527-50

2. K. Bezouška, G. Vlahas, O. Horváth, G. Jinochová, A. Fišerová, R. Giorda, W. H. Chambers, T. Feizi, M. Pospíšil, J. Biol. Chem., 269(24), (1994), 16945.

3. K. Bezouška, J. Sklenář, J. Dvořáková, V. Havlíček, M. Pospíšil, J. Thiem, V. Křen , Biochem. Biophys. Res. Commun., 238, (1997), 149.

This work was supported by Ministry of Education of the Czech Republic No. MSM 0021620808, by Institutional Research Concept No. AVOZ 50200510, by Grant Agency of the Czech Republic No. 204/06/0771 and 301/05/P567, and by Grant Agency of the Academy of Sciences of the Czech Republic No. A5020403.