Interactions of the NK cell receptors and endogenous ligands analyzed by Surface Plasmon Resonance (SPR).

PABLO NICOLAS ROMASANTA; JULIETA FERNANDEZ LYNCH; TODONE, MARCOS; BAUER, ANDREA B.; SOFÍA NOLI TRUANT; DE MARZI, MAURICIO C.; MALCHIODI, EMILIO L.; FERNANDEZ, MARISA M.

Ciudad de Tucuman

Congreso; XLI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE BIOFÍSICA.; 2012

Sociedad Argentina de Biofisica

To identify tumoral or infected cells, the NK cells have inhibitory and activating receptors that recognize MHC class I molecules, and non-self analogues present in target cells. In mice, the NK cells receptors belong to the Ly49 family. The recognition between activating Ly49s and self ligands modified by the murine cytomegalovirus (MCMV) protein m04 (Ly49P-MHC-I/m04)1, 2, 3 has been described. The inhibitory Ly49s interactions with MHC-I have also been reported4, 5. However, the recognition between activating Ly49s and MHC-I has not been investigated thoroughly. The aim of this work is to analyze the interaction between activating Ly49 (H, P, D, L) and the MHC-I molecule H2Dk through Surface Plasmon Resonance (SPR) employing BIAcore T100 equipment. Activating Ly49s (H, P, D, L), inhibitory Ly49s (G, I) and H2Dk, were expressed in E. coli BL21 as inclusion bodies and refolded in vitro yielding ~1 mg of purified protein per litre of culture. Site-specific biotinilation was achieved in the N-terminal region of the Ly49s to correctly orient them over the CM5 chip, which was previously used to immobilize streptavidin. Several concentrations of H2Dk were passed over the surfaces with Ly49 molecules immobilized. The KD for the interactions between the different Ly49s and H2Dk were as follows: H: no interaction; P: 0.64 μM; D: 36 μM; L: not determined; G: 5 μM; I: 20 μM. These results indicate that activating Ly49s are able to interact with H2Dk, despite the latter is not modified by m04. This biophysical study contributes with new evidence indicating that Ly49 receptors are involved in the NK cell education and generation of anergy for an optimal response to control pathogens.