Estudio de la acción del óxido nítrico sobre la molécula HLA-G y sobre sus receptores ILT2 e ILT4

Resumo

EFFECT OF NITRIC OXIDE ON HLA-G AND THE RECEPTORS ILT2 AND ILT4 ÁNGEL DÍAZ LAGARES FACULTY OF SCIENCES. UNIVERSITY OF NAVARRA. 2009 The non-classical human leucocyte antigen (HLA) -G is a suppresive protein that is mainly expressed in placenta and ectopically in inflammatory tissues. HLA-G presents several isoforms and can also suffer posttranslational modifications such as multimers linked by disulfide- binding. HLA-G isoform are presented in membrane or in circulation, and due to the interaction with inhibitory receptors ILT2, ILT4 and NKG2D, HLA-G exerts a suppressive function. As this action is usually carried out in a proinflammatory medium with a high production of nitric oxide (NO), the aim of this work was to investigate the regulation of HLA-G and the receptors ILT2 and ILT4 by NO. Furthermore, we investigated if multimers or nitration of soluble HLA-G can occur in vivo. For these purposes we analysed the expression, nitration and function of HLA-G, ILT2 and ILT4 following treatment with the NO donor DETA-NO or the peroxynitrite donor SIN-1 in various cell lines. In addition, 8 samples of patients with inflammatory diseases were selected based on HLA-G production and nitrite concentration. Results showed NO-dependent nitration of both cellular and soluble HLA-G protein, but not all HLA-G moieties underwent nitration. Endogenous biosynthesis of NO by both U-937-HLA-G1 and M8-HLA-G5 stable transfectants also caused HLA-G nitration. Both NO and peroxynitrite decreased total HLA-G cellular protein content and expression on the cell surface, while increasing HLA-G shedding into the culture medium. This effect was post-transcriptional and metalloprotease-dependent. Moreover, in the biological samples after SDS-PAGE under non-reducing conditions we observed bands of HLA-G at 45, 75 and 150 kDa. Under reducing conditions we detected a band of 25 kDa, and a ladder of bands at 50-100 kDa. However in these reducing conditions the band at 45 kDa was not visible. Immunoprecipitation with anti nitrotyrosine antibody showed that there was nitrated HLA-G at 45 kDa, and also several bands at 100-200 kDa. On the other hand, we observed that preincubation of ILT2-expressing Natural Killer cell line (NKL) with DETA-NO further decreased the cytotoxic lysis of K562-HLA-G compared to untreated NKL. However, this treatment increased significantly the cytotoxicity against K562-PcDNA cells. DETA-NO did not modify ILT2 and ILT4 mRNA expression or protein production at the cell surface. Immunoprecipitation analysis showed that ILT2 and ILT4 protein was nitrated in these conditions. Furthermore, mass spectrometry analysis showed that not all Tyr residues in ILT2 were modified by peroxynitrite, but the Ty-35, Tyr-76, and Tyr-99, which are involved in HLA-G binding, appeared nitrated. ILT2 nitration increases the suppressive effect elicited by HLA-G. In conclusion, in this work we have demonstrated that NO nitrates HLA-G protein and decreases HLA-G cell surface expression by increasing metalloprotease-dependent shedding. HLA-G is present in biological fluids mainly as complexes and as nitrated protein.