Báo cáo khoa học: Tyrosine nitration in the human leucocyte antigen-Gbinding domain of the Ig-like transcript 2 protein ppt

Tyrosine nitration in the human leucocyte antigen-Gbinding domain of the Ig-like transcript protein ´ Angel Dıaz-Lagares1, Estibaliz Alegre1, Ainhoa Arroyo1, Fernando J Corrales2 ´ ´ and Alvaro Gonzalez1 Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain Division of Hepatology and Gene Therapy, Proteomics Unit, CIMA, University of Navarra, Pamplona, Spain Keywords HLA-G; ILT2; inflammation; natural killer; nitration Correspondence ´ ´ A Gonzalez, Department of Biochemistry, ´ University Clinic of Navarra, Avenida de Pıo XII, 36, 31008 Pamplona, Spain Fax: +34 948 296500 Tel: +34 948 255400 E-mail: agonzaleh@unav.es (Received 21 February 2009, revised 26 May 2009, accepted June 2009) doi:10.1111/j.1742-4658.2009.07131.x Ig-like transcript (ILT2) is a suppressive receptor that participates in the control of the autoimmune reactivity This action is usually carried out in a proinflammatory microenvironment where there is a high production of free radicals and NO However, little is known regarding whether these conditions modify the protein or affect its suppressive functions The present study aimed to investigate the suppressive response of the ILT2 receptor under oxidative stress To address this topic, we treated the ILT2-expressing natural killer cell line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine (DETA-NO) We observed that DETA-NO caused ILT2 protein nitration MS analysis of the chimeric recombinant human ILT2-Fc protein after treatment with the peroxynitrite donor 3-(morpholinosydnonimine hydrochloride) (SIN-1) showed the nitration of Tyr35, Tyr76 and Tyr99, which are involved in human leucocyte antigen-G binding This modification is selective because other Tyr residues were not modified by SIN-1 Recombinant human ILT2-Fc treated with SIN-1 bound a significantly higher quantity of human leucocyte antigen-G than untreated recombinant human ILT2-Fc DETA-NO did not modify ILT2 mRNA expression or protein expression at the cell surface Preincubation of NKL cells with DETA-NO decreased the cytotoxic lysis of K562-human leucocyte antigen-G1 cells compared to untreated NKL cells (P < 0.05) but increased cytotoxicity against K562-pcDNA cells (P < 0.05) Intracellular tyrosine phosphorylation produced after human leucocyte antigen-G binding was not affected by DETA-NO cell pretreatment These results support the hypothesis that the ILT2–human leucocyte antigen-G interaction should have a central role in tolerance under oxidative stress conditions when other tolerogenic mechanisms are inhibited Structured digital abstract l MINT-7144982: ILT2 (uniprotkb:Q8NHL6) binds (MI:0407) to HLA-G (uniprotkb:P17693) by affinity technologies (MI:0400) Introduction Peripheral tolerance is an important part of the immune defence system, comprising a mechanism to avoid the uncontrolled spread of immune attacks and autoreactivity against normal cells Of particular Abbreviations DETA-NO, N-(4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine; HLA, human leucocyte antigen; ILT2, Ig-like transcript 2; nitroTyr, nitrotyrosine; NK, natural killer; rh, recombinant human; SIN-1, 3-(morpholinosydnonimine hydrochloride) FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4233 ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain interest is tolerance during pregnancy, where maternal immune cells not attack the fetus, even though the fetus can be considered immunologically as a semiallogenic graft as a result of the expression of paternal antigens [1] One of the molecules implicated in the immune tolerance is the Ig-like transcript (ILT2), also known as CD85j, LIR-1 and LILRB1, comprising an inhibitory receptor expressed on monocytes, dendritic cells, T cells, B cells and natural killer (NK) cells [2] ILT2 belongs to the Ig superfamily, where the extracellular domains D1 and D2 bind the a3 domain of both classical and nonclassical human leucocyte antigen (HLA)-I molecules [3], but with higher affinity to HLA-G than to classical HLA-I [4] The cytoplasmic tail contains immunoreceptor tyrosine-based inhibitory motifs [5], which trigger a cellular inhibitory response, such as the suppression of NK cytotoxicity [6] Interaction between HLA-G and ILT2 usually takes place in vivo in a proinflammatory microenvironment where free radicals are available that could modify this interaction Of special importance is NO, which is a very reactive free radical synthesized from l-arginine by the enzyme NOS [7] NO has pleiotropic immune actions controlling inflammation and tissue damage, including immune cell proliferation and function, and cytokine production [7,8] For example, NO increases macrophage and NK cell function [9,10] and downregulates the T helper cell response, favouring a T helper reaction [11] NO-derived metabolites peroxynitrite or nitrite, in conjunction with peroxidases, can react with tyrosine to produce nitrotyrosine (nitroTyr) at the inflammatory site [12] This modification can induce deep changes in the physicochemical properties of the proteins, affecting their stability or functionality [13] Furthermore, tyrosine nitration comprises a reversible reaction [14] that affects a limited number of proteins and few tyrosine residues, and it can influence different biological activities [13] For example, the immunosuppressive enzyme indoleamine 2,3-dioxygenase is inactivated by high concentrations of NO [15] NitroTyr has been detected in many disorders, such as preeclampsia [16], bacterial and viral infection, and chronic inflammation [17] To date, there is a scarcity of data available concerning how inflammatory stress affects the interaction between HLA-G and its receptors We recently reported that NO can nitrate HLA-G, increasing its metalloprotease-dependent shedding to the medium [18] This modified HLA-G conserves its suppressive properties, allowing the spread of the tolerogenic microenvironment To determine whether HLA-G 4234 receptors are also capable of responding pressive stimulus under oxidative stress, study aimed to investigate the effect of expression and function of the ILT2 receptor to the supthe present NO in the suppressive Results and Discussion NO modifies ILT2 protein by tyrosine nitration Protein nitration is a post-translational modification caused by NO derivates, that can modify protein structure and function [13] Initially, we wanted to analyze whether ILT2 was susceptible to being nitrated (Fig 1) After NKL cell treatment with N-(4-[1-(3-aminopropyl)2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine (DETA-NO) 100 lm for 24 h, we immunoprecipitated the cell lysate with anti-nitrotyrosine serum Western blotting using anti-ILT2 serum HP-F1 showed a band of approximately 90 kDa, which was not present in untreated control cells (Fig 1A) Similarly, this band did not appear in the control of specificity, where antinitrotyrosine serum was preincubated with 3-nitrotyro- Fig Immunoblot analyses of ILT2 nitration in NKL cells (A) and U-937 cells (B), untreated or treated with DETA-NO 100 lM or with SIN-1 100 lM Cell lysates were immunoprecipitated using anti-3nitrotyrosine serum The control (+) corresponds to a cell lysate of NKL cells A negative control was performed by preincubation of the antibody with 3-nitrotyrosine mM Immunoprecipitated proteins were separated by SDS ⁄ PAGE, blotted onto a nitrocellulose membrane, and then probed with HP-F1 anti-ILT2 serum A representative experiment out of three is shown FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ A Dıaz-Lagares et al sine mm before immunoprecipitation To determine whether endogenous NO production can also cause ILT2 nitration, we used the U-937 cell line, which produces NO that nitrates intracellular proteins [15,18] Interestingly, there was a band of nitrated ILT2 in the lane corresponding to untreated U-937 cells (Fig 1B) As a positive control of nitration, U-937 cells were treated with DETA-NO or 3-(morpholinosydnonimine hydrochloride) (SIN-1) 100 lm for 24 h These results show that ILT2 can undergo nitration, which should be related to the presence of exposed Tyr residues [19–21] To our knowledge, this is the first report of a post-translational modification of the ILT2 protein The other member of the ILT family, LILRA4, has also been found nitrated within the domain Ig-like C2-type in human tumour tissues [22] Although most of the effects of nitration cause functional loss [23], protein nitration can also elicit increased biological activity, such as in cathepsin D [24], or in the glucocorticoid receptor, where nitration leads to an increase in binding capacity [25] Identification of nitration site in the extracellular domain of ILT2 In the extracellular domain of ILT2, there are several Tyr residues that participate in the interaction with HLA-G [3,19] Because protein nitration is a phenomenon that cannot be predicted from the amino acid sequence, we were very interested in analyzing whether ILT2 nitration affected the Tyr residues in the hydrophobic interdomain that binds HLA-G To address this issue, we used a commercial recombinant human recombinant human (rh)ILT2-Fc chimera, which possess the extracellular domain and maintains the HLAG binding capacity [19] This protein was treated for h with the pure peroxynitrite donor SIN-1 mm As a negative control, we processed untreated rhILT2-Fc simultaneously After tryptic digestion, the presence of nitrotyrosine in the resultant peptides was analyzed by LC-MS ⁄ MS Under these experimental conditions, we analyzed 40% of the ILT2 extracellular domain (Fig 2A), including Tyr76 that is suggested to participate in HLA-G binding [19] We identified six peptides with nitrated Tyr that were not present in the untreated control These nitroTyr corresponded to positions Tyr35, Tyr76, Tyr77, Tyr99, Tyr229 and Tyr355 (Figs 2B–E and Table 1) In particular, the charged ions CQGGQETQEYR and the corresponding fragment y2, with m ⁄ z = 700.784, and the CYYGSDTAGR and the corresponding fragments y9 and b2, with m ⁄ z = 597.74, showed an increased mass of ILT2 nitration in the binding domain 45 Da as a result of the acquisition of a nitro group in Tyr35 and Tyr76, respectively However, not all rhILT2-Fc was nitrated because these peptides also appeared without nitration (Table 1) Furthermore, other residues analyzed (i.e Tyr235 and Tyr372) were resistant to nitration The fact that the nitration is partial is not surprising because, even for proteins that are easy targets for nitration, the relative yield of nitroTyr formation under inflammatory conditions is low [26] Because we were unable to sequence more than 38% of the Tyr residues, we cannot rule out the possibility that other tyrosines could also be nitrated Nevertheless, these data demonstrate that the binding domain of ILT2 could undergo nitration, which implies conformational changes ILT2 nitration increases HLA-G binding To determine whether treatment with NO modifies the interaction of ILT2 with HLA-G, we performed a binding assay against HLA-G, where the capture molecule was rhILT2-Fc pre-treated with different concentrations of SIN-1 As shown in Fig 3, SIN-1 treatment significantly increased rhILT2-Fc binding to HLA-G (150 ± 18%; HLA-G binding to SIN-1 mm treated rhILT2-Fc compared to untreated rhILT2-Fc; P < 0.05) As a positive control of HLA-G binding, we used the capture serum anti-HLA-G MEM-G ⁄ [18], which produced 315% of HLA-G binding compared to untreated rhILT2-Fc These results are in agreement with the MS analyses because Tyr76 participates directly in the interaction with HLA-G [3,19] and Tyr35 is located in the very vicinity of Tyr38 These modifications should affect the binding pocket directly Furthermore, Tyr99 stabilizes the angle between D1 and D2 domains, which is necessary for HLA-G binding [3,19], and the modification of this angle should also affect the interaction with HLA-G Effectively, tyrosine nitration causes a shift in the pKa of the tyrosine hydroxyl group and makes the nitrated tyrosine more hydrophobic and prone to move into more hydrophobic regions [13,26] These modifications could induce changes in protein structure and function that affect the affinity of the interaction between ILT2 and HLA-G NO does not affect ILT2 expression NO modulates the expression of multiple genes [7] To determine whether NO affects ILT2 expression, we treated NKL cells with increasing quantities of DETANO for 24 h Real-time RT-PCR analysis indicated FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4235 ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain Fig (A) Amino acid sequence coverage and sites of nitration of SIN-1-treated rhILT2-Fc, obtained by LC-MS ⁄ MS analysis Protein was nitrated with SIN-1, subjected to trypsin digestion, and peptides were separated on a reverse phase HPLC column online with ESI and ion trap MS The amino acid sequence coverage obtained by LC-MS ⁄ MS is shown in bold Nitrated peptides are underlined and nitrated Tyr are indicated by asterisks (B–E) Annotated mass spectra of peptides containing nitrotyrosine observed after the reaction of SIN-1 mM with rhILT2-Fc (F) Annotated mass spectra of the same peptide as in (E) but without nitrotyrosine residues that DETA-NO did not modify the transcriptional levels of ILT2 (Fig 4) Similarly, western blot analysis showed that DETA-NO did not change ILT2 protein content and flow cytometry analysis revealed no change in ILT2 cell surface expression We concluded that the effect of NO in the ILT2 receptor is limited to a post-translational modification ILT2 maintains its suppressive function in the presence of NO Finally, we aimed to determine whether the presence of NO under conditions known to nitrate ILT2 could affect the sensitivity to HLA-G Accordingly, we incubated NKL cells with DETA-NO 100 lm for 24 h and then performed a cytotoxicity assay using either K562pcDNA or K562-HLA-G1 as target cells (Fig 5A) 4236 The possible cytotoxic effect of NO was avoided because this compound was not present during the cytotoxic assay As previously described [2,6], we observed a significant decrease in the lysis of K562HLA-G1 cells compared to K562-pcDNA cells at a 50 : effector : target cell ratio (P < 0.05) Preincubation of NKL cells with DETA-NO increased K562pcDNA cell lysis (P < 0.05), whereas it significantly decreased K562-HLA-G1 cell lysis (P < 0.05) This increased NKL cytotoxicity against K562pcDNA after incubation with DETA-NO is in agreement with previous findings where NO released by macrophages was found to participate in the functional maturation of NK cells [7] However, these more activated NKL cells have an even lower killing function against K562-HLA-G1 cells It has been demonstrated that the inhibition of NKL cytotoxicity against FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain Fig (Continued) K562-HLA-G1 is a result of the interaction of HLA-G with ILT2 [2,27] We verified these data under our experimental conditions by preincubating NKL cells with the monoclonal anti-ILT2 serum GHI ⁄ 75 (10 lgỈmL)1) Blockade of the ILT2 receptor impaired HLA-G suppression of NKL cell cytotoxicity, regardless of whether it was treated or not with DETA-NO (33 ± 5% K562-HLA-G1 cell lysis) These results indicate that NO maintains, or even increases, ILT2mediated suppression in NKL cells After HLA-G binding, immunoreceptor tyrosinebased inhibitory motifs in the cytoplasmic tail of the ILT2 receptor become tyrosine phosphorylated, eliciting a suppressive response [4,5] The results shown in Fig 5A suggest that tyrosine phosphorylation is not modified by NO treatment because the suppression caused by ILT2–HLA-G interaction was not blocked by the addition of DETA-NO To further confirm these data, we studied intracellular phosphotyrosine formation in NKL cells after incubation with FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4237 ILT2 nitration in the binding domain ´ A Dıaz-Lagares et al Fig (Continued) 4238 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain Table Nitrated peptides from rhILT2-Fc Recombinant protein was untreated (control) or treated with SIN-1 mM Nitrated Tyr are shown in bold and marked with asterisks Score Nitrated tyrosine (domain) Tyr35 (D1) Tyr76 (D1) Tyr77 (D1) Tyr99 (D1) Tyr229 (D3) Tyr355 (D4) Peptide Control SIN-1 CQGGQETQEYR CQGGQETQEY*R CYYGSDTAGR CY*YGSDTAGR CYY*GSDTAGR SESSDPLELVVTGAYIK SESSDPLELVVTGAY*IK KPSLSVQPGPIVAPEE TLTLQCGSDAGYNR KPSLSVQPGPIVAPEETLT LQCGSDAGY*NR YQAEFPMGPVTSAHAGTYR Y*QAEFPMGPVTSAHAGTYR 13.78 – 7.77 – – 10.49 – 13.06 14.19 8.88 9.69 6.61 6.15 14.28 8.85 19.50 – 12.35 – 10.64 16.64 10.67 results indicate that NO does not affect tyrosine phosphorylation, which is related to our previous observation that ILT2 maintains its suppressive function in the presence of NO (Fig 5A) Modulation of ILT2–HLA-G interactions by NO could be especially important in the placenta, in which HLA-G is expressed [28], because the most important immune population comprises the NK cells [29] and there is a controlled state of inflammation with high NO production [30] NO causes metalloproteasedependent HLA-G shedding and nitrates both HLA-G [18] and ILT2, although also allowing these proteins to conserve their suppressive function These results suggest that the ILT2–HLA-G interaction is an important mechanism for controlling NK cell immune attacks under inflammatory oxidative stress, and under conditions where other suppressive molecules are inactivated [15] Experimental procedures Cell culture The NK cell line, NKL, the monocytic cell line, U-937, and the MHC class I-deficient human erythroleukaemia transfected cells, K562-HLA-G1 and K562-pcDNA (kindly provided by E D Carosella, SRHI-CEA, Paris, France), were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, mm glutamine, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin (Gibco BRL ⁄ Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 humidified atmosphere For NKL cells, 50 mL)1 rhIL-2 (Roche Molecular Biochemicals, Mannheim, Germany) was added to the culture medium NO donors were DETA-NO (Alexis Corporation, Lausane, Switzerland) SIN-1 (Alexis Corporation) The rhILT2-Fc chimera was purchased from R&D Systems (Abingdon, UK) Cellular viability measured by trypan blue exclusion was higher than 95% throughout the study Fig Effect of the peroxynitrite donor SIN-1 on the capability of rhILT2-Fc to bind HLA-G rhILT2-Fc was treated with increased concentrations of SIN-1 for h at 37 °C The results show the relative quantities of the HLA-G concentration compared to untreated control rhILT2-Fc (assigned a value of 100) and are expressed as the mean ± SD of three different experiments *P < 0.05 compared to untreated control rhILT2-Fc supernatants containing HLA-G for Flow cytometric analysis of intracellular phosphotyrosine using anti-phosphotyrosine serum showed that HLA-G caused a shift in the fluorescence compared to untreated control cells (Fig 5B) NKL cells preincubation with DETA-NO 100 lm for 24 h did not modify this HLA-G-induced tyrosine phosphorylation These Cytotoxic assay NKL cell cytotoxicity against the K562 cell line was evaluated in a standard h 51Cr release assay K562-HLA-G1 or K562-pcDNA transfected cells were incubated for h at 37 °C with 51Cr After two washes with RPMI-1640 medium, target cells were co-cultured with NKL effector cells for h at 37 °C NKL cells were previously stimulated with IL-2 (100 mL)1) for 24 h in presence or absence of DETA-NO 100 lm Co-culture was performed in triplicate and at several K562 : NKL ratios from : to : 50 After h, 50 lL of each supernatant were mixed with 250 lL of scintillation buffer (PerkinElmer, Waltham, MA, USA) in a 96-well plate and read in a b-radiation counter (Wallac 1450; Amersham Biosciences, Uppsala, Sweden) FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4239 ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain Fig ILT2 expression in NKL cells treated with different concentrations of DETA-NO for 24 h Upper: flow cytometry of ILT2 surface expression using anti-ILT2-PE serum Grey histograms represent control cells and open histograms represent cells treated with DETA-NO Grey lines represent irrelevant isotypic antibody Data are representative of three different experiments Lower left: HLA-G mRNA expression analyzed by real-time RT-PCR Data are shown as the relative quantities of ILT2 transcripts compared to control GAPDH expression The results are compared to untreated control cells (assigned a value of 1) and are expressed as the mean ± SD of three different experiments Lower right: western blot analysis of ILT2 expression Bands of ILT2, immunodetected with HP-F1 anti-ILT2 antibody, appeared at 90 kDa Loading control was performed using an antibody against b-actin, which produced a band at 42 kDa The data indicate the intensity of the HLA-G band related to the b-actin band and are representative of three different experiments Specific lysis level was calculated as the percentage 51Cr release from the maximum release: % specific lysis = 100 · [(sample c.p.m ) spontaneous release) ⁄ (maximum release ) spontaneous release)] The spontaneous release was the c.p.m measured in 51 Cr-labelled K562 cells cultured in medium without NKL cells The maximum release was achieved when 51Crlabelled K562 cells were incubated with Triton-X100 Blocking experiments of ILT2 were performed by incubating treated and untreated NKL cells with monoclonal anti-ILT2 serum GHI ⁄ 75 (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) for 30 at 37 °C before co-culturing them with K562 cells Flow cytometry For cell surface labelling, cells were incubated for 30 at °C in NaCl ⁄ Pi containing 20% human serum (SigmaAldrich, St Louis, MO, USA), and stained with PE conjugated anti-ILT2 serum (Beckman Coulter, Marseille, France) for 20 at °C After washing, cells were fixed in paraformaldehyde 1% For intracellular staining, cells were fixed with paraformaldehyde 1% for 10 at 37 °C and permeabilized with 90% methanol for 30 on ice After washing with NaCl ⁄ Pi-BSA 0.5%, cells were stained 4240 with Alexa Fluor 488-conjugated anti-phosphotyrosine serum (Beckman Coulter) for 30 min, washed with NaCl ⁄ Pi-BSA 0.5%, and resuspended in NaCl ⁄ Pi for flow cytometry analysis Control aliquots were stained with the isotype-matched mouse antibody (Beckman Coulter) Fluorescence was detected by an EPICS XL flow cytometer (Beckman Coulter) Real-time RT-PCR analysis Real-time PCR analysis was used to quantify variations in the amounts of ILT2 transcripts after cell treatment with DETA-NO Total RNA was extracted from 3–5 million NKL cells using RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions Residual DNA was eliminated by DNase I treatment (10–20 units per 100 lg; Roche Molecular Biochemicals) for h at 25 °C Reverse transcription was carried out using High-Capacity cDNA Archive Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA USA) Real-time PCR was performed using the TaqMan Gene Expression Assay (Applied Biosystems) on an ABI PRISM 7700 Sequence Detector (Applied Biosystems) and GAPDH expression was used as internal standard FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain precipitation was performed with a protein A-sepharose assay kit purchased from Pierce Biotechnology Inc (Rockford, IL, USA) according to the manufacturer’s instructions Western blotting Protein concentration was quantified by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) using BSA as standard After centrifugation at 10 000 g for min, 20 lg of total protein were denatured at 100 °C for in a protein sample buffer containing 125 mm Tris-ClH (pH 6.8), 4% SDS, 30% glycerol, 5% b-mercaptoethanol and 0.4% bromophenol Proteins were subjected to 10% PAGE under denaturing conditions (SDS ⁄ PAGE), with subsequent electroblotting transfer onto a nitrocellulose membrane The membrane was blocked with 5% nonfat dried milk in NaCl ⁄ Pi-Tween 0.1% for h at room temperature, and then incubated for h with HP-F1 anti-ILT2 serum (kindly provided by M Lopez´ ` Botet, Institut Municipal d’Investigacio Medica, Barcelona, Spain) diluted : 500 in NaCl ⁄ Pi-Tween, or anti-b-actin (Abcam, Cambridge, UK), diluted : 5000 in NaCl ⁄ Pi-Tween Immunoblot detection was performed using an horseradish peroxidase-conjugated anti-mouse antibody (dilution : 5000; Amersham Biosciences) and developed using the ECL kit (Amersham Biosciences) For incubation with additional antibodies, the membranes were previously stripped for 30 at 56 °C in 62.5 mm Tris (pH 6.8), 2% SDS and 100 mm b-mercaptoethanol Fig (A) Effect of DETA-NO on HLA-G-mediated inhibition of NKL cytotoxicity The data show the percentage (± SD) of specific lysis achieved by NKL cells during h of co-culture, with K562-pcDNA or K562-HLA-G1 cells as target cells, in a 50 : effector : target cell ratio NKL cells were previously incubated without or with DETA-NO 100 lM for 24 h The results are expressed as the mean of three different experiments performed in triplicate *P < 0.05 (B) Effect of HLA-G on phosphotyrosine formation in NKL cells preteated or not with DETA-NO Cells were cultivated for 24 h with or without DETA-NO 100 lM After cell washing, supernatants containing HLA-G were added and incubated for Cells were then fixed, perma permeabilized, and stained with anti-phosphotyrosine serum Dotted peaks represent irrelevant isotypic antibody The histograms shown are representative of four different experiments M.f.i., mean fluorescence intensity Nitrotyrosine immunoprecipitation Cells were lysed in NP40 0.5% in Tris-HCl buffer with protease inhibitors (Roche Applied Sciences, Mannheim, Germany) and incubated with anti-nitrotyrosine serum (Upstate Biotechnology, Lake Placid, NY, USA) at a dilution of : 230 for 30 [15] Preincubation of anti-nitrotyrosine serum with nitrotyrosine mm (Sigma-Aldrich) for h was used as control of immune specificity Immuno- LC-ESI-MS ⁄ MS analysis Fifteen micrograms of rhILT2-Fc fusion protein were treated with SIN-1 mm for h at 37 °C in continuous agitation Then, nitrated rhILT2-Fc was precipitated with trichloroacetic acid 20%, reduced with dithiotheitol 10 mm in ammonium bicarbonate 100 mm, and alkilated with iodoacetamide 55 mm The protein was resuspended in ammonium bicarbonate 50 mm and digested with ngỈlL)1 trypsin for h at 37 °C The rhILT2-Fc negative control was processed in the same way, except for the nitration treatment MS ⁄ MS analysis was performed as previously described [31] Microcapillary reversed phase LC was performed with a CapLCÔ (Waters, Milford, MA, USA) capillary system Reversed phase separation of tryptic digests was carried out with an Atlantis, C18, lm, 75 lm · 10 cm Nano EaseÔ fused silica capillary column (Waters) equilibrated in 5% acetonitrile and 0.2% formic acid After injection of lL of sample, the column was washed for with the same buffer and the peptides were eluted using a linear gradient of 5–50% acetonitrile over 45 at a constant flow rate of 0.2 lLỈmin)1 The column was coupled online to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters) The heated capillary temperature was 80 °C and the spray voltage was FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4241 ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain 1.8–2.2 kV MS ⁄ MS data were collected in an automated data-dependent mode The three most intense ions in each survey scan were sequentially fragmented by collisioninduced dissociation using an isolation width of 2.0 and a relative collision energy of 35 V Data processing was performed with masslynx, version 4.1 Database searching was carried out using proteinlynx global server 2.3 (Waters) and phenyx, version 2.5 (GeneBio, Geneva, Switzerland) The search was enzymatically constrained for trypsin and allowed for one missed cleavage site Further search parameters were: no restriction on molecular weight and isoelectric point; carbamidomethylation of cysteine; variable modification; and oxidation of methionine HLA-G binding assay rhILT2-Fc was treated with increasing concentrations of SIN-1 for h at 37 °C Polystyrene microtiter plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 10 lgỈmL)1 rhILT2-Fc, or with 10 lgỈmL)1 anti-HLA-G MEM G ⁄ (Exbio, Prague, Czech Republic) in NaCl ⁄ Pi overnight at °C Plates were washed with NaCl ⁄ PiTween 0.2%, and blocked with NaCl ⁄ Pi-BSA 3% for h Then, equal quantities of supernatant containing HLA-G were added and incubated for 90 at 37 °C After washing, anti-b2-microglobulin serum (Dako, Glostrup, Denmark) was added and incubated for h at 37 °C HLA-G binding was detected using EnVision+ Dual Link System-HRP (Dako) and 3,3¢,5,5¢-tetramethylbenzidine (Sigma-Aldrich) Colour development was stopped with HCl m and the absorbance was measured at 450 nm in a microplate reader Multiskan Ascent (Thermo Fisher Scientific, Waltham, MA, USA) Results were normalized to the absorbance obtained from the untreated control rhILT2-Fc Statistical analysis Data are expressed as the mean ± SD Statistical analysis was performed using the spss statistical program for Windows (SPSS Inc., Chicago, IL, USA) Results were compared with nonparametric Kruskal–Wallis and Mann– Whitney U-tests P < 0.05 was considered statistically significant Acknowledgements This work was supported by the Fondo de Investiga´ cion Sanitaria E.A was the recipient of a grant from ´ Fondo de Investigacion Sanitaria PI070298 and ´ A.D.L received a grant from Asociacion Amigos Universidad de Navarra and Caixanova The laboratory of Proteomic CIMA is member of the National Institute of Proteomics Facilities, ProteoRed 4242 References Moffett A & Loke C (2006) Immunology of placentation in eutherian mammals Nat Rev Immunol 6, 584–594 Colonna M, Navarro F, Bellon T, Llano M, Garcia P, Samaridis J, Angman L, Cella M & Lopez-Botet M (1997) A Common Inhibitory Receptor for Major Histocompatibility Complex Class I Molecules on Human Lymphoid and Myelomonocytic Cells J Exp Med 186, 1809–1818 Chapman TL, Heikema AP, West AP Jr & Bjorkman PJ (2000) Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2) Immunity 13, 727–736 Shiroishi M, Tsumoto K, Amano K, Shirakihara Y, Colonna M, Braud VM, Allan DSJ, 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Journal 27 6 (20 09) 423 3– 424 3 ª 20 09 The Authors Journal compilation ª 20 09 FEBS ´ A Dıaz-Lagares et al ILT2 nitration in the binding domain Fig (Continued) K5 62- HLA-G1 is a result of the interaction... discovered with a ILT2 nitration in the binding domain 23 24 25 26 27 28 29 30 31 nitrotyrosine affinity column and tandem mass spectrometry Anal Biochem 354, 27 9? ?28 9 Fujigaki H, Saito K, Lin F, Fujigaki