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

Tyrosine nitration in the human leucocyte antigen-G- binding domain of the Ig-like transcript 2 protein Angel Dı´az-Lagares1, Estibaliz Alegre1, Ainhoa Arroyo1, Fernando J. Corrales2 and A´ lvaro Gonza´ lez1

1 Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain 2 Division of Hepatology and Gene Therapy, Proteomics Unit, CIMA, University of Navarra, Pamplona, Spain

Keywords HLA-G; ILT2; inflammation; natural killer; nitration

Correspondence A´ . Gonza´ lez, Department of Biochemistry, University Clinic of Navarra, Avenida de Pı´o 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 4 June 2009)

doi:10.1111/j.1742-4658.2009.07131.x

Ig-like transcript 2 (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 condi- tions modify the protein or affect its suppressive functions. The present study aimed to investigate the suppressive response of the ILT2 receptor under oxi- dative stress. To address this topic, we treated the ILT2-expressing natural line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2- killer cell hydroxy-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 per- oxynitrite donor 3-(morpholinosydnonimine hydrochloride) (SIN-1) showed the nitration of Tyr35, Tyr76 and Tyr99, which are involved in human leuco- cyte antigen-G binding. This modification is selective because other Tyr resi- dues 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. Preincuba- tion 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 pretreat- ment. 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

tolerance is an important part of

Peripheral 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).

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receptors are also capable of responding to the sup- pressive stimulus under oxidative stress, the present study aimed to investigate the effect of NO in the expression and function of the ILT2 suppressive receptor.

Results and Discussion

NO modifies ILT2 protein by tyrosine nitration

interest is tolerance during pregnancy, where maternal immune cells do not attack the fetus, even though the fetus can be considered immunologically as a semiallo- genic 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 2 (ILT2), also known as CD85j, LIR-1 and LILRB1, comprising an inhibitory receptor expressed on monocytes, den- dritic 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 cytoplas- mic tail contains immunoreceptor tyrosine-based inhib- itory motifs [5], which trigger a cellular inhibitory response, such as the suppression of NK cytotoxicity [6].

Protein nitration is a post-translational modification caused by NO derivates, that can modify protein struc- ture 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 anti- nitrotyrosine serum was preincubated with 3-nitrotyro-

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 down- regulates the T helper 1 cell response, favouring a T helper 2 reaction [11].

NO-derived metabolites peroxynitrite or nitrite, in conjunction with peroxidases, can react with tyrosine to produce nitrotyrosine (nitroTyr) at the inflamma- tory site [12]. This modification can induce deep changes in the physicochemical properties of the pro- teins, affecting their stability or functionality [13]. Fur- thermore, 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 immunosup- pressive enzyme indoleamine 2,3-dioxygenase is inacti- vated by high concentrations of NO [15]. NitroTyr has been detected in many disorders, such as preeclampsia [16], bacterial and viral infection, and chronic inflam- mation [17].

receptors. We

Fig. 1. 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-3- nitrotyrosine serum. The control (+) corresponds to a cell lysate of NKL cells. A negative control was performed by preincubation of the antibody with 3-nitrotyrosine 1 mM. Immunoprecipitated pro- teins were separated by SDS ⁄ PAGE, blotted onto a nitrocellulose membrane, and then probed with HP-F1 anti-ILT2 serum. A repre- sentative experiment out of three is shown.

To date, there is a scarcity of data available con- cerning how inflammatory stress affects the interaction between HLA-G and its 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

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45 Da as a result of the acquisition of a nitro group in Tyr35 and Tyr76, respectively.

also

peptides

appeared without

that

the nitration is partial

sine 1 mm before immunoprecipitation. To determine whether endogenous NO production can also cause ILT2 nitration, we used the U-937 cell line, which pro- duces 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 trea- ted with DETA-NO or 3-(morpholinosydnonimine hydrochloride) (SIN-1) 100 lm for 24 h.

However, not all rhILT2-Fc was nitrated because these nitration (Table 1). Furthermore, other residues analyzed (i.e. Tyr235 and Tyr372) were resistant to nitration. The fact is not surprising because, even for proteins that are easy targets for formation the relative yield of nitroTyr nitration, 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

These results show that ILT2 can undergo nitra- tion, 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 4 in human tumour tis- sues [22]. Although most of the effects of nitration cause functional loss [23], protein nitration can also elicit increased biological activity, such as in cathep- sin 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

the

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 mole- cule was rhILT2-Fc pre-treated with different concen- trations 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 2 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 ⁄ 9 [18], which produced 315% of HLA-G binding com- pared to untreated rhILT2-Fc. These results are in agreement with the MS analyses because Tyr76 partici- pates 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.

that were not present

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 DETA- NO for 24 h. Real-time RT-PCR analysis indicated

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 phenome- non that cannot be predicted from the amino acid sequence, we were very interested in analyzing whether ILT2 nitration affected the Tyr residues in the hydro- phobic interdomain that binds HLA-G. To address this issue, we used a commercial recombinant human recombinant human (rh)ILT2-Fc chimera, which pos- sess the extracellular domain and maintains the HLA- G binding capacity [19]. This protein was treated for 3 h with the pure peroxynitrite donor SIN-1 2 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 ILT2 extracellular domain analyzed 40% of (Fig. 2A), including Tyr76 that is suggested to partici- pate in HLA-G binding [19]. We identified six peptides with nitrated Tyr 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 correspond- ing fragment y2, with m ⁄ z = 700.784, and the CY- YGSDTAGR and the corresponding fragments y9 and b2, with m ⁄ z = 597.74, showed an increased mass of

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Fig. 2. (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 2 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 lev- els 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.

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 K562- HLA-G1 cells compared to K562-pcDNA cells at a 50 : 1 effector : target cell ratio (P < 0.05). Preincuba- tion of NKL cells with DETA-NO increased K562- pcDNA cell lysis (P < 0.05), whereas it significantly decreased K562-HLA-G1 cell lysis (P < 0.05).

This

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 incu- bated NKL cells with DETA-NO 100 lm for 24 h and then performed a cytotoxicity assay using either K562- pcDNA or K562-HLA-G1 as target cells (Fig. 5A).

increased NKL cytotoxicity against K562- pcDNA after incubation with DETA-NO is in agree- ment with previous findings where NO released by macrophages was found to participate in the functional maturation of NK cells [7]. However, these more acti- vated NKL cells have an even lower killing function against K562-HLA-G1 cells. It has been demon- strated that the inhibition of NKL cytotoxicity against

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Fig. 2. (Continued).

After HLA-G binding,

anti-ILT2

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 serum GHI ⁄ 75 with the monoclonal (10 lgÆmL)1). Blockade of the ILT2 receptor impaired HLA-G suppression of NKL cell cytotoxicity, regard- less of whether it was treated or not with DETA-NO lysis). These results (33 ± 5% K562-HLA-G1 cell indicate that NO maintains, or even increases, ILT2- mediated suppression in NKL cells.

immunoreceptor tyrosine- based inhibitory motifs in the cytoplasmic tail of the ILT2 receptor become tyrosine phosphorylated, elicit- ing 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 phosphotyro- sine formation in NKL cells after incubation with

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Fig. 2. (Continued).

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Table 1. Nitrated peptides from rhILT2-Fc. Recombinant protein was untreated (control) or treated with SIN-1 2 mM. Nitrated Tyr are shown in bold and marked with asterisks.

results indicate that NO does not affect tyrosine phos- phorylation, which is related to our previous observa- tion that ILT2 maintains its suppressive function in the presence of NO (Fig. 5A).

Score

Control

SIN-1

Nitrated tyrosine (domain)

Peptide

Tyr35 (D1)

Tyr76 (D1) Tyr77 (D1)

Tyr99 (D1)

13.78 – 7.77 – – 10.49 – 13.06

14.19 8.88 9.69 6.61 6.15 14.28 8.85 19.50

CQGGQETQEYR CQGGQETQEY*R CYYGSDTAGR CY*YGSDTAGR CYY*GSDTAGR SESSDPLELVVTGAYIK SESSDPLELVVTGAY*IK KPSLSVQPGPIVAPEE TLTLQCGSDAGYNR

Tyr229 (D3)

–

10.64

Tyr355 (D4)

12.35 –

16.64 10.67

KPSLSVQPGPIVAPEETLT LQCGSDAGY*NR YQAEFPMGPVTSAHAGTYR Y*QAEFPMGPVTSAHAGTYR

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 metalloprotease- dependent HLA-G shedding and nitrates both HLA-G [18] and ILT2, although also allowing these proteins to conserve their suppressive function. These results sug- gest that the ILT2–HLA-G interaction is an important mechanism for controlling NK cell immune attacks under inflammatory oxidative stress, and under condi- tions 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 trans- fected cells, K562-HLA-G1 and K562-pcDNA (kindly pro- vided by E. D. Carosella, SRHI-CEA, Paris, France), were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomycin (Gibco BRL ⁄ Invitrogen, Carls- bad, CA, USA) at 37 (cid:2)C in a 5% CO2 humidified atmo- sphere. For NKL cells, 50 UÆmL)1 rhIL-2 (Roche Molecular Biochemicals, Mannheim, Germany) was added to the cul- ture medium. NO donors were DETA-NO (Alexis Corpora- tion, 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.

Cytotoxic assay

Fig. 3. Effect of the peroxynitrite donor SIN-1 on the capability of rhILT2-Fc to bind HLA-G. rhILT2-Fc was treated with increased con- centrations of SIN-1 for 3 h at 37 (cid:2)C. The results show the relative the HLA-G concentration compared to untreated quantities of 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.

in the fluorescence

NKL cell cytotoxicity against the K562 cell line was evalu- ated in a standard 4 h 51Cr release assay. K562-HLA-G1 or K562-pcDNA transfected cells were incubated for 1 h at 37 (cid:2)C with 51Cr. After two washes with RPMI-1640 med- ium, target cells were co-cultured with NKL effector cells for 4 h at 37 (cid:2)C. NKL cells were previously stimulated with IL-2 (100 UÆmL)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 1 : 6 to 1 : 50. After 4 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).

supernatants containing HLA-G for 5 min. Flow cyto- metric analysis of intracellular phosphotyrosine using serum showed that HLA-G anti-phosphotyrosine caused a shift compared to untreated control cells (Fig. 5B). NKL cells preincuba- tion with DETA-NO 100 lm for 24 h did not modify this HLA-G-induced tyrosine phosphorylation. These

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Fig. 4. 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 expres- sion 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 experi- ments. 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: for % specific lysis = 100 · [(sample c.p.m. ) spontaneous release) ⁄ (maximum release ) spontaneous release)].

with Alexa Fluor 488-conjugated anti-phosphotyrosine serum (Beckman Coulter) 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). Fluo- rescence was detected by an EPICS XL flow cytometer (Beckman Coulter). The spontaneous release was the c.p.m. measured in 51Cr-labelled K562 cells cultured in medium without NKL cells. The maximum release was achieved when 51Cr- labelled K562 cells were incubated with Triton-X100.

Real-time RT-PCR analysis

Blocking experiments of ILT2 were performed by incu- bating treated and untreated NKL cells with monoclonal anti-ILT2 serum GHI ⁄ 75 (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) for 30 min at 37 (cid:2)C before co-culturing them with K562 cells.

Flow cytometry

instructions

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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 1 h at 25 (cid:2)C. Reverse transcription was carried out using High-Capacity cDNA Archive Kit according to the manufacturer’s (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 Biosys- tems) and GAPDH expression was used as internal standard. For cell surface labelling, cells were incubated for 30 min at 4 (cid:2)C in NaCl ⁄ Pi containing 20% human serum (Sigma- Aldrich, St Louis, MO, USA), and stained with PE conju- gated anti-ILT2 serum (Beckman Coulter, Marseille, France) for 20 min at 4 (cid:2)C. After washing, cells were fixed in paraformaldehyde 1%. For intracellular staining, cells were fixed with paraformaldehyde 1% for 10 min at 37 (cid:2)C and permeabilized with 90% methanol for 30 min on ice. After washing with NaCl ⁄ Pi-BSA 0.5%, cells were stained

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precipitation was performed with a protein A-sepharose assay kit purchased from Pierce Biotechnology Inc. (Rock- ford, 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 5 min, 20 lg of total protein were denatured at 100 (cid:2)C for 5 min in a protein sample buffer containing 125 mm Tris-ClH (pH 6.8), 4% SDS, 30% glycerol, 5% b-mercap- toethanol and 0.4% bromophenol. Proteins were subjected (SDS ⁄ to 10% PAGE under denaturing conditions 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 1 h at room temperature, and then incubated for 2 h with HP-F1 anti-ILT2 serum (kindly provided by M. Lopez- Botet, Institut Municipal d’Investigacio´ Me` dica, Barcelona, Spain) diluted 1 : 500 in NaCl ⁄ Pi-Tween, or anti-b-actin (Abcam, Cambridge, UK), diluted 1 : 5000 in NaCl ⁄ Pi-Tween. Immunoblot detection was performed using an horseradish peroxidase-conjugated anti-mouse antibody (dilution 1 : 5000; Amersham Biosciences) and developed using the ECL kit (Amersham Biosciences). For incuba- tion with additional antibodies, the membranes were pre- viously stripped for 30 min at 56 (cid:2)C in 62.5 mm Tris (pH 6.8), 2% SDS and 100 mm b-mercaptoethanol.

LC-ESI-MS ⁄ MS analysis

Fig. 5. (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 4 h of co-culture, with K562-pcDNA or K562-HLA-G1 cells as target cells, in a 50 : 1 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 pret- eated or not with DETA-NO. Cells were cultivated for 24 h with or without DETA-NO 100 lM. After cell washing, supernatants con- taining HLA-G were added and incubated for 5 min. Cells were then fixed, perma permeabilized, and stained with anti-phosphotyro- sine serum. Dotted peaks represent irrelevant isotypic antibody. The histograms shown are representative of four different experi- ments. M.f.i., mean fluorescence intensity.

Nitrotyrosine immunoprecipitation

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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 dilu- tion of 1 : 230 for 30 min [15]. Preincubation of anti-nitroty- rosine serum with nitrotyrosine 1 mm (Sigma-Aldrich) for 1 h was used as control of immune specificity. Immuno- Fifteen micrograms of rhILT2-Fc fusion protein were trea- ted with SIN-1 2 mm for 3 h at 37 (cid:2)C in continuous agita- tion. 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 6 ngÆlL)1 trypsin for 5 h at 37 (cid:2)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 per- formed with a CapLC(cid:3) (Waters, Milford, MA, USA) cap- illary system. Reversed phase separation of tryptic digests was carried out with an Atlantis, C18, 3 lm, 75 lm · 10 cm Nano Ease(cid:3) fused silica capillary column (Waters) equilibrated in 5% acetonitrile and 0.2% formic acid. After injection of 6 lL of sample, the column was washed for 5 min with the same buffer and the peptides were eluted using a linear gradient of 5–50% acetonitrile over 45 min at a constant flow rate of 0.2 lLÆmin)1. The column was coupled online to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters). The heated capillary spray voltage was temperature was 80 (cid:2)C and the

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References

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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 collision- induced dissociation using an isolation width of 2.0 and a relative collision energy of 35 V. Data processing was per- formed 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. 3 Chapman TL, Heikema AP, West AP Jr & Bjorkman PJ (2000) Crystal structure and ligand binding proper- ties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2). Immunity 13, 727–736.

HLA-G binding assay

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Mutational analysis of immunoreceptor tyrosine-based inhibition motifs of the Ig-like transcript 2 (CD85j) leukocyte receptor. J Immunol 168, 3351–3359.

6 Rouas-Freiss N, Goncalves RM-B, Menier C, Dausset J & Carosella ED (1997) Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci USA 94, 11520–11525. 7 Bogdan C (2001) Nitric oxide and the immune response. Nat Immunol 2, 907–916.

rhILT2-Fc was treated with increasing concentrations of SIN-1 for 3 h at 37 (cid:2)C. Polystyrene microtiter plates (Gre- iner Bio-One, Frickenhausen, Germany) were coated with 10 lgÆmL)1 rhILT2-Fc, or with 10 lgÆmL)1 anti-HLA-G MEM G ⁄ 9 (Exbio, Prague, Czech Republic) in NaCl ⁄ Pi overnight at 4 (cid:2)C. Plates were washed with NaCl ⁄ Pi- Tween 0.2%, and blocked with NaCl ⁄ Pi-BSA 3% for 2 h. Then, equal quantities of supernatant containing HLA-G were added and incubated for 90 min at 37 (cid:2)C. After washing, anti-b2-microglobulin serum (Dako, Glostrup, Denmark) was added and incubated for 1 h at 37 (cid:2)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 1 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.

8 Berchner-Pfannschmidt U, Yamac H, Trinidad B & Fandrey J (2007) Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2. J Biol Chem 282, 1788–1796. 9 Cifone MG, D’Alo S, Parroni R, Millimaggi D,

Statistical analysis

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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

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Investigacio´ n Sanitaria PI070298

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This work was supported by the Fondo de Investiga- cio´ n Sanitaria. E.A. was the recipient of a grant from Fondo de and A.D.L. received a grant from Asociacio´ n Amigos Uni- versidad de Navarra and Caixanova. The laboratory of Proteomic CIMA is member of the National Insti- tute of Proteomics Facilities, ProteoRed.

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