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

11 339 0
  • Loading ...
    Loading ...
    Loading ...

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Tài liệu liên quan

Thông tin tài liệu

Ngày đăng: 07/03/2014, 02:20

Tyrosine nitration in the human leucocyte antigen-G-binding domain of the Ig-like transcript 2 proteinAngel Dı´az-Lagares1, Estibaliz Alegre1, Ainhoa Arroyo1, Fernando J. Corrales2and A´lvaro Gonza´lez11 Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain2 Division of Hepatology and Gene Therapy, Proteomics Unit, CIMA, University of Navarra, Pamplona, SpainIntroductionPeripheral tolerance is an important part of theimmune defence system, comprising a mechanism toavoid the uncontrolled spread of immune attacksand autoreactivity against normal cells. Of particularKeywordsHLA-G; ILT2; inflammation; natural killer;nitrationCorrespondenceA´. Gonza´lez, Department of Biochemistry,University Clinic of Navarra, Avenida de Pı´oXII, 36, 31008 Pamplona, SpainFax: +34 948 296500Tel: +34 948 255400E-mail: agonzaleh@unav.es(Received 21 February 2009, revised 26May 2009, accepted 4 June 2009)doi:10.1111/j.1742-4658.2009.07131.xIg-like transcript 2 (ILT2) is a suppressive receptor that participates in thecontrol of the autoimmune reactivity. This action is usually carried out in aproinflammatory microenvironment where there is a high production of freeradicals and NO. However, little is known regarding whether these condi-tions modify the protein or affect its suppressive functions. The present studyaimed to investigate the suppressive response of the ILT2 receptor under oxi-dative stress. To address this topic, we treated the ILT2-expressing naturalkiller cell line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-d iam ine (DETA-NO). Weobserved that DETA-NO caused ILT2 protein nitration. MS analysis of thechimeric recombinant human ILT2-Fc protein after treatment with the per-oxynitrite donor 3-(morpholinosydnonimine hydrochloride) (SIN-1) showedthe 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 withSIN-1 bound a significantly higher quantity of human leucocyte antigen-Gthan untreated recombinant human ILT2-Fc. DETA-NO did not modifyILT2 mRNA expression or protein expression at the cell surface. Preincuba-tion of NKL cells with DETA-NO decreased the cytotoxic lysis ofK562-human leucocyte antigen-G1 cells compared to untreated NKLcells (P < 0.05) but increased cytotoxicity against K562-pcDNA cells(P < 0.05). Intracellular tyrosine phosphorylation produced after humanleucocyte antigen-G binding was not affected by DETA-NO cell pretreat-ment. These results support the hypothesis that the ILT2–human leucocyteantigen-G interaction should have a central role in tolerance under oxidativestress conditions when other tolerogenic mechanisms are inhibited.Structured digital abstractlMINT-7144982: ILT2 (uniprotkb:Q8NHL6) binds (MI:0407)toHLA-G (uniprotkb:P17693)by affinity technologies (MI:0400)AbbreviationsDETA-NO, N-(4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine; HLA, human leucocyte antigen; ILT2, Ig-liketranscript 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 4233interest is tolerance during pregnancy, where maternalimmune cells do not attack the fetus, even though thefetus can be considered immunologically as a semiallo-genic graft as a result of the expression of paternalantigens [1]. One of the molecules implicated in theimmune tolerance is the Ig-like transcript 2 (ILT2),also known as CD85j, LIR-1 and LILRB1, comprisingan 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 theextracellular domains D1 and D2 bind the a3 domainof both classical and nonclassical human leucocyteantigen (HLA)-I molecules [3], but with higher affinityto HLA-G than to classical HLA-I [4]. The cytoplas-mic tail contains immunoreceptor tyrosine-based inhib-itory motifs [5], which trigger a cellular inhibitoryresponse, such as the suppression of NK cytotoxicity[6].Interaction between HLA-G and ILT2 usually takesplace in vivo in a proinflammatory microenvironmentwhere free radicals are available that could modify thisinteraction. Of special importance is NO, which is avery reactive free radical synthesized from l-arginineby the enzyme NOS [7]. NO has pleiotropic immuneactions controlling inflammation and tissue damage,including immune cell proliferation and function, andcytokine production [7,8]. For example, NO increasesmacrophage and NK cell function [9,10] and down-regulates the T helper 1 cell response, favouring a Thelper 2 reaction [11].NO-derived metabolites peroxynitrite or nitrite, inconjunction with peroxidases, can react with tyrosineto produce nitrotyrosine (nitroTyr) at the inflamma-tory site [12]. This modification can induce deepchanges in the physicochemical properties of the pro-teins, affecting their stability or functionality [13]. Fur-thermore, tyrosine nitration comprises a reversiblereaction [14] that affects a limited number of proteinsand few tyrosine residues, and it can influence differentbiological activities [13]. For example, the immunosup-pressive enzyme indoleamine 2,3-dioxygenase is inacti-vated by high concentrations of NO [15]. NitroTyr hasbeen detected in many disorders, such as preeclampsia[16], bacterial and viral infection, and chronic inflam-mation [17].To date, there is a scarcity of data available con-cerning how inflammatory stress affects the interactionbetween HLA-G and its receptors. We recentlyreported that NO can nitrate HLA-G, increasing itsmetalloprotease-dependent shedding to the medium[18]. This modified HLA-G conserves its suppressiveproperties, allowing the spread of the tolerogenicmicroenvironment. To determine whether HLA-Greceptors are also capable of responding to the sup-pressive stimulus under oxidative stress, the presentstudy aimed to investigate the effect of NO in theexpression and function of the ILT2 suppressivereceptor.Results and DiscussionNO modifies ILT2 protein by tyrosine nitrationProtein nitration is a post-translational modificationcaused by NO derivates, that can modify protein struc-ture and function [13]. Initially, we wanted to analyzewhether 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 immunoprecipitatedthe cell lysate with anti-nitrotyrosine serum. Westernblotting using anti-ILT2 serum HP-F1 showed a bandof approximately 90 kDa, which was not present inuntreated control cells (Fig. 1A). Similarly, this banddid not appear in the control of specificity, where anti-nitrotyrosine serum was preincubated with 3-nitrotyro-Fig. 1. Immunoblot analyses of ILT2 nitration in NKL cells (A) andU-937 cells (B), untreated or treated with DETA-NO 100 lM or withSIN-1 100 lM. Cell lysates were immunoprecipitated using anti-3-nitrotyrosine serum. The control (+) corresponds to a cell lysate ofNKL cells. A negative control was performed by preincubation ofthe antibody with 3-nitrotyrosine 1 mM. Immunoprecipitated pro-teins were separated by SDS ⁄ PAGE, blotted onto a nitrocellulosemembrane, and then probed with HP-F1 anti-ILT2 serum. A repre-sentative experiment out of three is shown.ILT2 nitration in the binding domain A. Dı´az-Lagares et al.4234 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBSsine 1 mm before immunoprecipitation. To determinewhether endogenous NO production can also causeILT2 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 thelane 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-(morpholinosydnoniminehydrochloride) (SIN-1) 100 lm for 24 h.These results show that ILT2 can undergo nitra-tion, which should be related to the presence ofexposed Tyr residues [19–21]. To our knowledge, thisis the first report of a post-translational modificationof the ILT2 protein. The other member of the ILTfamily, LILRA4, has also been found nitrated withinthe domain Ig-like C2-type 4 in human tumour tis-sues [22]. Although most of the effects of nitrationcause functional loss [23], protein nitration can alsoelicit increased biological activity, such as in cathep-sin D [24], or in the glucocorticoid receptor, wherenitration leads to an increase in binding capacity[25].Identification of nitration site in the extracellulardomain of ILT2In the extracellular domain of ILT2, there are severalTyr residues that participate in the interaction withHLA-G [3,19]. Because protein nitration is a phenome-non that cannot be predicted from the amino acidsequence, we were very interested in analyzing whetherILT2 nitration affected the Tyr residues in the hydro-phobic interdomain that binds HLA-G. To addressthis issue, we used a commercial recombinant humanrecombinant human (rh)ILT2-Fc chimera, which pos-sess the extracellular domain and maintains the HLA-G binding capacity [19]. This protein was treated for3 h with the pure peroxynitrite donor SIN-1 2 mm.Asa negative control, we processed untreated rhILT2-Fcsimultaneously. After tryptic digestion, the presence ofnitrotyrosine in the resultant peptides was analyzed byLC-MS ⁄ MS. Under these experimental conditions, weanalyzed 40% of the ILT2 extracellular domain(Fig. 2A), including Tyr76 that is suggested to partici-pate in HLA-G binding [19]. We identified six peptideswith nitrated Tyr that were not present in theuntreated control. These nitroTyr corresponded topositions Tyr35, Tyr76, Tyr77, Tyr99, Tyr229 andTyr355 (Figs 2B–E and Table 1). In particular, thecharged ions CQGGQETQEYR and the correspond-ing fragment y2, with m ⁄ z = 700.784, and the CY-YGSDTAGR and the corresponding fragments y9 andb2, with m ⁄ z = 597.74, showed an increased mass of45 Da as a result of the acquisition of a nitro group inTyr35 and Tyr76, respectively.However, not all rhILT2-Fc was nitrated becausethese peptides also appeared without nitration(Table 1). Furthermore, other residues analyzed (i.e.Tyr235 and Tyr372) were resistant to nitration. Thefact that the nitration is partial is not surprisingbecause, even for proteins that are easy targets fornitration, the relative yield of nitroTyr formationunder inflammatory conditions is low [26]. Because wewere unable to sequence more than 38% of the Tyrresidues, we cannot rule out the possibility that othertyrosines could also be nitrated. Nevertheless, thesedata demonstrate that the binding domain of ILT2could undergo nitration, which implies conformationalchanges.ILT2 nitration increases HLA-G bindingTo determine whether treatment with NO modifies theinteraction of ILT2 with HLA-G, we performed abinding 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-1treatment significantly increased rhILT2-Fc binding toHLA-G (150 ± 18%; HLA-G binding to SIN-1 2 mmtreated 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 inagreement 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 pocketdirectly. Furthermore, Tyr99 stabilizes the anglebetween D1 and D2 domains, which is necessary forHLA-G binding [3,19], and the modification of thisangle should also affect the interaction with HLA-G.Effectively, tyrosine nitration causes a shift in the pKaof the tyrosine hydroxyl group and makes the nitratedtyrosine more hydrophobic and prone to move intomore hydrophobic regions [13,26]. These modificationscould induce changes in protein structure and functionthat affect the affinity of the interaction between ILT2and HLA-G.NO does not affect ILT2 expressionNO modulates the expression of multiple genes [7]. Todetermine whether NO affects ILT2 expression, wetreated NKL cells with increasing quantities of DETA-NO for 24 h. Real-time RT-PCR analysis indicatedA. Dı´az-Lagares et al. ILT2 nitration in the binding domainFEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4235that DETA-NO did not modify the transcriptional lev-els of ILT2 (Fig. 4). Similarly, western blot analysisshowed that DETA-NO did not change ILT2 proteincontent and flow cytometry analysis revealed nochange in ILT2 cell surface expression. We concludedthat the effect of NO in the ILT2 receptor is limited toa post-translational modification.ILT2 maintains its suppressive function in thepresence of NOFinally, we aimed to determine whether the presenceof NO under conditions known to nitrate ILT2 couldaffect the sensitivity to HLA-G. Accordingly, we incu-bated NKL cells with DETA-NO 100 lm for 24 h andthen performed a cytotoxicity assay using either K562-pcDNA or K562-HLA-G1 as target cells (Fig. 5A).The possible cytotoxic effect of NO was avoidedbecause this compound was not present during thecytotoxic assay. As previously described [2,6], weobserved a significant decrease in the lysis of K562-HLA-G1 cells compared to K562-pcDNA cells at a50 : 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 significantlydecreased K562-HLA-G1 cell lysis (P < 0.05).This increased NKL cytotoxicity against K562-pcDNA after incubation with DETA-NO is in agree-ment with previous findings where NO released bymacrophages was found to participate in the functionalmaturation of NK cells [7]. However, these more acti-vated NKL cells have an even lower killing functionagainst K562-HLA-G1 cells. It has been demon-strated that the inhibition of NKL cytotoxicity againstFig. 2. (A) Amino acid sequence coverage and sites of nitration of SIN-1-treated rhILT2-Fc, obtained by LC-MS ⁄ MS analysis. Protein wasnitrated with SIN-1, subjected to trypsin digestion, and peptides were separated on a reverse phase HPLC column online with ESI and iontrap MS. The amino acid sequence coverage obtained by LC-MS ⁄ MS is shown in bold. Nitrated peptides are underlined and nitrated Tyr areindicated by asterisks. (B–E) Annotated mass spectra of peptides containing nitrotyrosine observed after the reaction of SIN-1 2 mM withrhILT2-Fc. (F) Annotated mass spectra of the same peptide as in (E) but without nitrotyrosine residues.ILT2 nitration in the binding domain A. Dı´az-Lagares et al.4236 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBSK562-HLA-G1 is a result of the interaction of HLA-Gwith ILT2 [2,27]. We verified these data under ourexperimental conditions by preincubating NKL cellswith the monoclonal anti-ILT2 serum GHI⁄ 75(10 lgÆmL)1). Blockade of the ILT2 receptor impairedHLA-G suppression of NKL cell cytotoxicity, regard-less of whether it was treated or not with DETA-NO(33 ± 5% K562-HLA-G1 cell lysis). These resultsindicate that NO maintains, or even increases, ILT2-mediated suppression in NKL cells.After HLA-G binding, immunoreceptor tyrosine-based inhibitory motifs in the cytoplasmic tail of theILT2 receptor become tyrosine phosphorylated, elicit-ing a suppressive response [4,5]. The results shown inFig. 5A suggest that tyrosine phosphorylation is notmodified by NO treatment because the suppressioncaused by ILT2–HLA-G interaction was not blockedby the addition of DETA-NO. To further confirmthese data, we studied intracellular phosphotyro-sine formation in NKL cells after incubation withFig. 2. (Continued).A. Dı´az-Lagares et al. ILT2 nitration in the binding domainFEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4237Fig. 2. (Continued).ILT2 nitration in the binding domain A. Dı´az-Lagares et al.4238 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBSsupernatants containing HLA-G for 5 min. Flow cyto-metric analysis of intracellular phosphotyrosine usinganti-phosphotyrosine serum showed that HLA-Gcaused a shift in the fluorescence compared tountreated control cells (Fig. 5B). NKL cells preincuba-tion with DETA-NO 100 lm for 24 h did not modifythis HLA-G-induced tyrosine phosphorylation. Theseresults indicate that NO does not affect tyrosine phos-phorylation, which is related to our previous observa-tion that ILT2 maintains its suppressive function inthe presence of NO (Fig. 5A).Modulation of ILT2–HLA-G interactions by NOcould be especially important in the placenta, in whichHLA-G is expressed [28], because the most importantimmune population comprises the NK cells [29] andthere is a controlled state of inflammation with highNO production [30]. NO causes metalloprotease-dependent HLA-G shedding and nitrates both HLA-G[18] and ILT2, although also allowing these proteins toconserve their suppressive function. These results sug-gest that the ILT2–HLA-G interaction is an importantmechanism for controlling NK cell immune attacksunder inflammatory oxidative stress, and under condi-tions where other suppressive molecules are inactivated[15].Experimental proceduresCell cultureThe NK cell line, NKL, the monocytic cell line, U-937, andthe 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), weregrown in RPMI-1640 medium supplemented with 10% fetalbovine serum, 2 mm glutamine, 100 UÆmL)1penicillin and100 lgÆmL)1streptomycin (Gibco BRL ⁄ Invitrogen, Carls-bad, CA, USA) at 37 °C in a 5% CO2humidified atmo-sphere. For NKL cells, 50 UÆmL)1rhIL-2 (Roche MolecularBiochemicals, Mannheim, Germany) was added to the cul-ture medium. NO donors were DETA-NO (Alexis Corpora-tion, Lausane, Switzerland) SIN-1 (Alexis Corporation). TherhILT2-Fc chimera was purchased from R&D Systems(Abingdon, UK). Cellular viability measured by trypan blueexclusion was higher than 95% throughout the study.Cytotoxic assayNKL cell cytotoxicity against the K562 cell line was evalu-ated in a standard 4 h51Cr release assay. K562-HLA-G1 orK562-pcDNA transfected cells were incubated for 1 h at37 °C with51Cr. After two washes with RPMI-1640 med-ium, target cells were co-cultured with NKL effector cellsfor 4 h at 37 °C. NKL cells were previously stimulated withIL-2 (100 UÆmL)1) for 24 h in presence or absence ofDETA-NO 100 lm. Co-culture was performed in triplicateand at several K562 : NKL ratios from 1 : 6 to 1 : 50.After 4 h, 50 lL of each supernatant were mixed with250 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).Table 1. Nitrated peptides from rhILT2-Fc. Recombinant proteinwas untreated (control) or treated with SIN-1 2 mM. Nitrated Tyrare shown in bold and marked with asterisks.Nitratedtyrosine (domain) PeptideScoreControl SIN-1Tyr35 (D1) CQGGQETQEYR 13.78 14.19CQGGQETQEY*R – 8.88CYYGSDTAGR 7.77 9.69Tyr76 (D1) CY*YGSDTAGR – 6.61Tyr77 (D1) CYY*GSDTAGR – 6.15SESSDPLELVVTGAYIK 10.49 14.28Tyr99 (D1) SESSDPLELVVTGAY*IK – 8.85KPSLSVQPGPIVAPEETLTLQCGSDAGYNR13.06 19.50Tyr229 (D3) KPSLSVQPGPIVAPEETLTLQCGSDAGY*NR– 10.64YQAEFPMGPVTSAHAGTYR 12.35 16.64Tyr355 (D4) Y*QAEFPMGPVTSAHAGTYR – 10.67Fig. 3. Effect of the peroxynitrite donor SIN-1 on the capability ofrhILT2-Fc to bind HLA-G. rhILT2-Fc was treated with increased con-centrations of SIN-1 for 3 h at 37 °C. The results show the relativequantities of the HLA-G concentration compared to untreatedcontrol rhILT2-Fc (assigned a value of 100) and are expressedas the mean ± SD of three different experiments. *P < 0.05compared to untreated control rhILT2-Fc.A. Dı´az-Lagares et al. ILT2 nitration in the binding domainFEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4239Specific lysis level was calculated as the percentage51Crrelease from the maximum release:% specific lysis = 100 · [(sample c.p.m. ) spontaneousrelease) ⁄ (maximum release ) spontaneous release)].The spontaneous release was the c.p.m. measured in51Cr-labelled K562 cells cultured in medium without NKLcells. The maximum release was achieved when51Cr-labelled K562 cells were incubated with Triton-X100.Blocking experiments of ILT2 were performed by incu-bating treated and untreated NKL cells with monoclonalanti-ILT2 serum GHI ⁄ 75 (Becton-Dickinson Biosciences,Franklin Lakes, NJ, USA) for 30 min at 37 °C beforeco-culturing them with K562 cells.Flow cytometryFor cell surface labelling, cells were incubated for 30 min at4 °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 °C. After washing, cells were fixedin paraformaldehyde 1%. For intracellular staining, cellswere fixed with paraformaldehyde 1% for 10 min at 37 °Cand permeabilized with 90% methanol for 30 min on ice.After washing with NaCl ⁄ Pi-BSA 0.5%, cells were stainedwith Alexa Fluor 488-conjugated anti-phosphotyrosineserum (Beckman Coulter) for 30 min, washed withNaCl ⁄ Pi-BSA 0.5%, and resuspended in NaCl ⁄ Pi for flowcytometry analysis. Control aliquots were stained with theisotype-matched mouse antibody (Beckman Coulter). Fluo-rescence was detected by an EPICS XL flow cytometer(Beckman Coulter).Real-time RT-PCR analysisReal-time PCR analysis was used to quantify variationsin the amounts of ILT2 transcripts after cell treatmentwith DETA-NO. Total RNA was extracted from 3–5million 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 °C. Reverse transcription was carried outusing High-Capacity cDNA Archive Kit according to themanufacturer’s instructions (Applied Biosystems, FosterCity, CA USA). Real-time PCR was performed using theTaqMan Gene Expression Assay (Applied Biosystems) onan ABI PRISM 7700 Sequence Detector (Applied Biosys-tems) and GAPDH expression was used as internalstandard.Fig. 4. ILT2 expression in NKL cells treated with different concentrations of DETA-NO for 24 h. Upper: flow cytometry of ILT2 surfaceexpression 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 at90 kDa. Loading control was performed using an antibody against b-actin, which produced a band at 42 kDa. The data indicate the intensityof the HLA-G band related to the b-actin band and are representative of three different experiments.ILT2 nitration in the binding domain A. Dı´az-Lagares et al.4240 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBSNitrotyrosine immunoprecipitationCells were lysed in NP40 0.5% in Tris-HCl buffer withprotease 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) for1 h was used as control of immune specificity. Immuno-precipitation was performed with a protein A-sepharoseassay kit purchased from Pierce Biotechnology Inc. (Rock-ford, IL, USA) according to the manufacturer’s instructions.Western blottingProtein concentration was quantified by the Bradfordassay (Bio-Rad Laboratories, Hercules, CA, USA) usingBSA as standard. After centrifugation at 10 000 g for5 min, 20 lg of total protein were denatured at 100 °Cfor 5 min in a protein sample buffer containing 125 mmTris-ClH (pH 6.8), 4% SDS, 30% glycerol, 5% b-mercap-toethanol and 0.4% bromophenol. Proteins were subjectedto 10% PAGE under denaturing conditions (SDS ⁄PAGE), with subsequent electroblotting transfer onto anitrocellulose membrane. The membrane was blockedwith 5% nonfat dried milk in NaCl ⁄ Pi-Tween 0.1% for1 h at room temperature, and then incubated for 2 h withHP-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 anhorseradish peroxidase-conjugated anti-mouse antibody(dilution 1 : 5000; Amersham Biosciences) and developedusing the ECL kit (Amersham Biosciences). For incuba-tion with additional antibodies, the membranes were pre-viously stripped for 30 min at 56 °C in 62.5 mm Tris (pH6.8), 2% SDS and 100 mm b-mercaptoethanol.LC-ESI-MS⁄MS analysisFifteen micrograms of rhILT2-Fc fusion protein were trea-ted with SIN-1 2 mm for 3 h at 37 °C in continuous agita-tion. Then, nitrated rhILT2-Fc was precipitated withtrichloroacetic acid 20%, reduced with dithiotheitol 10 mmin ammonium bicarbonate 100 mm, and alkilated withiodoacetamide 55 mm. The protein was resuspended inammonium bicarbonate 50 mm and digested with 6 ngÆlL)1trypsin for 5 h at 37 °C. The rhILT2-Fc negative controlwas processed in the same way, except for the nitrationtreatment. MS ⁄ MS analysis was performed as previouslydescribed [31]. Microcapillary reversed phase LC was per-formed with a CapLCÔ (Waters, Milford, MA, USA) cap-illary system. Reversed phase separation of tryptic digestswas carried out with an Atlantis, C18, 3 lm, 75 lm · 10cm Nano EaseÔ fused silica capillary column (Waters)equilibrated in 5% acetonitrile and 0.2% formic acid. Afterinjection of 6 lL of sample, the column was washed for5 min with the same buffer and the peptides were elutedusing a linear gradient of 5–50% acetonitrile over 45 minat a constant flow rate of 0.2 lLÆmin)1. The column wascoupled online to a Q-TOF Micro (Waters) using a PicoTipnanospray ionization source (Waters). The heated capillarytemperature was 80 °C and the spray voltage wasFig. 5. (A) Effect of DETA-NO on HLA-G-mediated inhibition of NKLcytotoxicity. The data show the percentage (± SD) of specific lysisachieved by NKL cells during 4 h of co-culture, with K562-pcDNAor K562-HLA-G1 cells as target cells, in a 50 : 1 effector : targetcell ratio. NKL cells were previously incubated without or withDETA-NO 100 lM for 24 h. The results are expressed as the meanof 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 orwithout DETA-NO 100 lM. After cell washing, supernatants con-taining HLA-G were added and incubated for 5 min. Cells werethen 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.A. Dı´az-Lagares et al. ILT2 nitration in the binding domainFEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 42411.8–2.2 kV. MS ⁄ MS data were collected in an automateddata-dependent mode. The three most intense ions in eachsurvey scan were sequentially fragmented by collision-induced dissociation using an isolation width of 2.0 and arelative collision energy of 35 V. Data processing was per-formed with masslynx, version 4.1. Database searchingwas carried out using proteinlynx global server 2.3(Waters) and phenyx, version 2.5 (GeneBio, Geneva,Switzerland). The search was enzymatically constrained fortrypsin and allowed for one missed cleavage site. Furthersearch parameters were: no restriction on molecular weightand isoelectric point; carbamidomethylation of cysteine;variable modification; and oxidation of methionine.HLA-G binding assayrhILT2-Fc was treated with increasing concentrations ofSIN-1 for 3 h at 37 °C. Polystyrene microtiter plates (Gre-iner Bio-One, Frickenhausen, Germany) were coated with10 lgÆmL)1rhILT2-Fc, or with 10 lgÆmL)1anti-HLA-GMEM G ⁄ 9 (Exbio, Prague, Czech Republic) in NaCl ⁄ Piovernight at 4 °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-Gwere added and incubated for 90 min at 37 °C. Afterwashing, anti-b2-microglobulin serum (Dako, Glostrup,Denmark) was added and incubated for 1 h at 37 °C.HLA-G binding was detected using EnVision+ Dual LinkSystem-HRP (Dako) and 3,3¢,5,5¢-tetramethylbenzidine(Sigma-Aldrich). Colour development was stopped withHCl 1 m and the absorbance was measured at 450 nm ina microplate reader Multiskan Ascent (Thermo FisherScientific, Waltham, MA, USA). Results were normalizedto the absorbance obtained from the untreated controlrhILT2-Fc.Statistical analysisData are expressed as the mean ± SD. Statistical analysiswas performed using the spss statistical program forWindows (SPSS Inc., Chicago, IL, USA). Results werecompared with nonparametric Kruskal–Wallis and Mann–Whitney U-tests. P < 0.05 was considered statisticallysignificant.AcknowledgementsThis work was supported by the Fondo de Investiga-cio´n Sanitaria. E.A. was the recipient of a grant fromFondo de Investigacio´n Sanitaria PI070298 andA.D.L. received a grant from Asociacio´n Amigos Uni-versidad de Navarra and Caixanova. The laboratoryof Proteomic CIMA is member of the National Insti-tute of Proteomics Facilities, ProteoRed.References1 Moffett A & Loke C (2006) Immunology of placentationin eutherian mammals. Nat Rev Immunol 6, 584–594.2 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 His-tocompatibility Complex Class I Molecules on HumanLymphoid and Myelomonocytic Cells. J Exp Med 186,1809–1818.3 Chapman TL, Heikema AP, West AP Jr & BjorkmanPJ (2000) Crystal structure and ligand binding proper-ties of the D1D2 region of the inhibitory receptorLIR-1 (ILT2). Immunity 13, 727–736.4 Shiroishi M, Tsumoto K, Amano K, Shirakihara Y,Colonna M, Braud VM, Allan DSJ, Makadzange A,Rowland-Jones S, Willcox B et al. (2003) Human inhib-itory receptors Ig-like transcript 2 (ILT2) and ILT4compete with CD8 for MHC class I binding and bindpreferentially to HLA-G. Proc Natl Acad Sci USA 100,8856–8861.5 Bellon T, Kitzig F, Sayos J & Lopez-Botet M (2002)Mutational analysis of immunoreceptor tyrosine-basedinhibition 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 therole of HLA-G in protecting the fetus from maternaluterine natural killer cytolysis. Proc Natl Acad Sci USA94, 11520–11525.7 Bogdan C (2001) Nitric oxide and the immuneresponse. Nat Immunol 2, 907–916.8 Berchner-Pfannschmidt U, Yamac H, Trinidad B &Fandrey J (2007) Nitric oxide modulates oxygensensing by hypoxia-inducible factor 1-dependentinduction of prolyl hydroxylase 2. J Biol Chem 282,1788–1796.9 Cifone MG, D’Alo S, Parroni R, Millimaggi D,Biordi L, Martinotti S & Santoni A (1999) Interleukin-2-activated rat natural killer cells express induciblenitric oxide synthase that contributes to cytotoxicfunction and interferon-gamma production. Blood 93,3876–3884.10 Coleman JW (2001) Nitric oxide in immunity andinflammation. Int Immunopharmacol 1, 1397–1406.11 Roozendaal R, Vellenga E, de Jong MA, TraanbergKF, Postma DS, de Monchy JGR & Kauffman HF(2001) Resistance of activated human Th2 cells to NO-induced apoptosis is mediated by g-glutamyltranspepti-dase. Int Immunol 13, 519–528.12 Singer II, Kawka DW, Scott S, Weidner JR, MumfordRA, Riehl TE & Stenson WF (1996) Expression ofinducible nitric oxide synthase and nitrotyrosine in colo-nic epithelium in inflammatory bowel disease. Gastro-enterology 111, 871–885.ILT2 nitration in the binding domain A. Dı´az-Lagares et al.4242 FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS[...]... 8009–8 022 22 Zhan X & Desiderio DM (20 06) Nitroproteins from a human pituitary adenoma tissue 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 S, Takahashi K, Martin BM, Chen CY, Masuda J, Kowalak J, Takikawa O et al (20 06) Nitration and Inactivation of. .. crystal structure of disulfide-linked HLA-G dimer J Biol Chem 28 1, 10439–10447 20 Ischiropoulos H (20 03) Biological selectivity and functional aspects of protein tyrosine nitration Biochem Biophys Res Commun 305, 776–783 21 Sacksteder CA, Qian WJ, Knyushko TV, Wang H, Chin MH, Lacan G, Melega WP, Camp DG II, Smith RD, Smith DJ et al (20 06) Endogenously nitrated proteins in mouse brain: links to neurodegenerative... & Radi R (20 08) Protein tyrosine nitration - functional alteration or just a biomarker? Free Radic Biol Med 45, 357–366 14 Gorg B, Qvartskhava N, Voss P, Grune T, Haussinger D & Schliess F (20 07) Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration FEBS Lett 581, 84–90 15 Lopez AS, Alegre E, Diaz A, Mugueta C & Gonzalez A (20 06) Bimodal effect of nitric oxide in the enzymatic... enzymatic activity of indoleamine 2, 3-dioxygenase in human monocytic cells Immunol Lett 106, 163–171 16 Myatt L, Rosenfield RB, Eis AL, Brockman DE, Greer I & Lyall F (1996) Nitrotyrosine residues in placenta Evidence of peroxynitrite formation and action Hypertension 28 , 488–493 17 Ohmori H & Kanayama N (20 05) Immunogenicity of an in ammation-associated product, tyrosine nitrated self-proteins Autoimmunity... antiinflammatory effects of novel steroid ligands J Immunol 171, 324 5– 325 2 Radi R (20 04) Nitric oxide, oxidants, and protein tyrosine nitration Proc Natl Acad Sci USA 101, 4003–4008 Menier C, Riteau B, Carosella ED & Rouas-Freiss N (20 02) MICA triggering signal for NK cell tumor lysis is counteracted by HLA-G1-mediated inhibitory signal Int J Cancer 100, 63–70 Kovats S, Main EK, Librach C, Stubblebine M, Fisher... expressed in human trophoblasts Science 24 8, 22 0 22 3 Sargent IL, Borzychowski AM & Redman CWG (20 06) NK cells and human pregnancy – an in ammatory view Trends Immunol 27 , 399–404 Schiessl B, Mylonas I, Hantschmann P, Kuhn C, Schulze S, Kunze S, Friese K & Jeschke U (20 05) Expression of endothelial NO synthase, inducible NO synthase, and estrogen receptors alpha and beta in placental tissue of normal,... Reviews 4, 22 4 22 9 18 Diaz-Lagares A, Alegre E, Lemaoult J, Carosella ED & Gonzalez A (20 09) Nitric oxide produces HLA-G nitration and induces metalloprotease-dependent shedding creating a tolerogenic milieu Immunology 126 , 436–445 19 Shiroishi M, Kuroki K, Ose T, Rasubala L, Shiratori I, Arase H, Tsumoto K, Kumagai I, Kohda D & Maenaka K (20 06) Efficient leukocyte IG-like receptor signaling and crystal... Immunol 176, 3 72 379 Zaragoza R, Torres L, Garcia C, Eroles P, Corrales F, Bosch A, Lluch A, Garcia-Trevijano ER & Vina JR (20 09) Nitration of cathepsin D enhances its proteolytic activity during mammary gland remodeling after lactation Biochem J 6, 6 Paul-Clark MJ, Roviezzo F, Flower RJ, Cirino G, Soldato PD, Adcock IM & Perretti M (20 03) Glucocorticoid receptor nitration leads to enhanced antiinflammatory... normal, preeclamptic, and intrauterine growth-restricted pregnancies J Histochem Cytochem 53, 1441–1449 Munoz J, Fernandez-Irigoyen J, Santamaria E, Parbel A, Obeso J & Corrales FJ (20 08) Mass spectrometric characterization of mitochondrial complex variants I NDUFA10 Proteomics 8, 1898–1908 FEBS Journal 27 6 (20 09) 423 3– 424 3 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 424 3 . compilation ª 20 09 FEBS 423 7Fig. 2. (Continued).ILT2 nitration in the binding domain A. Dı´az-Lagares et al. 423 8 FEBS Journal 27 6 (20 09) 423 3– 424 3 ª 20 09 The. 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
- Xem thêm -

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