DETA-NO did not modify ILT2 mRNA expression or protein expression at the cell surface.. Structured digital abstract l MINT-7144982 : ILT2 uniprotkb: Q8NHL6 binds MI:0407 to HLA-G unip
Trang 1binding 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
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
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 funccondi-tions 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 killer cell line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2-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 )
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).
Trang 2interest 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 inhibinhib-itory
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
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]
To date, there is a scarcity of data available
con-cerning 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
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 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-Fig 1 Immunoblot analyses of ILT2 nitration in NKL cells (A) and U-937 cells (B), untreated or treated with DETA-NO 100 l M or with SIN-1 100 l M 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 m M 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.
Trang 3sine 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
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
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
analyzed 40% of the ILT2 extracellular domain
(Fig 2A), including Tyr76 that is suggested to
partici-pate 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
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
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 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
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
Trang 4that 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
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)
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 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
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 m M with rhILT2-Fc (F) Annotated mass spectra of the same peptide as in (E) but without nitrotyrosine residues.
Trang 5K562-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 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
(33 ± 5% K562-HLA-G1 cell lysis) These results
indicate 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 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
Fig 2 (Continued).
Trang 6Fig 2 (Continued).
Trang 7supernatants containing HLA-G for 5 min Flow
cyto-metric 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
preincuba-tion with DETA-NO 100 lm for 24 h did not modify
this HLA-G-induced tyrosine phosphorylation These
results indicate that NO does not affect tyrosine phos-phorylation, which is related to our previous observa-tion that ILT2 maintains its suppressive funcobserva-tion 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 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,
atmo-sphere For NKL cells, 50 UÆmL)1rhIL-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
NKL cell cytotoxicity against the K562 cell line was evalu-ated in a standard 4 h51Cr release assay K562-HLA-G1 or K562-pcDNA transfected cells were incubated for 1 h at
med-ium, target cells were co-cultured with NKL effector cells for 4 h at 37C NKL cells were previously stimulated with
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)
Table 1 Nitrated peptides from rhILT2-Fc Recombinant protein
was untreated (control) or treated with SIN-1 2 m M Nitrated Tyr
are shown in bold and marked with asterisks.
Nitrated
tyrosine (domain) Peptide
Score Control SIN-1
KPSLSVQPGPIVAPEE TLTLQCGSDAGYNR
13.06 19.50 Tyr229 (D3) KPSLSVQPGPIVAPEETLT
LQCGSDAGY*NR
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 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.
Trang 8Specific lysis level was calculated as the percentage 51Cr
release from the maximum release:
release)⁄ (maximum release) spontaneous release)]
The spontaneous release was the c.p.m measured in
51Cr-labelled K562 cells cultured in medium without NKL
Cr-labelled K562 cells were incubated with Triton-X100
Blocking experiments of ILT2 were performed by
incu-bating treated and untreated NKL cells with monoclonal
co-culturing them with K562 cells
Flow cytometry
For cell surface labelling, cells were incubated for 30 min at
(Sigma-Aldrich, St Louis, MO, USA), and stained with PE
France) for 20 min at 4C After washing, cells were fixed
in paraformaldehyde 1% For intracellular staining, cells
and permeabilized with 90% methanol for 30 min on ice
After washing with NaCl⁄ Pi-BSA 0.5%, cells were stained
with Alexa Fluor 488-conjugated anti-phosphotyrosine
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)
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)
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 Biosys-tems) and GAPDH expression was used as internal standard
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 expresexpres-sion 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.
Trang 9Nitrotyrosine 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
dilu-tion of 1 : 230 for 30 min [15] Preincubadilu-tion of
anti-nitroty-rosine serum with nitrotyanti-nitroty-rosine 1 mm (Sigma-Aldrich) for
1 h was used as control of immune specificity
Immuno-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
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
PAGE), with subsequent electroblotting transfer onto a nitrocellulose membrane The membrane was blocked
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
Pi-Tween Immunoblot detection was performed using an
(dilution 1 : 5000; Amersham Biosciences) and developed using the ECL kit (Amersham Biosciences) For incuba-tion with addiincuba-tional antibodies, the membranes were pre-viously stripped for 30 min at 56C in 62.5 mm Tris (pH 6.8), 2% SDS and 100 mm b-mercaptoethanol
LC-ESI-MS⁄ MS analysis
Fifteen micrograms of rhILT2-Fc fusion protein were trea-ted with SIN-1 2 mm for 3 h at 37C 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
trypsin for 5 h at 37C The rhILT2-Fc negative control was processed in the same way, except for the nitration
described [31] Microcapillary reversed phase LC was per-formed with a CapLC (Waters, Milford, MA, USA) cap-illary system Reversed phase separation of tryptic digests
cm Nano Ease 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
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 l M 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 l M 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.
Trang 101.8–2.2 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
HLA-G binding assay
rhILT2-Fc was treated with increasing concentrations of
SIN-1 for 3 h at 37C Polystyrene microtiter plates
(Gre-iner Bio-One, Frickenhausen, Germany) were coated with
Then, equal quantities of supernatant containing HLA-G
HLA-G binding was detected using EnVision+ Dual Link
(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
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-cio´n Sanitaria E.A was the recipient of a grant from
Fondo de Investigacio´n Sanitaria PI070298 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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