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

Tyrosine nitration in the human leucocyte antigen-G-
binding domain of the Ig-like transcript 2 protein
Angel Dı
´az-Lagares
1
, Estibaliz Alegre
1
, Ainhoa Arroyo
1
, Fernando J. Corrales
2
and A
´
lvaro Gonza
´
lez
1
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:
(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
killer 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). 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)toHLA-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).
FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4233
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].
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.
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 FEBS
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.
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 pK
a
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
A. Dı
´
az-Lagares et al. ILT2 nitration in the binding domain

FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4235
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.
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.
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 FEBS
K562-HLA-G1 is a result of the interaction of HLA-G
with ILT2 [2,27]. We verified these data under our
experimental conditions by preincubating NKL cells
with the monoclonal anti-ILT2 serum GHI⁄ 75
(10 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).
A. Dı
´
az-Lagares et al. ILT2 nitration in the binding domain
FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4237
Fig. 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 FEBS
supernatants 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 function in
the presence of NO (Fig. 5A).
Modulation of ILT2–HLA-G interactions by NO
could be especially important in the placenta, in which
HLA-G is expressed [28], because the most important
immune population comprises the NK cells [29] and

there is a controlled state of inflammation with high
NO production [30]. NO causes 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 °C in a 5% CO
2
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
NKL cell cytotoxicity against the K562 cell line was evalu-
ated in a standard 4 h
51
Cr release assay. K562-HLA-G1 or
K562-pcDNA transfected cells were incubated for 1 h at
37 °C with
51
Cr. After two washes with RPMI-1640 med-
ium, target cells were co-cultured with NKL effector cells
for 4 h at 37 °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).
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
Tyr35 (D1) CQGGQETQEYR 13.78 14.19
CQGGQETQEY*R – 8.88
CYYGSDTAGR 7.77 9.69
Tyr76 (D1) CY*YGSDTAGR – 6.61
Tyr77 (D1) CYY*GSDTAGR – 6.15
SESSDPLELVVTGAYIK 10.49 14.28
Tyr99 (D1) SESSDPLELVVTGAY*IK – 8.85
KPSLSVQPGPIVAPEE
TLTLQCGSDAGYNR
13.06 19.50
Tyr229 (D3) KPSLSVQPGPIVAPEETLT
LQCGSDAGY*NR
– 10.64
YQAEFPMGPVTSAHAGTYR 12.35 16.64
Tyr355 (D4) Y*QAEFPMGPVTSAHAGTYR – 10.67
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.
A. Dı
´
az-Lagares et al. ILT2 nitration in the binding domain
FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4239
Specific lysis level was calculated as the percentage

51
Cr
release from the maximum release:
% specific lysis = 100 · [(sample c.p.m. ) spontaneous
release) ⁄ (maximum release ) spontaneous release)].
The spontaneous release was the c.p.m. measured in
51
Cr-labelled K562 cells cultured in medium without NKL
cells. The maximum release was achieved when
51
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
anti-ILT2 serum GHI ⁄ 75 (Becton-Dickinson Biosciences,
Franklin Lakes, NJ, USA) for 30 min at 37 °C before
co-culturing them with K562 cells.
Flow cytometry
For cell surface labelling, cells were incubated for 30 min at
4 °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 fixed
in paraformaldehyde 1%. For intracellular staining, cells
were fixed with paraformaldehyde 1% for 10 min at 37 °C
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
serum (Beckman Coulter) for 30 min, washed with
NaCl ⁄ Pi-BSA 0.5%, and resuspended in NaCl ⁄ Pi for flow

cytometry analysis. Control aliquots were stained with the
isotype-matched mouse antibody (Beckman Coulter). 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)
for 1 h at 25 °C. Reverse transcription was carried out
using High-Capacity cDNA Archive Kit according to the
manufacturer’s instructions (Applied Biosystems, Foster
City, CA USA). Real-time PCR was performed using the
TaqMan Gene Expression Assay (Applied Biosystems) on
an ABI PRISM 7700 Sequence Detector (Applied 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 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.
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 FEBS
Nitrotyrosine immunoprecipitation
Cells were lysed in NP40 0.5% in Tris-HCl buffer with
protease inhibitors (Roche Applied Sciences, Mannheim,
Germany) and incubated with anti-nitrotyrosine serum
(Upstate Biotechnology, Lake Placid, NY, USA) at a 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-
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 °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
to 10% PAGE under denaturing conditions (SDS ⁄
PAGE), with subsequent electroblotting transfer onto a
nitrocellulose membrane. The membrane was blocked
with 5% nonfat dried milk in NaCl ⁄ Pi-Tween 0.1% for
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 °C 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 37 °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 °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 CapLCÔ (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 EaseÔ 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
temperature was 80 °C and the spray voltage was
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.
A. Dı
´
az-Lagares et al. ILT2 nitration in the binding domain

FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4241
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.
HLA-G binding assay
rhILT2-Fc was treated with increasing concentrations of
SIN-1 for 3 h at 37 °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 °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 °C. After
washing, anti-b
2

-microglobulin serum (Dako, Glostrup,
Denmark) was added and incubated for 1 h at 37 °C.
HLA-G binding was detected using EnVision+ Dual Link
System-HRP (Dako) and 3,3¢,5,5¢-tetramethylbenzidine
(Sigma-Aldrich). Colour development was stopped with
HCl 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.
References
1 Moffett A & Loke C (2006) Immunology of placentation
in 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 Human
Lymphoid and Myelomonocytic Cells. J Exp Med 186,
1809–1818.
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.
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 ILT4
compete with CD8 for MHC class I binding and bind
preferentially 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-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.
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,
Biordi L, Martinotti S & Santoni A (1999) Interleukin-
2-activated rat natural killer cells express inducible
nitric oxide synthase that contributes to cytotoxic
function and interferon-gamma production. Blood 93,
3876–3884.
10 Coleman JW (2001) Nitric oxide in immunity and
inflammation. Int Immunopharmacol 1, 1397–1406.
11 Roozendaal R, Vellenga E, de Jong MA, Traanberg
KF, 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, Mumford
RA, Riehl TE & Stenson WF (1996) Expression of
inducible 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
13 Souza JM, Peluffo G & Radi R (2008) 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 (2007) Reversible inhibition of mamma-
lian glutamine synthetase by tyrosine nitration. FEBS
Lett 581, 84–90.
15 Lopez AS, Alegre E, Diaz A, Mugueta C & Gonzalez
A (2006) Bimodal effect of nitric oxide in the 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. Hyper-
tension 28, 488–493.
17 Ohmori H & Kanayama N (2005) Immunogenicity of
an inflammation-associated product, tyrosine nitrated
self-proteins. Autoimmunity Reviews 4, 224–229.
18 Diaz-Lagares A, Alegre E, Lemaoult J, Carosella ED &
Gonzalez A (2009) 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 (2006) Efficient leukocyte IG-like receptor
signaling and crystal structure of disulfide-linked
HLA-G dimer. J Biol Chem 281, 10439–10447.
20 Ischiropoulos H (2003) Biological selectivity and func-
tional 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. (2006) Endogenously nitrated pro-
teins in mouse brain: links to neurodegenerative disease.
Biochemistry 45, 8009–8022.
22 Zhan X & Desiderio DM (2006) Nitroproteins from a
human pituitary adenoma tissue discovered with a
nitrotyrosine affinity column and tandem mass
spectrometry. Anal Biochem 354, 279–289.
23 Fujigaki H, Saito K, Lin F, Fujigaki S, Takahashi K,
Martin BM, Chen CY, Masuda J, Kowalak J,
Takikawa O et al. (2006) Nitration and Inactivation of
IDO by Peroxynitrite. J Immunol 176, 372–379.
24 Zaragoza R, Torres L, Garcia C, Eroles P, Corrales F,
Bosch A, Lluch A, Garcia-Trevijano ER & Vina JR
(2009) Nitration of cathepsin D enhances its proteolytic
activity during mammary gland remodeling after lacta-
tion. Biochem J 6,6.
25 Paul-Clark MJ, Roviezzo F, Flower RJ, Cirino G,
Soldato PD, Adcock IM & Perretti M (2003) Gluco-
corticoid receptor nitration leads to enhanced anti-
inflammatory effects of novel steroid ligands. J Immunol
171, 3245–3252.
26 Radi R (2004) Nitric oxide, oxidants, and protein tyro-
sine nitration. Proc Natl Acad Sci USA 101, 4003–4008.
27 Menier C, Riteau B, Carosella ED & Rouas-Freiss N
(2002) MICA triggering signal for NK cell tumor lysis
is counteracted by HLA-G1-mediated inhibitory signal.
Int J Cancer 100, 63–70.
28 Kovats S, Main EK, Librach C, Stubblebine M, Fisher

SJ & DeMars R (1990) A class I antigen, HLA-G,
expressed in human trophoblasts. Science 248, 220–223.
29 Sargent IL, Borzychowski AM & Redman CWG (2006)
NK cells and human pregnancy – an inflammatory
view. Trends Immunol 27, 399–404.
30 Schiessl B, Mylonas I, Hantschmann P, Kuhn C, Schulze
S, Kunze S, Friese K & Jeschke U (2005) Expression of
endothelial NO synthase, inducible NO synthase, and
estrogen receptors alpha and beta in placental tissue of
normal, preeclamptic, and intrauterine growth-restricted
pregnancies. J Histochem Cytochem 53, 1441–1449.
31 Munoz J, Fernandez-Irigoyen J, Santamaria E, Parbel
A, Obeso J & Corrales FJ (2008) Mass spectrometric
characterization of mitochondrial complex variants I
NDUFA10. Proteomics 8, 1898–1908.
A. Dı
´
az-Lagares et al. ILT2 nitration in the binding domain
FEBS Journal 276 (2009) 4233–4243 ª 2009 The Authors Journal compilation ª 2009 FEBS 4243