Article

The Membrane-Proximal Immunoreceptor Tyrosine-Based Inhibitory Motif Is Critical for the Inhibitory Signaling Mediated by Siglecs-7 and -9, CD33-Related Siglecs Expressed on Human Monocytes and NK Cells

Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, University of Dundee, Dundee, United Kingdom.
The Journal of Immunology (Impact Factor: 4.92). 01/2005; 173(11):6841-9. DOI: 10.4049/jimmunol.173.11.6841
Source: PubMed
ABSTRACT
Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.

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Available from: Tony Avril, Dec 27, 2013
The Membrane-Proximal Immunoreceptor Tyrosine-Based
Inhibitory Motif Is Critical for the Inhibitory Signaling
Mediated by Siglecs-7 and -9, CD33-Related Siglecs Expressed
on Human Monocytes and NK Cells
1
Tony Avril,* Helen Floyd,* Frederic Lopez,
Eric Vivier,
and Paul R. Crocker
2
*
Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and
are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs
display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the
immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and
-9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are
able to inhibit the Fc
RI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under
these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing
phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP
fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the
inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2
phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9,
raising the possibility that “inside-out” signaling may occur to regulate ligand binding. The Journal of Immunology, 2004, 173:
6841– 6849.
S
iglecs
3
are sialic acid-binding Ig-like lectins characterized
by a homologous N-terminal V-set Ig-like domain and
varying numbers of C2-set Ig-like domains (1). In humans,
11 Siglecs have been described: sialoadhesin (Siglec-1, CD169),
CD22 (Siglec-2), and the myelin-associated glycoprotein (MAG or
Siglec-4) form one group distinct from the other that includes
CD33 (Siglec-3) and the more recently described CD33-related
Siglecs (Siglecs-5 to -11) (1, 2). The CD33-related Siglecs are
expressed differentially in the hemopoietic system (1). Some are
broadly expressed as for example Siglec-5 (CD170), which is
found in B cells, monocytes, and neutrophils (1, 3). Others have a
more restricted distribution, notably Siglec-8, which is present on
circulating eosinophils (1, 4). Another feature of this group is the
presence of two conserved tyrosine-containing motifs in the cyto-
plasmic region. The membrane-proximal motif (EI/LXYAXLXF)
conforms to the consensus immunoreceptor tyrosine-based inhibi-
tion motif (ITIM) I/L/S/VxYxxL/V (5, 6), whereas the membrane-
distal motif does not.
ITIMs have been found in the cytoplasmic tail of many inhib-
itory receptors, such as CD158/killer cell Ig-like receptor (KIR),
Ly49, and CD85/Ig-like transport/leukocyte inhibitory receptor
molecules expressed on various hemopoietic cells including
monocytes and NK cells (reviewed in Refs. 7 and 8). ITIMs
were functionally defined as specific amino acid sequences that,
once tyrosine-phosphorylated, provide a docking site for the Src
homology 2 (SH2)-domain bearing cytoplasmic phosphatases (8).
ITIM-related motifs have also been described in many other
inhibitory receptors (9). Their amino acid sequences differ from
the consensus ITIM sequence by the presence of a threonine in
position 2 or an isoleucine in position 2. For some of the
CD33-related Siglecs (CD33, Siglecs-5, -6, and -9), the membrane-
distal motif (TEYSEI/VK/R) is similar to the immunoreceptor
tyrosine-based switch motif (ITSM) TxYxxI/V (10, 11) found in
CD150/signaling lymphocyte activation molecule (SLAM), CD66/
carcinoembryonic Ag cell adhesion molecule, leukocyte-
associated Ig-like receptor-1 (LAIR-1), and CD31/PECAM-1 mol-
ecules (10, 12). The discovery of CD33-related Siglecs and the
presence of these two tyrosine motifs raise the possibility that
these proteins are involved in regulating cellular activation within
the immune system.
Siglecs-7 and -9 share more than 80% sequence identity
throughout the extracellular, transmembrane, and intracellular re-
gions (13) but differ in their expression pattern (1). Siglec-7 is
highly expressed on NK cells and weakly on monocytes and a
minor subpopulation of CD8 T lymphocytes (14, 15). In contrast,
Siglec-9 is broadly expressed with high expression on monocytes
and low-level expression on neutrophils and subpopulations of
NK, B, and T cells (13). They also differ in sialic acid binding
specificity: Siglec-7 possesses a unique preference for
(2,8)-
linked disialic acids and branched
(2,6)-linked sialic acids,
*Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, Univer-
sity of Dundee, Dundee, United Kingdom; and
Centre d’Immunologie de Marseille-
Luminy, Centre National de la Recherche Scientifique-Institut National de la Sante et
de la Recherche Me´dicale-Universite´delaMe´diterrane´e, Marseille, France
Received for publication July 21, 2004. Accepted for publication September 21, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Research in the PRC laboratory is supported by the Wellcome Trust.
2
Address correspondence and reprint requests to Dr. Paul R. Crocker, The Wellcome
Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, United King-
dom. E-mail address: p.r.crocker@dundee.ac.uk
3
Abbreviations used in this paper: Siglec, sialic acid-binding Ig-like lectin; ITIM,
immunoreceptor tyrosine-based inhibition motif; ITSM, immunoreceptor tyrosine-
based switch motif; KIR, killer cell Ig-like receptor; LAIR-1, leukocyte-associated
Ig-like receptor-1; MAG, myelin-associated glycoprotein; PAb, polyclonal antibody;
PTP, protein tyrosine phosphatase; RBL, rat basophilic leukemia; SAP, SLAM-as-
sociated protein; SH2, Src homology 2; SHP, SH2 domain-containing phosphatase;
SLAM, signaling lymphocyte activation molecule; TRITC, tetramethylrhodamine
isothiocyanate, WT, wild type.
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
Page 1
whereas Siglec-9 does not bind to
(2,8)-linked disialic acids but
prefers terminal
(2,3)- and
(2,6)-linkages (16, 17). Regardless
of the role of sialic acid recognition on their function, Siglec-7 acts
as an inhibitory receptor in human NK cells after engagement by
Abs (14) or binding with sialic acid-containing ligands (18) and
once phosphorylated can recruit the SH2 domain-bearing protein
tyrosine phosphatase (PTP) SHP-1 (14). Anti-Siglec-7 Abs also
inhibit the proliferation of myeloid cells (19). Concerning the
function of Siglec-9, there have been no reports so far, although its
high similarity with Siglec-7 strongly suggests Siglec-9 has a
similar inhibitory function.
In this study, we demonstrate for the first time that Siglec-9 acts
as an inhibitory receptor using the rat basophilic leukemia (RBL)
model, which has been widely used to analyze KIR functions in
particular. We show that Siglec-9, as well as Siglec-7, can inhibit
Fc
RI-mediated serotonin release of RBL cells and recruit the ty-
rosine phosphatases SHP-1 and SHP-2. We then show that the
membrane-proximal ITIM is essential for the inhibitory activities
of Siglecs-7 and -9, for their tyrosine phosphorylation, and for the
recruitment of the PTPs SHP-1 and SHP-2. Furthermore, mutation
of the membrane-proximal motif leads to an increase in their RBC
binding ability, indicating that signaling involving SHP-1 and
SHP-2 may modulate sialic acid recognition mediated by Siglecs-7
and -9.
Materials and Methods
Reagents, plasmids, and Abs
Unless otherwise specified, all reagents and chemicals were purchased
from Sigma-Aldrich (Poole, U.K.). [
3
H]Serotonin (hydroxytryptamine cre
-
atine sulfate, 5-[1,2-[
3
H](N)]) was obtained from NEN (Hounslow, U.K.).
Vibrio cholerae sialidase was obtained from Calbiochem (Nottingham, U.K.).
The cDNAs encoding human Siglecs-7 and -9 were described in Refs.
13 and 15). Site-directed mutagenesis of Siglecs-7 and -9 cDNAs was
performed with sets of mutagenic primers (Table I) using the QuickChange
kit (Stratagene, Amsterdam, The Netherlands) according to the manufac-
turer’s instructions. The tail deletion form of Siglecs-7 (7) and -9 (9)
were obtained by PCR using sets of primers described in Table I and
cloned into the pcDNA3 vector (Invitrogen, Paisley, U.K.). The cDNA-
encoding human tyrosine phosphatases SHP-1 and SHP-2 were a kind gift
from Benjamin Neel (Harvard Medical School, Boston, MA) and were
cloned into the pEGFP-N1 vector (Clontech, Basingstoke, U.K.). Catalyt-
ically inactive forms of SHP-1 (C453S, mutSHP-1) and SHP-2 (C459S,
mutSHP-2) were generated as above using sets of mutagenic primers de-
scribed in Table I. All the PCRs were performed using PFU Turbo DNA
polymerase (Stratagene), and the presence of introduced mutations was
confirmed by DNA sequencing.
The purified sheep polyclonal Ig and mAbs anti-human Siglec-7 (S7.5a)
and anti-human Siglec-9 (K8) were previously described (13, 18). The
GL183 (anti-CD158b/KIR2DL3) mAb was obtained from Immunotech
(Marseille, France). The IgE-3 mouse IgE was obtained from BD Bio-
sciences (Oxford, U.K.). The goat anti-mouse IgG F(ab)
2
was obtained
from Jackson ImmunoResearch (Luton Beds, U.K.). The FITC-conjugated
rabbit anti-mouse Ig F(ab)
2
was obtained from Dako (Ely, U.K.). The
rabbit polyclonal Abs C-19 anti-SHP-1 and C-18 anti-SHP-2 were obtained
from Santa Cruz Biotechnology (Calne, U.K.). The 4G10 anti-
phosphotyrosine hybridoma was a kind gift from Lars Nitschke (University of
Wurzburg, Germany). The HRP-conjugated anti-mouse, rabbit, and sheep IgG
Abs were obtained from Vector Laboratories (Peterborough, U.K.).
Cells
The RBL-2H3 cells expressing KIR2DL3 (20) were cultured in DMEM
supplemented with 10% FCS, penicillin (100 IU/ml), and streptomycin
(100
g/ml). Stable cell lines expressing wild-type (WT) and mutant forms
of Siglecs-7 and -9 were generated by electroporation and selection with
geneticin at 1 mg/ml (G418) (Roche, East Sussex, U.K.).
Flow cytometry
All incubations were conducted on ice. Cells were incubated with primary
mAbs (10
g/ml) for 30 min, washed, and incubated with the FITC-con-
jugated rabbit anti-mouse Ig F(ab)
2
for 30 min. Cells were analyzed using
a FACSCalibur (Becton Dickinson, Oxford, U.K.).
Serotonin release assay
The serotonin release assay was performed as described in Ref. 20. Briefly,
[
3
H]serotonin-labeled RBL cells were incubated with mouse IgE (at 0.1
g/ml) in the absence or presence of various amount of anti-MHC class I
molecules (OX-18), anti-Siglec-7 (S7.5a), anti-Siglec-9 (K8), or anti-
KIR2DL3 (GL183) mAbs. After washing, cells were incubated for 30 min
at 37°C with goat anti-mouse IgG F(ab)
2
(at 50
g/ml). Supernatants were
collected, and the released radioactivity was analyzed using the WinSpec-
tral liquid scintillation counter (Wallac, Milton Keynes, U.K.). Each assay
was set up in triplicate, and the results were expressed as a percentage of
specific serotonin release: (cpm test cpm spont)/(cpm max cpm
spont) 100, where cpm spont is the spontaneous release of [
3
H]serotonin
obtained with cells incubated in absence of Abs and cpm max is the max-
imum release of [
3
H]serotonin obtained with cells lysed using Triton
X-100. For comparative analysis, results were expressed as a mean of the
percentage of inhibition of serotonin release: 100 (% test 100/% IgE)
obtained in experiments performed with n different clones, where % IgE is
Table I. Primers used in this study
a
7Y1 Sense 5-GGAAAGAGAGATCCAGTTTGCACCCCTCAGC-3
Antisense 5-GCTGAGGGGTGCAAACTGGATCTCTCTTTCC-3
7Y2 Sense 5-GCCACCAACAATGAGTTCTCAGAGATCAAGATCC-3
Antisense 5-GGATCTTGATCTCTGAGAACTCATTGTTGGTGGC-3
7 Sense 5-TAATACGACTCACTATAGGG*-3
Antisense 5-TCTAGATCATCACCTGCAGGACCTCACTACAATGAAG-3
9Y1 Sense 5-GGAGAGCTCCAGTTTGCATCCCTCAGCTTCC-3
Antisense 5-GGAAGCTGAGGGATGCAAACTGGAGCTCTCC-3
9Y2 Sense 5-GGCCACTGACACCGAGTTCTCGGAGATCAAGATCC-3
Antisense 5-GGATCTTGATCTCCGAGAACTCGGTGTCAGTGGCC-3
9 Sense 5-TAATACGACTCACTATAGGG*-3
Antisense 5-TCTAGATCATCACCTGCAGGACCTCACTACAACGAAG-3
SHP-1 Sense 5-CTCGAGATGGTGAGGTGGTTTCACCG-3
Antisense 5-AAGCTTCTTCCTCTTGAGGGAACCC-3
mutSHP-1 Sense 5-GGGCCCATCATCGTGCACTCCAGCGCCGGCATCGGCCGC-3
Antisense 5-GCGGCCGATGCCGGCGCTGGAGTGCACGATGATGGGCCC-3
SHP-2 Sense 5-CTCGAGATGACATCGCGGAGATGG-3
Antisense 5-AAGCTTTCTGAAACTTTTCTGCTGTTGC-3
mutSHP-2 Sense 5-GGGCCGGTCGTCGTCCACTCCAGTGCTGGAATTGGCCGG-3
Antisense 5-CCGGCCAATTCCAGCACTGGAGTGCACCACCACCGGCCC-3
a
Nucleotide changes introduced for changing tyrosine to phenylalanine (for 7Y1, 7Y2, 9Y1, and 9Y2 constructs) and
cysteine to serine (for mutSHP-1 and mutSHP-2 constructs) are in bold. Nucleotide changes that introduce restriction enzyme
sites to facilitate cloning (XbaI for 7 and 9 and XhoI/HindIII for SHP-1 and SHP-2) are in italic. Simple underline, initiation
codon; double underline, stop codon; asterisk, T7 primer.
6842 MUTATIONAL ANALYSIS OF THE TYROSINE-BASED MOTIFS IN SIGLECS-7 AND -9
Page 2
the percentage of specific serotonin release obtained with cells incubated
with mouse IgE alone.
Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting were performed as previously
described (21). Briefly, cells were treated or not with pervanadate (0.5 mM)
for 10 min at 37°C. Pre-cleared cell lysates were immunoprecipitated with
S7.5a (anti-Siglec-7) or K8 (anti-Siglec-9) mAbs and then analyzed by
immunoblotting with sheep anti-Siglecs-7 or -9 polyclonal Abs (PAbs),
with 4G10 mAb anti-phosphotyrosine or with rabbit PAbs C-19 anti-
SHP-1 or C-18 anti-SHP-2.
Fluorescence microscopy
Siglecs-7 and -9 RBL clones were transiently transfected with SHP-1-GFP,
SHP-2-GFP, mutSHP-1-GFP, or mutSHP-2-GFP cDNA by electroporation
and cultured overnight on coverships (BDH, Lutterworth, U.K.). Cells
were then stained with anti-Siglec-7 or anti-Siglec-9 sheep PAbs (10
g/
ml) for 30 min at 4°C and incubated for 30 min at 37°C with tetrameth-
ylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-sheep Ig
F(ab)
2
to generate patched Siglecs. Cells were then treated or not with
pervanadate (0.5 mM) for 10 min at 37°C, fixed with 4% paraformalde-
hyde, and analyzed by fluorescence microscopy. For the Fc
RI-mediated
activation of RBL cells, GFP-transfected cells were stained with mIgE (0.1
g/ml) and S7.5a or K8 mAbs (10
g/ml) for 30 min at 4°C, incubated
with goat anti-mouse Ig F(ab)
2
(50
g/ml) for 30 min at 37°C and then
labeled with anti-Siglec-7 or anti-Siglec-9 sheep PAbs (10
g/ml) and
TRITC-conjugated rabbit anti-sheep Ig F(ab)
2
(10
g/ml) for 15 min at
37°C to create clusters. Images were acquired using the AxioVision im-
aging system (Imaging Associates, Bicester, U.K.) and a Zeiss immuno-
fluorescence microscope (Jena, Germany).
RBC binding assays
RBC binding assays were performed as described in (13). Briefly, RBL
cells were treated or not with sialidase for1hat37°C, washed, and incu-
bated with human RBC for1hat4°C. Unbound RBC were gently washed,
and cells were fixed with 0.25% glutaraldehyde and rosetting assessed by
microscopy. To quantify binding, the percentage of RBL cells forming
rosettes (defined as RBL cells binding 5 RBC) was scored from counting
at least 200 RBL cells per field in five different fields per experiment.
Results are expressed as a mean percentage, obtained from three indepen-
dent experiments.
Statistics
Values represent the mean SD of n different experiments. Student’s t test
was applied using a two-tailed distribution of two samples of equal or
unequal variances.
Results
Cross-linking of Siglecs-7 and -9 inhibits Fc
RI-mediated
serotonin release in RBL cells
It has been shown previously that Siglec-7 acts as a NK cell in-
hibitory receptor in cytotoxicity assays (14, 18). In contrast, there
have been no reports on the inhibitory function of Siglec-9. To
investigate the potential inhibitory functions of Siglecs-7 and -9 in
parallel, we used the well-defined RBL model in which co-engage-
ment of inhibitory receptors and the activatory receptor, F
RI, in-
hibits serotonin secretion by RBL cells incubated with mIgE. We
generated RBL cell lines stably transfected with Siglec-7 or -9
cDNA (designated RBL-7WT and RBL-9WT, respectively) using
parental RBL-2H3 cells that had already been transfected with the
well-characterized inhibitory receptor KIR2DL3. The expression
of KIR2DL3 provided a useful internal reference control in the
inhibition assays. As shown in Fig. 1A on a representative clone,
expression of Siglec-7 or -9, KIR2DL3, MHC class I (used as a
negative control in the inhibition assays), and Fc
RI molecules
assessed by binding of IgE was demonstrated by flow cytometry
analysis (Fig. 1A). RBL-7WT and -9WT clones were then incu-
bated with a range of dilutions of mAbs anti-MHC class I mole-
cules (irrelevant mAb), anti-KIR2DL3 (positive control), anti-
Siglec-7 or anti-Siglec-9 in the presence of mIgE and used in a
serotonin release assay. As described previously (20), cross-link-
ing of KIR2DL3 with Fc
RI dramatically reduced the level of
serotonin release of RBL-7WT and -9WT cells in a dose-depen-
dent manner, whereas no significant effect was observed in the
presence of an irrelevant mAb that bound MHC class I molecules
(Fig. 1B). In contrast, cross-linking of Siglecs-7 and -9 substan-
tially reduced the serotonin release of RBL-7WT and -9WT cells
FIGURE 1. Inhibitory function of Siglecs-7 and -9
in RBL cells. A, RBL-7WT and -9WT cells were
stained or not (open histograms) with OX-18 (anti-
MHC class I molecules), S7.5a (anti-Siglec-7), K8 (anti-
Siglec-9), GL183 (anti-KIR2DL3) mAbs or mIgE
(IgE-3) (shaded histograms), followed by FITC-conju-
gated rabbit anti-mouse Ig F(ab)
2
, and analyzed by
flow cytometry. B,[
3
H]Serotonin-labeled RBL-7WT
and RBL-9WT cells were incubated with mIgE alone
() or with serial dilutions with an irrelevant mAb
OX-18 (f), S7.5a (Œ), K8 (), or GL183 () mAbs
and then challenged with goat anti-mouse Ig F(ab)
2
.
The serotonin released in supernatants was measured.
Results shown are expressed as the mean percentage of
specific serotonin release of triplicate wells from one
experiment representative of at least three independent
experiments.
6843The Journal of Immunology
Page 3
(Fig. 1B). These results shows that Siglec-9 is as potent as Siglec-7
in delivering inhibitory signals to RBL cells.
Activation of RBL cells leads to the recruitment of the
phosphatases SHP-1 and SHP-2 by Siglecs-7 and -9
To address the question of whether Siglecs-7 and -9 can recruit the
phosphatases SHP-1 and SHP-2 under conditions of activation in
RBL cells, we transfected RBL-7WT and -9WT cells with SHP-
1-GFP or SHP-2-GFP cDNA and performed co-localization ex-
periments using fluorescence microscopy. Transfected cells were
stained with anti-Siglec-7 or -9 sheep PAbs and then incubated
with TRITC-conjugated rabbit anti-sheep Ig F(ab)
2
for1hat
37°C to create patches of Siglec-7 and -9. The cells were then
treated or not with pervanadate to inhibit tyrosine phosphatases
FIGURE 2. Co-localization of WT and catalytically
inactive SHP-1-GFP and SHP-2-GFP with Siglecs-7
and -9 in resting and activated RBL cells. RBL-7WT (A
and B) and -9WT (C and D) were transfected with SHP-
1-GFP (A and C, lane 1–3), SHP-2-GFP (B and D, lane
1–3), mutSHP-1-GFP (A and C, lane 4), or mutSHP-2-
GFP (B and D, lane 4). After overnight culture, cells
were stained with anti-Siglec-7 or -9 sheep PAbs and
incubated for1hat37°C with TRITC-conjugated rab-
bit anti-sheep Ig F(ab)
2
. Cells were then treated or not
with pervanadate, fixed, and analyzed by fluorescence
microscopy. For the Fc
RI-mediated activation of RBL
cells, GFP-transfected cells were stained with mIgE and
S7.5a or K8 mAbs followed by goat anti-mouse Ig
F(ab)
2
, then labeled with anti-Siglec-7 (A and C, lane
3)or-9(B and D, lane 3) sheep PAbs followed by
TRITC-conjugated rabbit anti-sheep Ig F(ab)
2
. SHP1-,
mut-SHP-1-, SHP-2-, or mutSHP-2-GFP clusters are
indicated by the arrows.
FIGURE 3. Role of the two tyrosine motifs on the
tyrosine phosphorylation of Siglecs-7 and -9 and re-
cruitment of the PTPs SHP-1 and SHP-2 in RBL cells.
Parental RBL (A and B, lane 1), Siglec-7 (A), and Si-
glec-9 (B) RBL clones were untreated or treated with
pervanadate, lysed, and immunoprecipitated with S7.5a
(anti-Siglec-7, A) or K8 (anti-Siglec-9, B) mAbs. The
immunoprecipitates were then analyzed by immuno-
blotting with anti-Siglec-7 (A, first blot) or -9 (B, first
blot) sheep PAbs, 4G10 mAb (anti-phosphotyrosine, A
and B, second blot), C-19 rabbit PAb (anti-SHP-1,
A and B, third blot), or C-18 rabbit PAb (anti-SHP-2, A
and B, fourth blot).
6844 MUTATIONAL ANALYSIS OF THE TYROSINE-BASED MOTIFS IN SIGLECS-7 AND -9
Page 4
and increase phosphorylation, fixed, and analyzed. As shown in
Fig. 2, no co-localization of Siglec-7 or -9 with SHP-1 or SHP-2
was observed with untreated cells, whereas SHP-1 and SHP-2 co-
localized with Siglecs-7 and -9 when the cells were treated with
pervanadate. To investigate if the co-localization occurs in a more
physiological situation, mIgE and anti-Siglec-7 or -9 mAbs were
Table II. Constructs used in this study
a
proximal distal
motif motif
7WT 428 SSGEEREIQYAPLSFHKGEPQDLSGQEATNNEYSEIKIPK 467
7Y1 (Y437F) –––––––––F––––––––––––––––––––––––––––––
7Y2 (Y460F) ––––––––––––––––––––––––––––––––F–––––––
7Y1Y2 (Y437F/Y460F) –––––––––F––––––––––––––––––––––F–––––––
7WT 363 ALVFLSFCVIFIVVRSCRKKSARPAADVGDIGMKDANTIR 402
7 (R380) ALVFLSFCVIFIVVRSCR* 380
9WT 424 SSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR 463
9Y1 (Y433F) –––––––––F––––––––––––––––––––––––––––––
9Y2 (Y456F) ––––––––––––––––––––––––––––––––F–––––––
9Y1Y2 (Y433F/Y456F) –––––––––F––––––––––––––––––––––F–––––––
9WT 357 AKVFLSFCVIFVVVRSCRKKSARPAAGVGDTGIEDANAVR 396
9 (R374) ALVFLSFCVIFVVVRSCR* 374
a
Cytoplasmic tail sequences are shown for Siglecs-7 WT and -9 WT with amino acid substitutions shown for the mutants.
Double underlines, putative tyrosine motifs; single underlines, predicted trans-membrane regions; asterisks, stop codon.
FIGURE 4. Role of the two tyrosine motifs on the inhibitory function of Siglecs-7 and -9 in RBL cells. A, Siglecs-7 and -9 RBL clones were analyzed
for the expression of MHC class I molecules, Siglec-7, Siglec-9, KIR2DL3, and the binding of mIgE by flow cytometry as described in the legend to Fig.
1. B, Clones expressing similar levels of Siglecs-7 and -9 were used in inhibition assays as described in Fig. 1. C, The bar charts show the mean percentage
of inhibition observed with n different clones using mAbs at 10
g/ml (, p 0.05 vs RBL-WT).
6845The Journal of Immunology
Page 5
cross-linked using GFP-transfected cells in a similar way as in the
inhibition assay, and then clusters were created by adding sheep
PAbs anti-Siglec-7 or -9 followed by TRITC-conjugated rabbit
anti-sheep Ig F(ab)
2
. As shown in Fig. 2, SHP-1 and SHP-2 co-
localized with Siglecs-7 and -9 when the cells were activated via
engagement of the Fc
RI receptor.
We also used catalytically inactive forms of SHP-1-GFP and
SHP-2-GFP (mutSHP-1-GFP and mutSHP-2-GFP), which retain
the ability to bind their substrates but are unable to dephosphory-
late them. As shown in Fig. 2, Siglecs-7 and -9 co-localized with
mutSHP-1 and mutSHP-2 without any activation of RBL cells.
To investigate SHP-1 and SHP-2 association biochemically, pa-
rental RBL, RBL-7WT, and -9WT cells were treated or not with
pervanadate and Siglec-7 or -9 immunoprecipitates from RBL ly-
sates were then analyzed by immunoblotting with anti-Siglec-7 or
-9 PAbs, anti-phosphotyrosine mAb, anti-SHP-1, or anti-SHP-2
PAbs (Fig. 3). As expected, specific bands of 77 and 78 kDa
corresponding to Siglecs-7 and -9, respectively were observed with
untreated and pervanadate-treated RBL-7WT and -9WT lysates,
whereas no signal was detectable with parental RBL lysates (Fig.
3). Tyrosine-phosphorylated Siglecs-7 and -9 and co-immunopre-
cipitated SHP-1 and SHP-2 were readily detectable in pervana-
date-treated RBL-7WT and -9WT lysates, but no signal was seen
with untreated RBL-7WT, -9WT, or the parental RBL lysates
(Fig. 3).
Taken together, these results indicate that Siglecs-7 and -9 can
be phosphorylated by tyrosine kinases and then recruit the tyrosine
phosphatases SHP-1 and SHP-2.
The membrane-proximal tyrosine motif is necessary for the
inhibitory function, tyrosine phosphorylation, and recruitment of
the PTPs SHP-1 and SHP-2 by Siglecs-7 and -9
To examine the roles of individual tyrosine motifs within the cy-
toplasmic tail of Siglecs-7 and -9 on the inhibitory function of
these molecules, we generated tyrosine to phenylalanine mutants
in both the membrane-proximal and membrane-distal tyrosine mo-
tifs either individually or in combination (Table II). Truncated
forms of Siglecs-7 and -9 in which the cytoplasmic tails were
deleted were also used (Table II). RBL clones expressing similar
levels of Siglec-7 or -9 were selected for comparative analysis in
inhibition assays (Fig. 4A). Comparable levels of expression of
MHC class I molecules, KIR2DL3, and Fc
RI were also confirmed
by flow cytometry (Fig. 4A). Using RBL-7Y2 and -9-Y2 cells in
which the proximal tyrosine motif only was retained, strong inhi-
bition was still observed following co-cross-linking of Siglecs-7
and -9 with Fc
RI (Fig. 4, B and C). In contrast, mutation of the
membrane-proximal motif in RBL-7Y1, -7Y1Y2, -9Y1, or
-9Y1Y2 or truncation of the cytoplasmic tail resulted in a complete
loss of inhibition (Fig. 4, B and C). These results demonstrate that
the membrane-proximal tyrosine motif is both necessary and suf-
ficient to mediate the inhibitory function of Siglecs-7 and -9.
To investigate how the mutations of the tyrosine motifs affect
phosphorylation of Siglecs-7 and -9 and their capacity to recruit
the PTPs SHP-1 and SHP-2, RBL mutants were treated with per-
vanadate and Siglec-7 or -9 immunoprecipitates from RBL lysates
were analyzed by immunoblotting with anti-Siglec-7 or -9 PAbs,
anti-phosphotyrosine mAb, anti-SHP-1, or anti-SHP-2 PAbs (Fig.
3). As expected, no tyrosine phosphorylation was detected with
RBL-7Y1Y2, RBL-7, RBL-9Y1Y2, and RBL-9 lysates (Fig.
3). In addition, mutation of the membrane-proximal tyrosine motif
completely abrogated the tyrosine phosphorylation, whereas sub-
stantial phosphorylation was retained with the mutation of the
membrane-distal motif (Fig. 3). Concerning the recruitment of the
PTPs, all mutations completely abrogated the recruitment of
SHP-1, whereas only the membrane-distal mutation could still re-
cruit significant levels of SHP-2 (Fig. 3).
To confirm our findings in co-localization experiments, Si-
glecs-7 and -9 RBL mutants were transfected with mutSHP-1-GFP
FIGURE 5. Co-localization of catalytically inactive
SHP-2-GFP with Siglecs-7 and -9 in RBL clones. Si-
glec-7 (A) and Siglec-9 (B) RBL clones were trans-
fected with mutSHP-2-GFP cDNA. After overnight
culture, cells were stained with anti-Siglec-7 or -9
sheep PAbs as described in the legend to Fig. 2. Un-
treated cells were then analyzed by fluorescence mi-
croscopy. MutSHP-2-GFP clusters are indicated by the
arrows.
FIGURE 6. Role of the two tyrosine motifs on the RBC binding activity of Siglecs-7 and -9. Siglec-7 (A) and Siglec-9 (B) RBL clones were untreated
(gray bars) or sialidase-treated (black bars) and then incubated with human RBC. After washing, cells were fixed and the percentage of RBC cells with
rosettes were scored by microscopy as described in Materials and Methods. Results are expressed as the mean percentage obtained in three independent
experiments (, p 0.05 vs RBL-WT).
6846 MUTATIONAL ANALYSIS OF THE TYROSINE-BASED MOTIFS IN SIGLECS-7 AND -9
Page 6
and mutSHP-2-GFP and patches of Siglec-7 or -9 were created.
Cells were then fixed and analyzed by fluorescence microscopy.
No co-localization of Siglec-7 or -9 with mutSHP-1 was observed
with any of RBL mutants (data not shown). However, in approx-
imately one-third of RBL-7Y2 or RBL-9Y2 cells, Siglecs-7 and -9
clearly co-localized with mutSHP-2 (Fig. 5), whereas no associa-
tion was observed with the other Siglecs mutants (Fig. 5). These
results demonstrate that the membrane-proximal tyrosine motif is
essential for the tyrosine phosphorylation of Siglecs-7 and -9 and
the recruitment of the PTPs SHP-1 and SHP-2, whereas tyrosine
phosphorylation and association with SHP-2 can still occur after
mutation of the distal-motif of Siglecs-7 and -9.
The proximal tyrosine motif modulates the sialic acid
recognition of Siglecs-7 and -9
As members of the Siglec family, Siglecs-7 and -9 have the ability
to recognize sialic acid residues, either in cis on the Siglec-ex-
pressing cell, or in trans on other cells (reviewed in Ref. 1). In a
recent study, we have shown that the Siglec-7 binding site is
masked on NK cells, but can be unmasked following sialidase
treatment to destroy the cis-interacting sialic acids (18). Human
RBC are a very convenient indicator of sialic acid-dependent bind-
ing of most Siglecs, including Siglecs-7 and -9 (13, 15). To in-
vestigate if mutations in the tyrosine motifs affect recognition of
sialylated glycans by Siglecs-7 and -9, RBL-7WT, -9WT, and re-
spective mutants were treated or not with sialidase and tested in
RBC binding assays. As expected, no rosettes occurred with un-
treated RBL cells (Fig. 6), suggesting that cis-interactions between
Siglec-7 or -9 and sialic acids occurs on the membrane of RBL
cells and abrogate any trans-interaction. In contrast, sialidase-
treated RBL-7WT and -9WT mediated robust RBC binding activ-
ity. A striking increase in binding was observed with Y1 and Y1Y2
mutants, but no effect was observed with Y2 mutants (Fig. 6).
These results indicate that the membrane-proximal tyrosine motif
modulates the ligand binding activity of Siglecs-7 and -9. One
explanation could be that the Y1 and Y1Y2 mutations directly
affect the distribution of Siglecs-7 and -9 on the membrane of the
cells. To investigate this possibility, RBL-7WT, -7Y1Y2, -9WT,
and -9Y1Y2 cells were treated or not with sialidase, fixed, stained
with anti-Siglec-7 or -9 mAbs, and analyzed by confocal micros-
copy. No obvious difference was observed between RBL-7WT or
-9WT cells and RBL-7Y1Y2 or -9Y1Y2 cells, respectively (data
not shown).
Discussion
In this study, we show for the first time that Siglec-9 can function
as an inhibitory receptor. Taken altogether with the fact that
Siglec-9 contains an ITIM within its cytoplasmic domain, inhibits
the Fc
RI-mediated activation of RBL cells, is tyrosine-phospho-
rylated after pervanadate-treatment, and recruits the PTPs SHP-1
and SHP-2 after pervanadate-treatment and Fc
RI-mediated acti-
vation, it fulfills the criteria for inclusion in the inhibitory receptor
superfamily (5, 7, 8).
Previous reports on CD33 (22–24) and mouse Siglec-E (25, 26),
demonstrated the importance of the membrane-proximal tyrosine
motif in recruitment of SHP-1 and SHP-2 by co-immunoprecipi-
tation, but this study on Siglecs-7 and -9 provides the first analysis
of the role of tyrosine-based motifs in the inhibitory function for
CD33-related Siglecs. Mutation of the membrane-proximal ty-
rosine motif that conforms to the consensus ITIM led to a complete
abrogation of tyrosine-phoshorylation of the receptors, recruitment
of the PTPs SHP-1 and SHP-2, and the inhibition of Fc
RI-medi-
ated activation of RBL cells. Similar results have been obtained
with CD158/KIR molecules that contain two ITIMs, demonstrat-
ing that the proximal ITIM is critical for the inhibition of T cell
activation by CD158a/KIR2DL1 (27), the Fc
RI-mediated activa-
tion of RBL cells by CD158b/KIR2DL3 (28) or NK cytotoxicity
function by CD158b/KIR2DL3 (29), CD158e1/KIR3DL1 (30),
and CD158/KIR2DL5 (31). Some of these studies also showed that
tyrosine phosphorylation of these receptors and recruitment of the
PTPs were dependent on the membrane-proximal motif (28–30).
In addition, mutation of the membrane-proximal ITIM of LAIR-1
molecule, which also has two tyrosine-based motifs, resulted in
abrogation of its inhibitory function in NK cytotoxicity using a NK
cell line (32), whereas both motifs contributed equally to the in-
hibition function using RBL cells. Both motifs contributed to the
inhibition mediated by NKG2A, which has also two ITIMs, using
the RBL model (33), with a dominant role of the membrane-distal
motif probably due to the opposite orientation of the ITIMs in type
II vs type I transmembrane proteins (33). Interestingly, the fact that
tyrosine phosphorylation of Siglecs-7 and -9 did not occur after
mutation of the membrane-proximal motif strongly suggests that
this motif is the main binding site of tyrosine kinases. However,
we cannot exclude the possibility that the anti-phosphotyrosine
mAb used is unable to detect the tyrosyl-phosphorylated mem-
brane-distal motif present in the Y1 mutants. Although the identity
of the kinase(s) involved remains unknown, it is worth noting that
Siglec-3/CD33 can be phosphorylated in vitro and in vivo by Src
family kinase c-Src (22) and Lck (24), the membrane-proximal
motif playing a key role (24).
We have also shown that mutation of the membrane-distal motif
did not affect the inhibitory function of Siglecs-7 and -9. Interest-
ingly, with this mutation, we were still able to observe tyrosine
phosphorylation and SHP-2 recruitment using both immunopre-
cipitation- and fluorescence microscopy-based approaches,
whereas SHP-1 was not recruited. Although an involvement of
other signaling partners cannot be excluded, it is tempting to spec-
ulate that the inhibition observed after mutation of the membrane-
distal motif is mediated by SHP-2. Such a mechanism involving
SHP-2 rather than SHP-1 in inhibitory signaling has been sug-
gested in a previous study with KIR2DL3 (28) and has been dem-
onstrated recently with KIR2DL4 in RBL cells (34) and KIR3DL1
and KIR2DL5 in NK cells (30, 31). Based on these studies and the
work on LAIR-1 (32), it has been proposed that the conserved
sequence, VTYAQL, is sufficient for the recruitment of SHP-2 and
the inhibitory capacity of the receptor (32). The involvement of
SHP-2 rather than SHP-1 in the inhibitory function could also
apply to Siglecs-7 and -9, although they have different ITIM se-
quences (IQYAPL and LQYASL, respectively) compared with the
proposed consensus. Studies using peptides from KIR molecules
indicated that the position 2 from the tyrosine was critical for
SHP-1 and SHP-2 binding (5, 35, 36). Furthermore, a study using
peptides from mast cell function-associated Ag, which has a single
ITIM, clearly demonstrates that the nature of the amino acid res-
idue present at position 2 is important for differential binding of
SHP-1 or SHP-2 (37). Indeed binding of SHP-1 was only observed
with the native phospho-peptide SIpYSTL and the mutation serine
to valine, whereas binding of SHP-2 was maintained after mutation
of the serine to several amino acid residues and in particular to
isoleucine and leucine (37), residues present in Siglecs-7 and -9
ITIMs. Taken together, our results support the concept that SHP-1
requires two functional tyrosine-based motifs, whereas SHP-2 tol-
erates only one ITIM (28, 29, 32).
Another feature of some CD33-related Siglecs is that the mem-
brane-distal motif is similar to the ITSM TxYxxI/V found in
CD150/SLAM and SLAM-related molecules (10, 11, 38). It has
been shown that the ITSM of CD150 is involved in recruitment of
SLAM-associated protein (SAP) or EAT-2 in T and B cells (10,
6847The Journal of Immunology
Page 7
39 41). This prevented an interaction with the phosphatase SHP-2
(10, 39, 41, 42) and facilitated the recruitment of the phosphatase
SHIP (10, 43) or the Src family kinases FynT (43). Although this
issue has not been addressed directly in this study, it is unlikely
that this ITSM serves as a docking site for SAP-family adaptor
molecules. Indeed Siglec-10, which contains two membrane-prox-
imal ITIMs and one membrane-distal ITSM-like motif, does not
bind to the SAP molecule in a three-hybrid system (44).
Finally, we have shown here that mutation of the membrane-
proximal ITIM of Siglecs-7 and -9 leads to an increase in the sialic
acid-dependent binding of RBC, whereas mutation of the mem-
brane-distal motif has no effect. Similar results have been reported
previously with Siglec-3/CD33 using transiently transfected COS
cells (22) and may be a general feature of human CD33-related
Siglecs. The fact that SHP-2 was only recruited with Y2 mutants,
which displayed low levels of RBC binding similar to the WT
forms of Siglecs-7 and -9, raised the possibility that interactions of
this phosphatase could negatively regulate the ligand binding ac-
tivity of Siglecs-7 and -9. Binding of sialic acid residues by mo-
nomeric Siglecs is generally of very low affinity and therefore
clustering of receptors within the plasma membrane is essential for
high-avidity interactions with ligands on other cells. Although we
failed to see gross changes in distribution of Siglecs-7 or -9 com-
paring WT and mutant forms of the proteins expressed on trans-
fected RBL cells by confocal light microscopy, there could be
subtle changes that would require higher resolution imaging tech-
niques for their visualization.
In conclusion, we demonstrate that Siglec-9 is a new member
the inhibitory receptor superfamily and that the membrane-proxi-
mal ITIM is essential for the inhibitory function of both Siglecs-7
and -9 molecules.
Note added in proof. A recent publication demonstrated the in-
hibitory activity of Siglec-7 and Siglec-9 in transfected Jurkat cells
(Ikehara, Y., S. K. Ikehara, and J. C. Paulson. 2004. Negative
regulation of T cell receptor signaling by Siglec-7 (p70/AIRM)
and Siglec-9. J. Biol. Chem. 279:43117.).
Acknowledgments
We thank Benjamin Neel for the kind gift of SHP-1 and SHP-2 cDNA,
Lars Nitshke for providing the anti-phosphotyrosine mAb, Mathias Lucas
for the mouse IgE Abs, Claire Jones for the sheep anti-Siglec-9 Abs,
Gavin Nicoll and Kevin Lock for the anti-Siglec-7 Abs, Jiquan Zhang for
the anti-Siglec-9 mAb, and Andrew Ferenbach and Faye Wesley for the
SHP-1 and SHP-2-GFP constructs.
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    • "Although the mechanism of signaling transduction in mollusks is still veiled, it is proposed that the ITIM motifs in the intracellular domain of CgSiglec-1 serve as an inhibitory regulator for the initiation of downstream signal cascading. Similarly, previous studies have shown that ITIM in CD33-related siglecs dominates the recruitment of SRC homology 2 (SH2)-domain containing protein tyrosine phosphatases (SHP1 and SHP2) (Avril et al., 2004; Balaian et al., 2003), leading to the modulation of resultant Ca 2þ flux and the inhibition of signaling cascade (Paul et al., 2000). In the uniform manner, the mammal CD22 could serve as inhibitory in B-cell activation by setting a threshold of tolerance (Nitschke, 2005), and CD33-related siglecs could negatively regulate the cellular activation of haemopoietic and immune cells (Crocker and Varki, 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: Sialic acid binding immunoglobulin-type lectin (siglec) belongs to the immunoglobulin superfamily (IgSF), which acts as regulator involved in glycan recognition and signal transduction in the immune and nervous systems. In the present study, a siglec gene (designated CgSiglec-1) was characterized from the Pacific oyster, Crassostrea gigas. The cDNA of CgSiglec-1 was of 1251 bp encoding a predicted polypeptide of 416 amino acids. CgSiglec-1 was composed of two I-set immunoglobulin (Ig) domains, one transmembrane (TM) domain and two ITIM motifs, sharing a sequence similarity with vertebrate CD22 homologs. The mRNA expression of CgSiglec-1 could be detected in all the selected tissues, with the highest level in hemocytes and labial palps. The confocal analysis revealed that CgSiglec-1 mainly distributed on the cytoplasmic membrane of the oyster hemocytes. In addition, the mRNA transcripts of CgSiglec-1 in hemocytes increased significantly (4.29-fold to that of control group, p < 0.05) after Vibrio splendidus stimulation. The recombinant CgSiglec-1 protein (rCgSiglec-1) could bind to poly sialic acid (pSIAS), lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner. The blockade of CgSiglec-1 by specific polyclonal antibodies could enhance the LPS-induced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17. The results collectively indicated that CgSiglec-1 could act as a bridge molecule between invader recognition and signal transduction cascade, and modulate the immune response by inhibiting various important processes of immunity in oyster.
    Full-text · Article · Mar 2016 · Developmental and comparative immunology
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    • "The postulated inhibitory activity of Siglec 9 on the immune response through host and bacterial sialoglycans recognition [16,17] prompted us to verify its expression on monocyte subsets at baseline and after IVIg. In HD and in CVID, Siglec 9 expression was evident on all monocyte subsets , and it was higher on classical and on intermediate than on nonclassical monocytes (Fig. 3A and B). "
    [Show abstract] [Hide abstract] ABSTRACT: Intravenous IgG administration induces significant modifications in the innate and adaptive compartment of the immune system including the monocyte/macrophage system. We analyzed the in vivo effect of IgG administered at replacement dosages on the frequency of monocytes subsets, on the modulation of CD11b and sialic acid-binding immunoglobulin-like lectin receptor (Siglec 9) expression and on monocytes production of reactive oxygen species. We showed that patients with Common Variable Immune Deficiency have an increased frequency pro-inflammatory intermediate CD14(++)CD16(+) monocytes and an increased expression of CD11b and Siglec 9 on monocytes. IgG administered at replacement dosages exerted an in vivo anti-inflammatory effect as shown by a reduction of circulating monocytes, of intermediate pro-inflammatory monocytes, of CD11b and Siglec 9 expression and of ex vivo monocytes oxidative burst. Nevertheless, intravenous IgG administration did not affect the monocyte functional ability to respond to a bacterial stimulation in terms of CD11b and Siglec 9 expression and reactive oxygen species production. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · International immunopharmacology
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    • "It has been reported that tumor-produced MUC1 bound to Sig- lec-9, leading to negative immunomodulation [23]. Siglec-9 is an inhibitory receptor expressed on immune cells and the binding of Siglec-9 to its ligands induces inhibition of NK cell and T cell antitumor immunity [24,25]. We hypothesized that a factor inhibiting the binding of Siglec-9 to MUC1 interferes the transmission of the immunosuppression signal, leading to prevent antitumor immunity , and transplanted MM46-MUC1 mammary tumor cells to sSig- lec-9 Tg mice intraperitoneally. "
    [Show abstract] [Hide abstract] ABSTRACT: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.
    Preview · Article · Jun 2014 · Biochemical and Biophysical Research Communications
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