From the § Howard Hughes Medical Institute, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, December 26, 2000
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ABSTRACT |
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We describe the molecular cloning and
characterization of a novel myeloid inhibitory siglec, MIS, that
belongs to the family of sialic acid-binding immunoglobulin-like
lectins. A full-length MIS cDNA was obtained from murine bone
marrow cells. MIS is predicted to contain an extracellular region
comprising three immunoglobulin-like domains (V-set amino-terminal
domain followed by two C-set domains), a transmembrane domain and a
cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory
motif (ITIM)-like sequences. The closest relative of MIS in the siglec
family is human siglec 8. Extracellular regions of these two siglecs
share 47% identity at the amino acid level. Southern blot analysis
suggests the presence of one MIS gene. MIS is expressed in
the spleen, liver, heart, kidney, lung and testis tissues. Several
isoforms of MIS protein exist due to the alternative splicing. In a
human promonocyte cell line, MIS was able to bind Src homology
2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2. This
binding was mediated by the membrane-proximal ITIM of MIS. Moreover,
MIS exerted an inhibitory effect on Fc Leukocyte activation is controlled by a dynamic balance
between stimulatory and inhibitory signals through activating and inhibitory receptors, respectively. Engagement of the B and T cell
antigen receptors or Fc receptors on specific cells activates a cascade
of biochemical events required for cellular activation. However, the
termination of such activation signals also represents a critical
component of the natural immune response. Deficiency in inhibitory
pathways results in profound immune defects characterized by both
decreased activation thresholds and hyperresponsive phenotypes, which
often lead to autoimmunity and inflammation (1-4). In contrast, prevalence in signaling through an inhibitory receptor can result in
abrogation of signal transduction and cell unresponsiveness (5).
Inhibitory receptors belong to either of two structural types, the
immunoglobulin superfamily with type I membrane orientation or the
C-type lectin superfamily with type II membrane orientation (reviewed
in Ref. 6). Some inhibitory receptors are ubiquitously expressed (7),
while others display a more restricted pattern of expression (8-11).
Most inhibitory receptors contain one or more characteristic sequences
(V/IXYXXL/V) in the cytoplasmic domain
termed an immunoreceptor tyrosine-based inhibitory motif (ITIM)1 (6). Phosphorylation
of the tyrosine residue within the ITIM allows the binding of
protein-tyrosine phosphatases SHP-1 and/or SHP-2 or the inositol
phosphatase SHIP through their SH2 domains (12-14). When recruited to
the complex, these phosphatases act to block signal transduction by
dephosphorylating key proteins or lipids of a signaling cascade. Thus,
regardless of the structural type of an inhibitory receptor, the
inhibitory mechanism is similar.
The siglec family is composed of sialic acid-binding
immunolgobulin-like type I lectins including sialoadhesin (siglec 1), CD22 (siglec 2), CD33 (siglec 3), MAG, myelin-associated glycoprotein (siglec-4a), the structurally and functionally related SMP, Schwann cell myelin protein (siglec 4b), and the recently cloned human siglecs 5, 6, 7/p75/AIRM1, and 8 (15-22). Siglec family members have
variable numbers of extracellular immunoglobulin-like domains (ranging
from two in CD33 to 17 in sialoadhesin) with the characteristic amino-terminal V-set domain followed by C2-set domains (23, 24). The
common feature of these proteins is their recognition of sialic acid
residues on cell surface glycoproteins and glycolipids, which is
mediated by the amino-terminal V-set domain of a siglec (23, 24).
Although recognition of sialic acid residues is a hallmark feature of
all siglecs, the specificity of binding has been shown for certain
family members in terms of their preferences for the position of a
sialic acid residue on N-linked oligosaccharides as well as
in the binding of ligands expressed on different cell types (23, 25).
Each siglec is expressed in a highly restricted fashion (15-22),
implying a specific function for each family member. Indeed, it is
believed that sialoadhesin is involved in regulation of macrophage
function (25); MAG is implicated in myelinogenesis (26), while CD22
serves to inhibit signaling through the B-cell antigen receptor via
binding of SHP-1 tyrosine phosphatase to its ITIMs (27, 28), and the
lectin function of CD22 is thought to be important in recruiting CD22
to the B-cell antigen receptor (29). Although the function of the other
family members remains unknown, the presence of ITIM sequences in the
cytoplasmic tails of some of human siglecs renders them potential
inhibitory receptors. Recently, human CD33 has been characterized as an
inhibitory receptor by virtue of its ability to bind tyrosine
phosphatases SHP-1 and SHP-2 (30, 31).
Although nine human siglec proteins have been discovered, the mouse
siglec family has fewer members to date: sialoadhesin, MAG, SMP, CD22,
and CD33. Structurally, sialoadhesin, MAG, SMP, and CD22 are very
similar in mice and humans. In contrast, human and mouse CD33, although
somewhat similar in their extracellular domains, are strikingly
different in their transmembrane and cytoplasmic regions. Human CD33
contains two ITIM sequences in the cytoplasmic tail, whereas mouse CD33
does not. Recent characterization of several ITIM-containing human
siglecs implies that there must be murine ITIM-containing siglecs. Here
we describe the molecular cloning and characterization of a novel
murine ITIM-containing myeloid cell-restricted inhibitory siglec, MIS.
Cells and Antibodies--
EL4 and WEHI231 cells, murine T and B
cell lines, respectively, the human monocytic cell line, U937, and
human epithelioid carcinoma cell line, HeLa, were obtained from the
ATCC (Manassas, VA). The murine B cell line, A20, was a generous gift
from Dr. A. C. Chan (Washington University, St. Louis, MO). Murine
monocyte/macrophage cell lines, J774 and WEHI265.1, as well as NK cell
lines, KY-1 and KY-2, and the P815 mastocytoma cell line were generous
gifts from Dr. W. M. Yokoyama (Washington University, St. Louis,
MO). The murine T cell hybridoma, 3A9H, was a generous gift from Dr. P. M. Allen (Washington University, St. Louis, MO). EL4, A20, WEHI231, J774, and p815 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 0.1 mM nonessential amino acids, and 1 mM sodium
pyruvate. WEHI265.1 cells were maintained in RPMI 1640 medium
supplemented with 10% FCS. KY-1 and KY-2 cells were maintained in RPMI
1640 medium supplemented with 10% FCS, 100 units/ml
interleukin-2. All media were supplemented with 2 mM
L-glutamine, 100 µM 2-mercaptoethanol, 100 units/ml penicillin and 100 µg/ml streptomycin. U937 cells were
maintained in RPMI 1640 medium supplemented with 10% FCS and
L-glutamine. HeLa cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% bovine calf serum and
L-glutamine.
Mouse bone marrow cells were obtained by flushing the femurs of
euthanized mice with phosphate-buffered saline. Murine
peritoneal macrophages were aspirated from the peritoneal cavity of the
euthanized mice 2 days following intraperitoneal thioglycolate injection.
Antibodies used in this study included an anti-phosphotyrosine
monoclonal antibody (mAb), 4G10 (Upstate Biotechnology, Inc. Lake
Placid, NY), the anti-Flag M2 mAb (Sigma), the anti-SHP2 mAb (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), the anti-Fc RT-PCR--
Total RNA was isolated from 1 × 107 mouse bone marrow cells, peritoneal macrophages, or
various murine cell lines using RNAzolTM (Tel-Test,
Inc., Friendswood, TX) according to the manufacturer's instructions.
Three micrograms of isolated total RNA were reverse transcribed in 20 µl of buffer composed of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTP mix, 500 ng of
oligo(dT)12-18 primer (Life Technologies, Inc.) or 2 pmol
of gene-specific primer, and 200 units of SuperScript transcriptase
(Life Technologies). Three microliters of the resultant cDNA were
used in each PCR containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.0 mM MgCl2, 0.2 µM dNTP mix, 10 pmol of each oligonucleotide primer, and
1 unit of Taq polymerase (Promega). PCR amplification was
performed for 35 cycles. Each cycle consisted of denaturing at 94 °C
for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C
for 1.5 min. After the last cycle, the sample was maintained at
72 °C for 10 min. Amplification of cDNA ends was performed using
5'-RACE and 3'-RACE (Life Technologies, Inc.) according to the
manufacturer's instructions. Primers used in each PCR are listed in
Table I. PCR-amplified cDNA fragments were cloned into pCR2.1
vector (Invitrogen, Carlsbad, CA) and sequenced.
Southern and Northern Blot Analysis--
Genomic DNA was
isolated from C57Bl/6 mouse tail according to standard methods. A
360-base pair PCR-amplified fragment of MIS corresponding to the first
Ig-like domain of the molecule was labeled by random priming in the
presence of [32P]dCTP and [32P]dGTP
(Amersham Pharmacia Biotech) and used as a probe. Hybridization was
performed in 5× saline sodium citrate (SSC; 150 mM NaCl,
15 mM trisodium citrate), 5× Denhardt's solution, 1%
(w/v) SDS, 50% (v/v) formamide, 100 µg/ml denatured herring sperm
DNA at 42 °C. The blot was washed at 65 °C in 0.2× SSC and 0.1%
(w/v) SDS.
A mouse multiple tissue Northern blot (CLONTECH,
Palo Alto, CA) was hybridized with the full-length radiolabeled MIS
cDNA, a 0.7-kb SmaI fragment of mCD33A cDNA, and a
murine cDNA Reagents and Cell Transfection--
The mouse SHP-1
(C453S) cDNA has been previously described (32). MIS constructs
were made as fusion proteins composed of the extracellular and
transmembrane domains of vesicular stomatitis virus G protein (VSVG)
and the cytoplasmic domain of MIS (amino acids 377-466). Substitutions
of tyrosines by phenylalanines were introduced by PCR site-directed
mutagenesis. All mutations were confirmed by DNA sequencing. SHP-1 and
SHP-2 cDNAs were cloned into pBluescript (Stratagene) behind the T7
promoter; CD33 cDNAs were cloned into pGEM-3Z (Promega) behind the
T7 promoter. Transfection of HeLa cells was performed using LipofectAce
(Life Technologies) as previously described (33). FLAG epitope tags
were inserted at the amino terminus of MIS by PCR site overlap
extension and into an MscI site at the carboxyl terminus of
the MIS. MIS-Flag cDNA was cloned into the pLZRS retroviral
GFP-containing vector (kindly provided by Dr. D. Link, Washington
University). Ecotropic Phoenix cells were transfected with retroviral
constructs using the calcium phosphate method, and cell supernatants
were used to infect U937 cells. Green fluorescent protein-positive
cells were purified by cell sorting (Cytomation). MIS-Flag cell surface expression was verified by fluorescence-activated cell sorting analysis
following staining with biotinylated anti-Flag antibody followed by
phycoerythrin-conjugated streptavidin (Jackson Laboratories).
Immunoprecipitation and Immunoblot Analysis--
Retrovirally
transfected U937 cells overexpressing MIS and transfected HeLa cells
were washed with phosphate-buffered saline and, where indicated,
treated with 5 mM sodium pervanadate in phosphate-buffered
saline for 10 min at room temperature. Cells were lysed in 1 ml of
lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 7.5), 150 mM NaCl, 5 mM NaEDTA, 10 mM sodium
fluoride, 10 mM sodium molybdate, 1 mM sodium
vanadate, 5 mM iodoacetamide, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM phenylarsine oxide) on ice for 10 min. The lysates were
precleared by centrifugation at 12,000 rpm for 10 min at 4 °C. An
aliquot was removed as the crude lysate sample, and the reminder of the
lysate was subject to immunoprecipitation with the appropriate
antibody. Immunoprecipitates and crude lysates were resolved by
SDS-polyacrylamide gel electrophoresis under reducing conditions, and
immunoblot analysis was performed. Proteins were visualized using
enhanced chemiluminescence (Amersham Pharmacia Biotech) according to
manufacturer recommendations.
[Ca2+]i Measurement--
To ensure
high expression of Fc Molecular Cloning of MIS--
Considering the sequence similarity
between the extracellular domains of mouse and human CD33 and assuming
at least partial similarity between the transmembrane regions of human
CD33 and a hypothetical mouse inhibitory receptor, we performed a
search within a mouse expressed sequence tag data base using the human CD33 transmembrane amino acid sequence in the query. The search resulted in the identification of one murine cDNA clone with 39% sequence identity to the transmembrane region of human CD33 (Fig. 1A). Moreover, there were two
ITIMs in the sequence of the mouse clone similar to those of human
CD33. The cDNA encoded for the transmembrane and cytoplasmic
regions of the protein and also contained a portion of the
3'-untranslated region. RT-PCR was used to obtain a full-length
cDNA from murine bone marrow cells. The design of the forward
primer (mCD33.1; Table I) was based upon
highly homologous stretches of cDNA within the first Ig-like domain
in mouse and human CD33. The reverse primer (M1; Table I) was based
upon the sequence from the expressed sequence tag clone. A 1.4-kb PCR
product was obtained and sequenced. To retrieve the 5'-untranslated
region and leader peptide, a 5'-RACE strategy was employed using the M1
primer for the first strand cDNA synthesis followed by sequential
two rounds of PCR amplification with a universal abridged primer and a
set of nested primers, M2 and M3 (Table I). To obtain the 3'-end,
3'-RACE was performed using oligo(dT) primer for the first strand
synthesis and M4 (Table I) and universal primer for the PCR
amplification. In an independent experiment, the full-length cDNA
was amplified from total RNA isolated from mouse bone marrow cells
using M5 and M6 primers deduced from the 5'-RACE and 3'-RACE sequences
(Table I).
Sequence analysis of the full-length cDNA revealed that the protein
had an extracellular domain of 331 residues, a hydrophobic transmembrane domain of 27 residues and a cytoplasmic tail of 93 amino
acid residues (Fig. 1B). The sequence begins with a putative hydrophobic signal peptide. The extracellular portion of the molecule contains three Ig-like domains consisting of an
NH2-terminal V-set domain followed by two C2-set domains.
In addition, there are 10 potential N-glycosylation sites
(Asn-X-Ser/Thr) in the extracellular region of the protein.
The cytoplasmic tail contains two potential ITIM sequences. Considering
(i) the myeloid cell-restricted pattern of expression of the newly
isolated protein (see below), (ii) the similarity of its extracellular
domain to the extracellular domains of siglec family members, and (iii)
the presence of ITIM-like tyrosine-based signaling motifs, this protein
was named MIS (a myeloid inhibitory
siglec).
Phylogenetic analysis of the siglecs revealed that MIS, siglec 8, and
siglec 7 cluster together, with MIS being most closely related to human
siglec 8. The similarity between MIS protein and other family members
ranges from 31% with mCD33 to 47% with human siglec 8. Amino acid
sequence alignment of MIS and three other siglecs, human siglecs 3 and
8 and murine CD33, is shown in Fig.
2A. Although human siglec 8 is
the closest relative of MIS and shares 57% identity with MIS in the
extracellular portion of the molecule, their cytoplasmic tails are
quite different. There are no ITIM sequences in the siglec 8 cytoplasmic region, whereas MIS possess two ITIM-like sequences. To
this end, the cytoplasmic tail of MIS is more similar to those of the
inhibitory human siglecs, siglecs 3, 5, 6, and 7 (33, 29, 29, and 33%
identity, respectively) (Fig. 2B). Thus, these data indicate
that MIS belongs to the siglec family and represents a potential
inhibitory receptor.
A Single MIS Gene--
To determine whether MIS belongs to a
family of closely related genes or represents a single MIS
gene, genomic DNA from a C57BL/6 mouse was digested with a panel of
restriction enzymes and subjected to Southern blot analysis. The probe
employed represented an exon corresponding to the first V-set
immunoglobulin-like domain of MIS. Only one band was observed for each
enzyme digestion (Fig. 3), suggesting the
presence of only one MIS gene in the mouse genome.
Tissue- and Cell-restricted Expression of MIS--
To determine
the expression pattern of MIS in adult mouse tissues, Northern blot
analysis was performed using the full-length MIS cDNA as a probe.
As shown in Fig. 4A, four
mRNA species of ~1.6, 1.8, 2.2, and 3.0 kb were detected in
different tissues. A transcript of 2.2 kb was expressed (in descending
order) in spleen, liver, heart, kidney, and lung (in the latter two
tissues this transcript could only be detected after prolonged
exposure). In addition to the 2.2-kb transcript, two smaller
transcripts were detected in these tissues. The 1.8-kb transcript was
expressed in liver and kidney, whereas 1.6-kb transcript was expressed
in the spleen and heart. When Northern blot analysis was performed using probes corresponding to the first and third MIS exons that code
for the first and third Ig-like domains, respectively, only a 2.2-kb
band was observed. Conversely, analysis with a probe corresponding to a
cytoplasmic exon revealed multiple bands (data not shown). These data,
together with the results of Southern blot analysis, indicate that
there are several MIS variants due to alternative splicing of a single
MIS gene. One transcript of 3.0 kb was observed in testis.
The MIS message could not be detected in murine brain and skeletal
muscle.
To determine the pattern of cell expression of MIS, a RT-PCR approach
was utilized. cDNA was generated from different mouse cell lines,
primary bone marrow cells, and peritoneal macrophages, and the
expression of MIS, mCD33, and MIS Exists as Various Isoforms--
Alternative mRNA splicing
is a common feature among Ig superfamily genes. Since Northern blot
analysis of MIS suggested the existence of multiple mRNAs, we next
examined the possibility that multiple isoforms of MIS did, indeed,
exist. Mouse total RNA from bone marrow cells was reverse transcribed
using the MIS-specific primer, M1, and then three different sets of
primers corresponding to either the first Ig-like domain (M7 and M8),
the first and the second Ig-like domains (M7 and M9), or all three
Ig-like domains of the molecule (M7 and M10) were used to PCR-amplify
cDNA fragments (Fig. 5A).
Amplification with the former two sets of primers each gave rise to a
single products of the expected size. However, the third set of primers
(M7 and M10) yielded two products, one of the predicted size (the
higher band on the gel) and a second, which was 0.28 kb smaller (Fig.
5B). Sequencing of the smaller PCR product revealed the
absence of the second Ig-like domain in the extracellular region.
Combined with the detection of multiple mRNAs by Northern blot
analysis, these data suggest that at least two isoforms of MIS exist
due to alternative splicing of mRNA within the second Ig-like
extracellular domain.
MIS Associates with Protein-tyrosine Phosphatases SHP-1 and
SHP-2--
Since there are two tyrosine-based signaling motifs in the
cytoplasmic tail of MIS, the ability of MIS to associate with the SHP-1
and SHP-2 protein-tyrosine phosphatases was examined. A Flag
epitope-tagged form of MIS was expressed in the U937 human monocytic
cell line (Fig. 6). The broad molecular
weight range for MIS, as detected by SDS-polyacrylamide gel
electrophoresis, was most likely due to glycosylation in the
extracellular region of the protein (there are 10 potential
N-glycosylation sites in the extracellular region of the
molecule). In resting cells, MIS was phosphorylated on tyrosine
residues, as detected by immunoblotting with an anti-phosphotyrosine
mAb (Fig. 6, lane 3). Treatment of the cells with
pervanadate, a tyrosine phosphatase inhibitor, resulted in a dramatic
increase in tyrosine phosphorylation of MIS (Fig. 6, compare
lanes 3 and 4). Since MIS contains two
ITIM sequences, it was found to co-immunoprecipitate with both SHP-1 and SHP-2. While SHP-1 and SHP-2 co-precipitated with MIS in resting cells (Fig. 6, lane 3), the degree of association
correlated with the extent of MIS tyrosine phosphorylation (Fig. 6,
compare lanes 3 and 4). Phosphorylated
forms of both phosphatases were found to co-precipitate with MIS in
pervanadate-stimulated cells, since (i) the lower band on
phosphotyrosine immunoblot (lane 4) co-migrates with SHP-1 as was judged by reprobing the phosphotyrosine blot with the
SHP-1 antibody and (ii) multiple bands detected by SHP-2 antibody in
MIS immunoprecipitate (lane 4) most likely
represent differently phosphorylated species of SHP-2 (detecting these
species by phosphotyrosine immunoblot analysis was not possible because of masking effect of tyrosine phosphorylated MIS band). Thus, the data
obtained indicate that MIS shows a basal level of tyrosine phosphorylation in unstimulated cells and is associated with protein tyrosine phosphatases SHP-1 and SHP-2. This association increases with
an increase in the tyrosine phosphorylation status of MIS.
The Proximal ITIM of MIS Is Essential for Binding to
Protein-tyrosine Phosphatases SHP-1 and SHP-2--
To examine which
ITIM-like sequence of MIS mediates phosphatase binding, fusion proteins
were made that contained the extracellular and transmembrane regions of
VSVG and either the wild type cytoplasmic tail of MIS (YY) or mutants
where tyrosyl residues were mutated to phenylalanines either
individually (FY, first tyrosine mutated; YF, second tyrosine mutated)
or in combination (FF). The chimeric proteins were overexpressed in
HeLa cells together with either a catalytically inactive SHP-1C453S
mutant or wild type SHP-1 or SHP-2. To ensure a high level of tyrosine
phosphorylation of cellular proteins, the cells were treated with
pervanadate prior to cell lysis. Equivalent levels of the SHP-1 and
SHP-2 expression was achieved in each individual cell culture (Fig.
7, A-C, bottom panels), and a comparable amount of each chimeric protein
was immunoprecipitated as judged by an immunoblot analysis with the anti-VSVG antibody. In HeLa cells co-transfected with catalytically inactive SHP-1, chimeric proteins bearing the wild type cytoplasmic tail of MIS (YY) and the YF mutant were found to be phosphorylated on
tyrosyl residues with the level of phosphorylation of the YF mutant
being reduced when compared with that of wild type (Fig. 7A,
compare lanes 5 and 3). In contrast,
both FY and FF mutants were not found to be tyrosine phosphorylated
(Fig. 7A, lanes 4 and 6).
This suggests that (i) there is a sequential order of tyrosine
phosphorylation of the ITIMs of MIS where tyrosine 431 needs to be
phosphorylated prior to phosphorylation of tyrosine 454 and (ii) both
tyrosines are likely to be phosphorylated in wild type MIS. Compatible
with the correlation between the tyrosine phosphorylation of MIS and
its association with SHP-1, the amount of SHP-1 that co-precipitated
with the YF mutant was reduced when compared with that of wild type
(Fig. 7A, compare lanes 5 and 3 of SHP-1 immunoblot). In addition, no binding of SHP-1 was
detected in the FY and FF mutants that were not tyrosine-phosphorylated (Fig. 7A, lanes 4 and 6).
In cells co-transfected with wild type SHP-1 or SHP-2, the level of
tyrosine phosphorylation of wild type MIS was similar to that in cells
co-transfected with SHP-1C453S mutant (Fig. 7, A-C, compare
lanes 1 and 3). In cells
co-transfected with wild type SHP-1, tyrosine phosphorylation of the YF
mutant was not detectable (Fig. 7B, lane
5), whereas in cells co-transfected with wild type SHP-2,
the YF mutant was found to be phosphorylated on tyrosine although its
tyrosine phosphorylation level was reduced to just a trace amount (Fig.
7C, lane 5). This suggests that the tyrosine of MIS membrane-proximal ITIM may serve as substrate for SHP-1
and SHP-2. In agreement with its phosphorylation status, no SHP-1 was
associated with YF mutant. In contrast, in cells co-transfected with
SHP-2, despite the low level of tyrosine phosphorylation of the YF
mutant, the amount of associated SHP-2 was only slightly reduced
compared with the wild type (Fig. 7C, compare
lanes 5 and 3), suggesting that SHP-2
associates with the MIS membrane-proximal ITIM more readily than does
SHP-1. No tyrosine phosphorylation or SHP-1 and SHP-2 binding was
detected for both FY and FF mutants. Thus, MIS associates with both
SHP-1 and SHP-2, and the proximal ITIM is absolutely required for
phosphatase binding.
MIS Exerts an Inhibitory Effect on Ca2+ Mobilization
following Fc Although nine human Siglecs have been characterized to date, there
are fewer murine family members (siglecs 1-4). With the exception of
mCD22, there are no other murine siglecs that possess ITIM sequences.
Here we describe a novel murine inhibitory siglec that contains an
extracellular region comprised of three Ig-like domains, a
transmembrane region and a cytoplasmic tail with two ITIM-like
sequences. Since this newly identified protein showed a pattern of
expression restricted to myeloid cells, had ITIM sequences (a
characteristic feature of an inhibitory receptor), and had an
extracellular region similar to that of some siglecs, we have named
this protein MIS (a myeloid inhibitory
siglec). MIS associates with protein-tyrosine phosphatases
SHP-1 and SHP-2 via its tyrosine-based signaling motifs and is able to
inhibit signaling initiated through the activating receptor on monocytes.
Although formally MIS has not been shown to bind sialic acid residues,
the inclusion of this protein in the siglec family is justified by the
structure of its extracellular region (characteristic amino-terminal
V-set domain followed by C-set domains) and the fact that the amino
acid residues involved in ligand binding in the other siglecs (24) are
preserved in the V-set domain of MIS.
The presence of ITIM sequences implies the inhibitory nature of a
molecule (35). There are two potential ITIMs in the cytoplasmic tail of
MIS protein. The proximal ITIM that is formed around tyrosine 431 (IHY431ATL) contains the SHP-1-binding ITIM consensus
sequence (I/V/LXYXXL/V) (35, 36) and is
similar to those present in some of the human siglecs (17-22) as well
as in other inhibitory receptors that associate with SHP-1, including
killer cell inhibitory receptor, immunoglobulin-like transcript-3, and
leukocyte-associated inhibitory receptor-1 (10, 37, 38). The distal
tyrosine-based sequence of MIS is formed around tyrosine 454, TEY454SEL. It contains a threonine residue in the
Human CD33 has been characterized as an inhibitory receptor on myeloid
cells based upon its ability to bind inhibitory phosphatases, to
inhibit signaling initiated from an activating receptor, and to repress
dendritic cell development (31, 30, 46). The other siglec family member
that binds SHP-1, p75/AIRM1, has been shown to inhibit the
proliferation of normal or leukemic myeloid cells (47). Similarly, an
inhibitory effect of MIS on Ca2+ mobilization may be due to
the recruitment of SHP-1 and SHP-2 to the activating complex.
The similar structure of ITIM-containing cytoplasmic tails of different
siglecs dictates the similarity in their inhibitory function. The
specificity of function of siglecs may be determined by their unique
extracellular regions and/or by their specific expression patterns. Our
data indicate that at least two MIS isoforms with either three or two
extracellular Ig-like domains exist. In the KIR family, the splicing of
one or more Ig-like domains may result in altered ligand recognition
(48). In the Siglec family, the amino-terminal V-set domain is
responsible for ligand binding (24); therefore, the loss of the second
Ig-like domain in a smaller MIS isoform most likely will not affect its
ligand binding ability. Sialylation of sialoadhesin and CD22 has been found to regulate their ligand binding activity (49, 50). In a
smaller MIS isoform, the splicing out of the second Ig-like domain that
contains the majority of N-glycosylation sites may result in
a lower overall oligosaccharide content and thus in decreased levels of
sialylation of the molecule. Therefore, the ligand binding of MIS
isoforms may be differently regulated. As far as the expression pattern
is concerned, the early onset of MIS expression in bone marrow cells
suggests the possibility that it may be involved in the regulation of
monocyte development.
RI receptor-induced calcium
mobilization. These data suggest that MIS can play an inhibitory role
through its ITIM sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI mAb
(Medarex, Inc., Annandale, NJ), rabbit anti-vesicular stomatitis virus
serum (Access Biomedical, San Diego, CA), cross-linking goat anti-mouse
IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA), and horseradish peroxidase-conjugated secondary antibodies (Cappel
Organon Teknika Corp., West Chester, PA). Rabbit anti-SHP-1 antiserum
was previously described (32).
-actin cDNA control probe at 42 °C as described above.
Following hybridization, the blot was washed at 65 °C in 0.2× SSC
and 0.1% (w/v) SDS.
RI receptors, parent U937 cells and those
overexpressing MIS were treated with 300 units/ml of the recombinant
human
-interferon (a gift from Dr. R. Schreiber, Washington
University, MO) for 72 h prior to the analysis. Cells were loaded
with 3 mM Fura-2AM (Molecular Probes, Inc. Eugene, OR) in
RPMI 1640 plus 10% FCS for 40 min in the dark at 37 °C. Cells
(6 × 106) were washed once and resuspended in RPMI
1640 plus 10% FCS. Primary antibody was added at a concentration of 8 µg/ml, and cells were incubated on ice for 40 min. Cells were washed
three times in phosphate-buffered saline and resuspended in 2 ml of calcium buffer (25 mM Hepes, pH 7.4, 125 mM
NaCl, 5 mM KCl, 1 mM
Na2HPO4, 1 mg/ml D-glucose, 1 mg/ml
bovine serum albumin, 1 mM CaCl2, 0.5 mM MgCl2). Changes in fluorescence, using
excitation wavelengths of 340 and 480 nm and the emission wavelength of
510 nm, were measured with a spectrofluorimeter (F-2000, Hitachi
Instruments, Danbury, CT) equipped with a thermostatic cuvette holder
maintained at 37 °C. Cells were warmed to 37 °C for 5 min prior
to analysis. Secondary cross-linking goat anti-mouse IgG antibody (10 µg/ml) was added to the cuvette at 30 s. Intracellular calcium
concentrations were calculated as described (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Molecular cloning of MIS. A,
alignment of the amino acid sequence of the transmembrane region of
human CD33 and the deduced amino acid sequence of the mouse expressed
sequence tag clone (GenBankTM accession number AA275188).
Identical amino acids in the transmembrane regions are
shaded. ITIM sequences are underlined.
B, deduced amino acid sequence of MIS. The leader peptide is
underlined with a dashed line. Potential
N-glycosylation sites are shaded. The
transmembrane region is underlined with a solid
line. ITIM-like sequences are boxed. The
arrows indicate the start of the Ig-like domains.
Oligonucleotide primers used for cloning and RT-PCR analysis of MIS
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Fig. 2.
MIS is a member of the siglec family.
A, alignment of the amino acid sequences of MIS
(GenBankTM accession number AF329269), human siglec 8 (AF195092), human siglec 3 (M23197), and murine CD33 A isoform
(AAB30843). Amino acids that are identical among all aligned siglecs
are shaded in black; in addition, amino acids that are
identical in MIS and human siglec 8 are shaded in
gray. The start of individual Ig-like domains, as well as
the transmembrane and cytoplasmic domains are indicated
above the sequence. Charged amino acid residues
in the transmembrane domain of mCD33 are indicated by
asterisks. ITIM-like sequences are underlined.
B, a schematic representation of members of siglec family
excluding siglecs 1-4 that are homologous in mice and humans.
Black circles, ITIMs; rectangles,
transmembrane regions.
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Fig. 3.
Southern blot analysis of MIS. Genomic
DNA from C57Bl/6 mouse analyzed using the probe corresponding to the
first Ig-like domain of MIS.
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Fig. 4.
Tissue and cell expression of MIS.
A, Northern blot analysis of various mouse tissue mRNAs
using a full-length MIS cDNA probe or mouse -actin probe. The
arrows indicate various MIS mRNA species. B,
RT-PCR analysis of the murine bone marrow cells (lane
1), peritoneal macrophages (lane 2), T
cell lines, EL-4 (lane 3), 3A9H (lane
4), B cell lines, A20 (lane 5),
WEHI-231 (lane 6), monocyte/macrophage cell lines
J774 (lane 7), WEHI-265.1 (lane
8), NK cells KY-1 (lane 9), KY-2
(lane 10), mastocytoma P815 (lane
11), and a mock reaction (lane 12).
cDNA was reverse transcribed using oligo(dT) primer and amplified
using gene-specific sets of primers, mCD33.2 and mCD33.3 for mCD33 and
M4 and M6 for MIS.
-actin was analyzed using gene-specific sets of primers (Fig. 4B). MIS was detected in
bone marrow cells, peritoneal macrophages, and monocyte/macrophage cell
lines (upper panel) but was not detected in
lymphocytes including T cells (lanes 3 and
4), B cells (lanes 5 and
6), or NK cells (lanes 10 and
11) or a mastocytoma, P815, cell line. The overall pattern
of MIS expression was similar to that of mCD33 (middle panel). The efficiency of cDNA synthesis was equivalent
in all of the samples as judged by the expression of
-actin (Fig.
4B, bottom panel). Hence, the
expression of MIS is restricted to cells of myeloid origin.
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Fig. 5.
MIS exists as two alternatively spliced
isoforms. A, a diagram of MIS protein. The
arrowheads indicate the localization of the sequences from
which the primers (M7-M10) were designed. TM, transmembrane
domain; black rectangles represent ITIMs.
B, RT-PCR analysis of murine bone marrow cells. First strand
cDNA was reverse transcribed from bone marrow RNA using
MIS-specific primer M1. Lane 1, 100-base pair DNA
ladder; lane 2, PCR amplification using M7 and M8
primers; lane 3, PCR amplification using M7 and
M9 primers; lane 4, PCR amplification using M7
and M10 primers; lane 5, control without the
template cDNA. The arrow indicates the band
corresponding to the smaller MIS isoform without the second Ig-like
domain.
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Fig. 6.
MIS associates with the SHP-1 and SHP-2
protein tyrosine phosphatases. U937 cells were retrovirally
infected with either empty vector (vect.) or with
Flag-tagged MIS (MIS-Flag). Cells were either left untreated or
stimulated with sodium pervanadate (PV). Following
treatment, cells were lysed in 1% Nonidet P-40 lysis buffer. Lysates
were subjected to immunoprecipitation with anti-FLAG antibody.
Immunoprecipitates and crude lysates were resolved by 7.5%
SDS-polyacrylamide gel electrophoresis and immunoblotted with the 4G10,
anti-Flag, and anti-SHP-2 mAbs and an antiserum against SHP-1.
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Fig. 7.
The proximal ITIM of MIS is essential for
binding to SHP-1 and SHP-2. The wild type cytoplasmic tail of MIS
(YY) and the mutants Y431F (FY), Y454F (YF), and YY431,454FF (FF) were
expressed as VSVG fusion proteins (G/MIS) in HeLa cells together with
catalytically inactive SHP-1 C453S (A), wild type SHP-1
(B), or wild type SHP-2 (C). Cells were treated
with pervanadate for 10 min and then lysed in 1% Nonidet P-40 lysis
buffer; cell lysates were immunoprecipitated with VSVG antibody.
Immunoprecipitates and crude lysates were resolved by 7.5%
SDS-polyacrylamide gel electrophoresis, and blots were probed with
antibodies to VSVG, SHP-1 (A and B), or SHP-2
(C) and phopshotyrosine.
RI Receptor Ligation--
Upon cross-linking of
Fc
RI, an activating receptor expressed on monocytes, a signaling
cascade is initiated that includes tyrosine phosphorylation and rapid
Ca2+ mobilization. To ascertain whether MIS acts as an
inhibitory receptor, the effect of its co-ligation with Fc
RI on
Ca2+ mobilization in monocytes was examined. Flag-MIS was
expressed in a human monocytic cell line, U937, by retroviral infection and its expression was detected by cell surface staining (data not
shown). Cells were preincubated with recombinant human
-interferon for 72 h to ensure a high level of Fc
RI
expression. After loading with the fluorescent dye, Fura-2AM, cells
were incubated with primary anti-Fc
RI and biotin-conjugated
anti-Flag mAbs either individually or in combination. Ca2+
mobilization was measured following receptor cross-linking with secondary anti-mouse IgG antibody. Co-ligation of MIS with Fc
RI resulted in a dramatic decrease in Ca2+ rise (Fig.
8A), indicating an inhibitory
effect of MIS. A similar pattern of Ca2+ mobilization was
observed when experiments were performed in the presence of 4 mM Ca2+ chelator, EGTA, to eliminate the impact
of extracellular Ca2+ (Fig. 8B). Cross-linking
of the Fc
RI on U937 cells expressing MIS (solid
line) or in control cells infected with empty vector (dashed line) resulted in similar increase in
Ca2+ mobilization (Fig. 8C), indicating that the
potential to flux calcium is not affected in MIS-expressing U937 cells.
Together, these data suggest that the inhibitory effect of MIS on
Ca2+ flux is due to blocking Ca2+ mobilization
from the intracellular stores.
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Fig. 8.
Calcium mobilization induced by
Fc RI receptor cross-linking is inhibited with
MIS co-ligation. U937 cells retrovirally infected with an empty
vector (dashed line) or MIS-Flag
(solid line) were preloaded with Fura-2AM and
incubated for 30 min either with antibody to Fc
RI in combination
with anti-Flag antibody (A and B) or with
anti-Fc
RI antibody alone (C).
[Ca2+]i was measured by fluorimetry. Secondary
cross-linking anti-mouse antibody was added at 30 s. In some
experiments, EGTA (4 mM) was added at time 0 (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-position and is similar to the SAP-docking site
(TIYXXV/I) found in 2B4 and SLAM, activating receptors
present on NK cells and T/B cells, respectively (39, 40). SAP was shown
to compete with protein-tyrosine phosphatase SHP-2 for binding to SLAM
and 2B4 receptors, thus regulating their function (41, 42). Similar
tyrosine-based signaling motifs are also present in a human inhibitory
receptor, siglec 3 (TEY342SEV) and in the SHP substrate 1, SIRP/SHPS1 (TEY453ASI), which were shown to bind both SHP-1
and SHP-2 phosphatases (31, 43). Our results demonstrate that MIS
associates with SHP-1 and SHP-2 protein-tyrosine phosphatases.
Moreover, this association is sustained in nonactivated cells most
likely due to a basal level of constitutive phosphorylation of MIS
ITIMs. Similar constitutive tyrosine phosphorylation of ITIM sequences and association with a SHP-2-containing tyrosine phosphatase was observed in the PIR-B (44) and in the SHPS-1-inhibitory receptor (43).
Constitutive tyrosine phosphorylation of the tyrosine-based signaling
motifs suggests an association of MIS with a tyrosine kinase. Indeed,
such an association has been shown in case with PIR-B and SHPS-1 with
Lyn and PYK2 kinases, respectively (44, 45). The nature of a kinase
that may associate with MIS requires further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Terry Woodford-Thomas, Dr. Andrew C. Chan, and Dr. Wayne M. Yokoyama for critical reading of the manuscript.
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Note Added in Proof |
---|
Since this article was submitted, a paper has been published (51) describing mSiglec-E (GenBank accession number AF317298) with an identical sequence to that of MIS.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grant GM56455 (to M. L. T.) and the Human Frontiers Science Program (to M. L. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of Dr. Matthew L. Thomas.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF329269.
To whom correspondence should be addressed: Washington
University School of Medicine, Department of Internal Medicine,
Rheumathology Division, P.O. Box 8045 S. Euclid Ave., St. Louis, MO
63110. Tel.: 314-362-9013; Fax: 314-454-0175; E-mail:
ulyanova@pathology.wustl.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M011650200
1 To whom correspondence should be addressed: ITIM, immunoreceptor tyrosine-based inhibitory motif; FCS, fetal calf serum; mAb, monoclonal antibody; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; RACE, rapid amplification of cDNA ends; kb, kilobase pair(s); VSVG, vesicular stomatitis virus G protein.
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REFERENCES |
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