Báo cáo Y học: A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33 docx

A new siglec family member, siglec-10, is expressed in cells of the
immune system and has signaling properties similar to CD33
Gena Whitney
1
, Shulin Wang
1
, Han Chang
2
, Ke-Yi Cheng
1
, Pin Lu
1
, Xia D. Zhou
1
, Wen-Pin Yang
2
,
Murray McKinnon
1
and Malinda Longphre
1
1
Inflammation and Pulmonary Drug Discovery Department, and
2
Applied Genomics Department, Bristol-Myers Squibb Pharmaceutical
Research Institute, Princeton, NJ, USA
The siglecs (sialic acid-binding Ig-like lectins) are a distinct
subset of the Ig superfamily with adhesion-molecule-like
structure. We describe here a novel member of the siglec
protein family that shares a similar structure including five
Ig-like domains, a transmembrane domain, and a cyto-

plasmic tail containing two ITIM-signaling motifs. Siglec-
10 was identified through database mining of an asthmatic
eosinophil EST library. Using the Stanford G3 radiation
hybrid panel we were able to localize the genomic sequence
of siglec-10 within the cluster of genes on chromosome
19q13.3-4 that encode other siglec family members. We
have demonstrated that siglec-10 is an immune system-
restricted membrane-bound protein that is highly expressed
in peripheral blood leukocytes as demonstrated by Northern,
RT-PCR and flow cytometry. Binding assays determined
that the extracellular domain of siglec-10 was capable of
binding to peripheral blood leukocytes. The cytoplasmic tail
of siglec-10 contains four tyrosines, two of which are
embedded in ITIM-signaling motifs (Y597 and Y667) and
are likely involved in intracellular signaling. The ability of
tyrosine kinases to phosphorylate the cytoplasmic tyrosines
was evaluated by kinase assay using wild-type siglec-10
cytoplasmic domain and Y!F mutants. The majority of
the phosphorylation could be attributed to Y597 and Y667.
Further experiments with cell extracts suggest that Src
homology region 2 domain-containing protein tyrosine
phosphatase (SHP)-1 interacts with Y667 and SHP-2
interacts with Y667 in addition to another tyrosine. This is
very similar to CD33, which also binds the phosphatases
SHP-1 and SHP-2, therefore siglec-10, as CD33, may be
characterized as an inhibitory receptor.
Keywords: sialoadhesin; CD33; inhibitory receptor; phos-
phatase; siglec.
A recently defined group of immunoglobulin superfamily
proteins expressed on a variety of cell types have been

described as having binding properties that may mediate cell
adhesion and cell signaling through recognition of sialyated
cell surface glycans [1,2]. This protein family was recently
termed siglec for sialic acid-binding Ig-like lectins and is
comprised of sialoadhesin (siglec-1) [3], CD22 (siglec-2)
[4], CD33 (siglec-3) [5], myelin-associated glycoprotein
(MAG, siglec-4a) [6], Schwann cell myelin protein (SMP,
siglec-4b) [4], OB-BP2 (siglec-5) [7], OB-BP1 (siglec-6)
[8], siglec-7 [9], siglec-8 [10], and siglec-9 [11,12].
Although expression of certain siglecs (e.g. CD33) has
long been observed and utilized for diagnosis of some
malignant disorders [13], the precise biological functions of
the siglec protein family are not well understood. However,
because of their structure and expression patterns, siglec
proteins are hypothesized to be involved in diverse
biological processes such as hematopoiesis, neuronal
development and immunity [2].
A trait shared by many of the siglec proteins is a
cytoplasmic tail containing ITIM signaling motifs capable
of recruiting both activating [e.g. Src homology region 2
domain-containing protein tyrosine phosphatase (SHP)-2]
and inhibitory (e.g. SHP-1) phosphatases [14]. Interaction of
extracellular domain of siglecs with their cognate binding
partners and the recruitment of SHP phosphatases may
modulate cell signaling. Aruffo et al. [15] demonstrated that
CD22 on B-cells downregulates T-cell activation via the
T-cell receptor through association with CD45RO. Like-
wise, Falco et al. [16] have shown an inhibitory role for
siglec-7 in NK-mediated cytotoxicity. These two studies are
the only evidence to date of a functional significance for

siglecs in immune modulation. However, these two
examples of siglec function may reflect a natural mechanism
that dampens an immune response to foreign entities
deemed not a threat or prevents an autoimmune response.
Recent genomic sequencing efforts have led to the
identification of a cluster of siglec genes on human
chromosome 19q13.3-4 including siglecs-3, -5, -6, -7, -8,
and -9. The novel siglec that is presented here is also a
member of this ‘leukocyte receptor cluster’ as termed by
Wende et al. [17]. Although these siglec genes are clustered
and are probably the result of evolutionary gene duplication,
they appear to maintain strict differences in expression,
suggesting that they may have a very important,
nonredundant role in hematopoietic cells.
Note: G. Whitney and S. Wang share first authorship.
Correspondence to G. Whitney, Bristol–Myers Squibb Pharmaceutical
Research Institute, Mail Stop K24-03 PO Box4000 Princeton,
NJ 08543–4000, USA.
(Received 9 April 2001, revised 5 July 2001, accepted
25 September 2001)
Abbreviations: SHP, Src homology region 2 domain-containing protein
tyrosine phosphatase; DMEM, Dulbecco’s modified Eagle’s medium;
HBSS, Hanks buffered salt solution; HRP, horse-radish peroxidase;
LOD, log of odds; 2,3
0
-PAA, 2,3
0
-sialyllactose; 2,6
0
-PAA, 2,6

0
sialyllactose; SCGF, stem cell growth factor; SIA, sialic acid; GST,
glutathione S-transferase; SMP, Schwann cell myelin protein.
Eur. J. Biochem. 268, 6083–6096 (2001) q FEBS 2001
Described here is a novel member of the siglec protein
family that shares a similar structure including five Ig-like
domains, a transmembrane domain, and a short cytoplasmic
domain containing two ITIM-signaling motifs. Our
objectives were to first, determine the cell-specific
expression pattern of this novel siglec. Second, to assign a
chromosomal location to the siglec-10 gene and third, to
look for cell-type specific binding of the protein that
might provide clues as to its in vivo function. Lastly,
to characterize signaling properties of the cytoplasmic
domain that might provide some insight into the function of
siglec-10 in immune responses.
EXPERIMENTAL PROCEDURES
Database searching
Siglec-10 nucleotide sequences and electronic Northern
expression data were obtained by searching a proprietary EST
database (Incyte, Palo Alto, CA, USA) for gene sequences that
exhibit elevated expression in diseased immune tissues. A
total of 995 libraries containing a total of 4 079 076 clones
were examined. Siglec-10 mRNA was upregulated in
eosinophils from asthmatic patients. The Incyte clones
(526604, 527595, 652995, 1709963, 3421048; in pSPORT
vector, Gibco/BRL, Grand Island, NY, USA) for several
splice variants were obtained from Incyte and sequenced.
Sequencing and alignment
Individual clone colonies were cultured and DNA was

isolated using a Qiagen BioRobot 9600 (Hilden, Germany).
The purified DNA was then cycle sequenced using dye
terminator chemistries and subsequently separated and
detected by electrophoresis through acrylamide gels run on
ABI 377 sequencers (PerkinElmer, Foster City, CA).
PHRED
[18,19] was used as the base caller and the PHRAP algorithm
[18,19] was used for assembly of separate sequences into
contiguous pieces. Assemblies were edited using
CONSED
[20] to manually inspect quality and to design primers
for closing sequence gaps and achieve contiguity. The
nucleotide sequences of the siglec-10 cDNA clones were
analyzed in all three ORFs on both strands to determine the
predicted amino-acid sequence of the encoded protein. The
nucleotide sequence analysis was performed with
SEQWEB
version 1.1 (Genetics Computer Group, Wisconsin,
Madison, WI, USA) using the
TRANSLATE tool to predict
the amino-acid sequences,
STRUCTURE ANALYSIS tool for
predicting the motifs, and the
PILEUP tool for sequence
comparison in
GCG (Unix version 9.1, 1997). A comparison
of each of the clones suggested that these cDNA clones
included sequences that encoded proteins having sequence
homology with human CD33 (siglec-3). These nucleotide
sequences were designated siglec-10 and the protein

sequences were designated siglec-10.
Northern blot hybridization
Northern blots containing < 1–2 mg of poly(A)1 RNA per
lane of human tissues were obtained from Clontech. The
RNA had been run on a denaturing formaldehyde 1%
agarose gel, transferred to a nylon membrane by Northern
blotting and fixed by UV irradiation. RNA size markers were
also run on these blots as size indicators. Oligonucleotide
probes were constructed by PCR using full-length siglec-10
as a reference sequence (Table 1.). The L3 probe includes
nucleotide sequences common among the splice variants
from position 713–1445. The S1 probe includes splice
variant sequences common among siglec-10c and siglec-
10d from nucleotide position 545–710. The S2 probe
includes splice variant sequences common among siglec-
10b and siglec-10c from nucleotide position 1330 –1567.
All three probes were amplified from the siglec-10c
sequence (e.g. 652995). Additionally, a b-actin probe was
used as a control (Clontech). Probes were individually
labeled with [
32
P]dCTP by random priming, purified on a
Chromospin 100 column (Clontech) and checked for
labeling effeciency by scintillation counting. The membranes
were prehybridized in ExpressHyb solution (Clontech) at
68 8C for 30 min with continuous shaking. The denatured
radioactive probe (2 million c.p.m. per ml) was then added
with fresh ExpressHyb solution and the membrane was
incubated with continuous shaking for 4 h at 68 8C. The blot
was then washed under stringent conditions using

2 Â NaCl/Cit containing 0.05% SDS followed by a wash
with 0.1 Â NaCl/Cit containing 0.1% SDS at 50 8C. An
image was acquired using a PhosphorImager 445 SI
(Molecular Dynamics, Sunnyvale, CA, USA).
Chromosomal localization
The human chromosomal map location of siglec-10 was
determined using the Stanford G3 Radiation Hybrid Panel
(Stanford University). A primer pair was chosen that would
allow amplification of a portion of the transmembrane
domain. The PCR conditions were: 95 8C for 5 min,
followed by 30 cycles of 95 8C/56 8C/72 8C, for 30 s each
Table 1. PCR primers used to make probes for Northern blots.
Primer
pair
Corresponding
nucleotides Sequence
Resulting probe
length (bp)
L3 713–733 5
0
-TGCTCAGCTTCACGCCCAGAC-3
0
732
1436–1445 3
0
-TGCACGGAGAGGCTGAGAGA-5
0
S1 545–563 5
0
-CTCAGAAGCCTGATGTCTA-3

0
166
693–710 3
0
-GAGAAGTGGGAGGTCGTT-5
0
S2 1458–1476 5
0
2CTGCTGGGCCCCTCCTGC-3
0
237
1676–1695 3
0
-GACGTTCCAGGCCTCACAG-5
0
6084 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
followed by 72 8C for 10 min. The primer sequences for G3
PCR were: TMD (1720 –1738), 5
0
-tgcagctgccagataaga-3
0
and (2065–2083) 3
0
-GGCTTGAGTGGATGATTT-5
0
; the
product generated was 363 bp.
Blood collection and processing
Blood was collected from each informed volunteer in
EDTA-treated tubes. The blood was underlaid with Ficoll

and centrifuged for 25 min at 550 g. The interface
containing a mixed white cell population was removed to
a new tube and washed twice in the wash buffer (RPMI
containing EDTA plus 10 mg
:
mL
21
polymycin B), and
centrifuged for 8 min at 550 g between washes. Further
purification was required for some experiments where
specific white cell populations were needed. Elutriation by
standard methods was carried out to obtain purified
monocytes and lymphocytes. Granulocytes (neutrophils
and eosinophils) were obtained from the red cell fraction by
adding one-third volume elutriation buffer and one-third
volume 4.5% dextran sulfate then allowing the red blood
cells to settle for 30 min. The supernatant was harvested
and centrifuged for 8 min at 500 g. The cells were then
resuspended in 0.2 Â NaCl/P
i
to induce red blood cell lysis
for 1 min and then 1.8 Â NaCl/P
i
is added. If eosinophils
were required, the cells were then centrifuged for 8 min at
500 g and resuspended in buffer containing CD16 micro-
beads (Miltenyi Biotech, Auburn, CA, USA). After a 30-min
incubation on ice, the cells were passed through a magnetic
bead column. Eosinophils passed through the column were
collected, and neutrophils retained on the column were

eluted with NaCl/P
i
. B-cells were purified from elutriated
monocytes by retention on a nylon column (Wako Chemical
Co., Japan).
Cell lines
MB, PM, and TJ are EBV-transformed B-cells that were
generated within Bristol–Myers Squibb by standard
transformation methods. B-cell lymphoblastomas Ramos,
Raji, Daudi and HSB-2, a T-cell lymphoblastoma Jurkat, a
erythroblastic leukemia cell line HEL and monocytic cell
lines U973 and HL60 were all purchased from the American
Type Culture Collection (Rockville, MD, USA).
Full-length protein expression
Incyte clone 652995 in pSPORT vector containing the
complete 3
0
end of the siglec-10 gene was digested with
Eco RI and Bbr PI and the larger plasmid fragment
(< 6.4Kb) was gel-purified. A second Incyte clone,
3421048, containing the complete 5
0
end, was restriction
digested with Eco RI and Bbr PI, then gel purified
(< 820 bp). The insert was then ligated into the pSPORT
vector and the resulting full-length siglec-10 clone was
designated 995-2 and the sequence was verified against the
other siglec-10 clones. This 995-2 clone was then restricted
with Eco RI and Not I and ligated into a similarly digested
pcDNA3 vector for full-length expression.

Ig and glutathione
S
-tranferase (GST) fusion proteins
Two siglec–human Ig fusion expression plasmids were
constructed by ligating the extracellular sequence of clone
995-2 into a Bristol–Myers Squibb proprietary expression
vector pd19 based on puc19 which contains a portion of the
human R g-chain downstream of a multiple cloning site.
The extracellular domain of siglec-10 was amplified using
primers containing linker sequences with restriction sites for
HindIII, Bgl II and Nco I. The amplified fragment was
cloned into pd19 by digesting the fragment with HindIII and
Bgl II and the plasmid with HindIII and Bam HI. The
fragment was ligated into the plasmid and the integrity of
the insertion was validated by digesting the plasmid
construct with either HindIII/Nco I to check the extracellular
domain of siglec or with HindIII/Xba I to check the entire
fusion construct. The siglec-10–hIg fusion protein was
expressed in COS7 cells by DEAE-dextran transient
transfection. COS7 cells were transfected with 1 mg
:
mL
21
DNA in Dulbecco’s modified Eagle’s medium (DMEM)
containing 1% DEAE-dextran (Sigma), 0.125% chloroquine
(Sigma) and 10% NuSerum (Beckton–Dickinson, Franklin
Lakes, NJ, USA) for 4 h followed by a 2-min treatment with
10% dimethylsulfoxide NaCl/P
i
. After 4 –7 days, the COS7

supernatant was passed over a Protein A trisacryl column
(Pierce, Rockford, IL, USA) at a rate of 1 mL
:
min
21
. The
fusion protein was then eluted with 0.1
M acetic acid
(pH 4.5) and immediately neutralized with 2
M Tris/base to
a final pH of 8.0. Siglec-10–hIg was then dialysed against
NaCl/P
i
.
To construct the GST fusion of the siglec-10 cytoplasmic
domain (GST–siglec-10cyto-wt) the cytoplasmic domain
(KRRTQTE…VFQ) of siglec-10 was amplified from a
phytohaemagglutinin-activated Jurkat cDNA library by
PCR. The fragment was subcloned into pGEX4T-3
(Pharmacia Biotech) via Eco RI/Xho I. The sequence of the
PCR clone matched 100% to the original Incyte pSport1
sequence. In addition, Y !F mutants were generated at
positions 597, 641, 667, and 691.
To construct the GST fusions of the tandem SH2 domains
of each SHP-1 and SHP-2, the sequences corresponding to
amino acids 2–232 of each SHP-1 and SHP-2 were
amplified from a PHA-activated Jurkat cDNA library by
PCR. The fragments were each subcloned into pGEX4T-3
(Pharmacia Biotech) via Eco RI/Xho I.
All GST-fusion proteins were expressed in Escherchia

coli and the protein purified according to Pharmacia
protocol based on the method of Smith & Johnson [21].
Antibody generation
Balb/c mice were immunized with an intraperitoneal
injection of siglec-10–hIg fusion protein in Ribi Adjuvant
(Corixa, Hamilton, MT, USA) once every 3 weeks. Three
days prior to sacrifice, the mice were boosted with an IV
injection of siglec-10 –hIg. Splenocytes were aseptically
harvested, washed, and mixed 10 : 1 with mouse myeloma
cells (P3X, ATCC, Rockville, MD, USA) in the presence of
50% poly(ethylene glycol) 1500 (Roche) to induce fusion.
Those clones producing antibodies selective for siglec-10–
hIg but not to other hIg, as screened by ELISA, were
expanded in roller flasks. The purified monoclonal
antibodies were further screened by Western blot of
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6085
6086 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
siglec-10–hIg and other similar fusion proteins. A third
screen for antibody specificity was performed using FACS
analysis of COS7 cells that were transfected with full-length
siglec-10 expression construct.
Western blotting for siglec-10
Ten micrograms of cell lysates (Triton X-100 soluble
protein fraction) from several cell lines and peripheral blood
cell preparations were mixed with sample buffer and
resolved by SDS/PAGE (4– 20% gradient gel) and trans-
ferred to nitrocellulose by standard Western blotting
techniques. The blots were then stained with monoclonal
anti-(siglec-1) Ig followed by a secondary goat HRP-
conjugated anti-(mouse IgG) Ig (Biosource Int., Camarillo

CA, USA). Stained proteins were imaged by adding a
chemiluminescent detection reagent (Renaissance, NEN Bio
Products, Boston, MA) using a PhosphorImager 445 SI
(Molecular Dynamics, Sunnyvale, CA, USA).
FACS analysis
Mixed white blood cell populations and hematopoietic cell
lines were obtained to determine the binding specificity for
siglec-10. Cells were suspended in binding buffer (DMEM
containing 1% w/v BSA) with siglec-10–hIg fusion protein,
mALCAM–hIg fusion protein (hIg Rg control), or CD5 hIg
fusion protein (hIg Eg control) at a concentration of 5 mg
protein per 1 Â 10
6
cells. Anti-(rabbit IgG) Ig (Sigma) was
also added at 100 mg per million cells to prevent nonspecific
binding of the Ig tail on the fusion proteins to Fc receptors.
The mixture was incubated on ice for 1 h followed by
washing twice with binding buffer. Cells were centrifuged at
500 g for 5 min between each wash. Fluorescein-conjugated
anti-hIg Ig (Jackson Immunoresearch, West Grove, PA,
USA) and/or phycoerythrin-conjugated anti-CD20 Ig, anti-
CD14 Ig, and anti-CD4 Ig (Beckton– Dickinson, San
Jose,CA, USA) were added on ice for 30 min. The cells
were analyzed on a Becton–Dickinson FACSort using
CELL
QUEST
software. Cells were live-gated and red/green color
was compensated.
Polyacrylamide glycoconjugate binding assays
COS7 cells were transiently transfected (see above for

transfection protocol) with full-length siglec-10 (995– 2 in
pcDNA3 vector) or sham transfected (vector alone), and
were plated in 96-well plates within 24 h of transfection and
allowed to attach for 18– 22 h. Half of the plated cells were
treated with 0.01 U sialidase (Calbiochem, La Jolla, CA) for
1 h at 37 8C because the treatment has been shown to
remove cell surface sialic acids that possibly mask the
binding site for other siglec family members [12]. The cells
were then washed with DMEM containing 1% BSA and
incubated with saturating concentrations (20 mg
:
mL
21
)ofa
polyacrylamide polymer containing biotin and carbohydrate
(lactose, 3
0
-sialyllactose or 6
0
-sialyllactose, GlycoTech
Corp., Rockville, MD, USA). In a parallel cell-free
experiment, Immulon plates were coated with purified
siglec-10–hIg fusion protein (200 ng
:
well
21
) and incubated
with 20 mg
:
mL

21
of the polyacrylamide polymers. After
1 h, plates were washed and treated with streptavidin–
horse-radish peroxidase (HRP) (Vector Laboratories,
Burlingame, CA, USA) in DMEM for 30 min. After a
final wash, 3,3
0
,5,5
0
-tetramethylbenzidine peroxidase sub-
strate (KPL, Gaithersburg, MD, USA) was added and the
plates were developed at room temperature. The reaction
was stopped with 0.1 N HCl and absorbance at 450 nm was
determined on a spectrophotometer.
Cell binding assays
To determine whether distinct blood cell populations or
various cell lines would bind to siglec-10 when immobilized
on a solid support, 96-well Immulon plates (PGC,
Gaithersburg, MD, USA) were coated with siglec-10–hIg
fusion protein (200 ng
:
well
21
) overnight. The plate was
then blocked for 1 h with DMEM containing 1% BSA.
Blood cells and cell lines were labeled with calcein-AM
(5 mL per 10
8
cells, Molecular Probes, Eugene, OR, USA)
for 30 min at 37 8C. Cells were then washed twice in Hanks

buffered salt solution (HBSS) and added to the blocked plate
(4 Â 10
5
per well in 200 mL) at 37 8C for 30 min. The plate
was then gently washed with HBSS and 100 mL HBSS was
added to each well. Fluorescence was read on a CytoFluor
4000 (PerSeptive Biosystems, Framingham, MA, USA) at
485 excitation/530 emission.
COS7 cells were also transiently transfected with a
pcDNA3 plasmid containing a full-length siglec-10 by
DEAE-dextran method. Sham-transfected COS7 were
treated the same but without pDNA in the transfection
protocol. Twenty-four hours after transfection, COS7 cells
were lifted from the plates with 0.02% EDTA and replated in
six-well plates containing DMEM with 10% fetal bovine
serum at a density of 2 Â 10
5
per well. Binding assays were
planned for between 48 and 60 h post-transfection. Blood
cells and cell lines were labeled with calcein-AM (5 mL per
10
8
cells) for 30 min at 37 8C. Red blood cells, mixed white
blood cells, Ramos and Daudi (B-cell lines), HL60 and
K562 (monocytic cell lines, and Jurkats (T-cell line) were
suspended in DMEM containing 0.25% BSA. Some
cells were also pretreated with sialidase (Calbiochem,
0.1 U
:
mL

21
for 30 min at 37 8C followed by three washes
with DMEM plus 0.25% BSA). One milliliter of blood cell
or cell line suspension was added to each well and incubated
at 37 8C for 30 min with gentle rocking. The plates were
then washed gently three times with NaCl/P
i
plus 0.25%
BSA and fixed with 0.25% glutaraldehyde. For better
contrast, the cells were stained lightly with Wrights and
Geimsa stains (Diff Quik, Dade, Puerto Rico). To quantify
binding, the percentage of COS7 cells binding two or more
of the added cell type was determined from 10 fields in each
Fig. 1. Nucleic acid and amino-acid sequence for siglec-10. The full-length sequence was derived from five overlapping Incyte clones. Predicted
domain structure illustrates the splice variants and full-length siglec-10. The 697 amino-acid sequence for siglec-10 was predicted based on the
longest open reading frame. The two spliced regions are indicated in black, the cryptic splice acceptor site is underlined, the transmembrane domain is
in bold and amino acids in the ITIM motifs in the cytoplasmic domain are boxed. The intron/exon boundaries as determined from accession no.
AC008750 are indicated with an arrow and the domain numbers above reflect the predicted secondary structure illustrated in C.
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6087
treatment and observed at 100 Â magnification (at least
100 cells from each treatment were scored). Results were
expressed as the percentage of COS7 cell binding and
all binding to transfected cells was compared to sham-
transfected controls. In addition to the binding assay with
adherent transfected COS7 cells, a binding assay with
adherent endothelial cells was also performed. HUVEC
cells (ATCC) were plated and grown to confluence in
96-well plates. COS7 cells were transfected with full-length
siglec-10 by the DEAE/dextran method and allowed to
recover for 36 h. Transfected and sham-transfected COS7

cells were then lifted from the plates with 0.02% EDTA
labeled with calcein AM (as detailed above) and washed. An
aliquot of COS7 and an aliquot of HUVEC cells were
pretreated with sialidase (0.01 U
:
mL
21
for 30 min at
37 8C). COS7 cells, suspended in binding buffer, were then
added to HUVEC cells and incubated for 2 h at 37 8C. After
several washes with HBSS, fluorescence in each well was
measured.
Kinase assays
To determine possible signaling interactions of the siglec-10
cytoplasmic domain with major signaling molecules in the
cell, kinase assays were performed using representatives of
the four major types of tyrosine kinases (Lck, Jak3, Emt and
ZAP-70) known to associate with receptors similar in nature
to siglec-10. All four kinases were expressed as His-tagged
fusions and purified according to the Pharmingen Baculo-
virus Expression Vector System (Pharmingen, San Diego,
CA, USA). GST–cyto-wt (wild-type), GST–LAT-control
(an adapter protein with 10 tyrosines available for
phosphorylation), GST–cyto-Y597F (Y!F mutation at
the 597 position), GST– cyto-Y641F (Y!F mutation at the
641 position), GST– cyto-Y667F (Y !F mutation at the 667
position), GST– cyto-Y691F (Y!F mutation at the 691
position), and GST alone proteins were coated on 96-well
ELISA plates at 4 mg
:

mL
21
in sodium carbonate pH 9 for
16 h at room temperature. Plates were washed and then
treated with Blocking Reagent (Hitachi Genetics Systems,
Alameda, CA, USA). Kinase reactions were carried out in a
50-mL volume for 1 h at room temperature. The kinase
buffer contained 25 m
M Hepes pH 7.0, 6.25 mM MnCl
2
,
6.25 m
M MgCl
2
, 0.5 mM sodium vanadate, 7.5 mM ATP
and twofold dilutions of the tyrosine kinases starting at
0.25 mg
:
mL
21
. Plates were washed and phospho/tyrosine
content detected with antiphospho-Tyr (PY99) HRP (Santa
Cruz, Santa Cruz, CA.) at 1 : 1000 and peroxidase substrate
(KPL). Absorbance at 650/450 was detected.
Western blotting for SHP proteins
To determine if the cytoplasmic domain of siglec-10 binds
SHP-1 and SHP-2 in cell lysates, 10 mg of GST fusion
protein 1 tyrosine phosphorylation were incubated with
300 mL of cell lysate (Triton X-100-soluble fraction
5 Â 10

7
unstimulated cells) at 4 8C overnight. The GST
fusion protein complexes were captured with 50 mLof
glutathione–Sepharose beads (Amersham–Pharmacia Bio-
tech) for 1 h at 4 8C. The beads were washed three times
with ice-cold lysis buffer, and bound proteins were eluted in
SDS reducing sample buffer and resolved by SDS/PAGE.
The separated proteins were transferred to nitrocellulose by
standard Western blotting techniques. The blots were then
stained with either mouse anti-(SHP-1) Ig or mouse
anti-(SHP-2) (Transduction Laboratories, Laboratories,
Lexington, KY, USA) followed by a secondary goat HRP-
conjugated anti-(mouse IgG) Ig (Biosource Int., Camarillo,
CA, USA). Stained proteins were imaged by adding a
chemiluminescent detection reagent (Renaissance, NEN Bio
Products, Boston, MA, USA) and exposing to film (Kodak).
ITIM peptide binding to SHP proteins
A biotinylated siglec-10 phosphopeptide (660–678)
ESQEELHpYATLNFPGRVPR (ITIM667) was produced
by W. M. Keck Biotechnology Resource Center (New
Haven, CT, USA). Phosphopeptide (4 mg
:
mL
21
) in block-
ing reagent (Hitachi Genetics Systems) was bound to a
strepavidin-coated ELISA plate (Pierce). Plates were
Fig. 2. PILEUP analysis of siglec-5, -8 and -10. Siglec-10 is 69%
homologous at the amino-acid level to a closely related protein siglec-5
(accession no. NM003830). The homology is exceptionally high (86%)

in the fifth Ig-like domain. Siglec-8 (accession no. NM014442) was also
compared because it is also expressed on eosinophils but is not very
homologous to siglec-10 except for the first Ig-like (SIA binding)
domain. Homologous regions are indicated by dark bolding. Conserved
cysteines are marked with an asterisk.
6088 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
washed and then twofold dilutions of the GST fusion
proteins, GST alone, GST– SHP-1SH2SH2 or GST–SHP-
2SH2SH2 or GST–ZAP-70SH2SH2 were added and
incubated for 1 h at room temperature. Polyclonal anti-
GST Ig (prepared in-house by procedures similar to those
detailed for anti-siglec Ig production) was added at
1 : 1000, HRP-conjugated anti-(rabbit IgG) Ig (Biosource)
at 1 : 2000, and signal detected with peroxidase substrate
(KPL).
RESULTS
Sequence and chromosomal location
The full-length sequence of 3024 bp was derived by
aligning five clones and four splice variants, containing
overlapping sequence (Incyte clones: 526604, 527595,
652995, 1709963, 342148, Fig. 1A). The full-length cDNA
sequence and predicted amino-acid sequence of siglec-10
Table 2. Expression of siglec-10 on hematopoietic cell lines and primary leukocytes. Biotinylated monoclonal anti-siglec-10 was added to cells
followed by treatment with fluoresceine isothiocyanate (FITC)-conjugated streptavidin. The antibody was chosen based on immunoreactivity to
COS7 cells transfected with siglec-10 as determined by FACS. For peripheral blood mononuclear cell preparations (PBMC), a secondary
phosphatidylethanolamine-conjugated antibody was used to distinguish subpopulations. The percentage of total cells with increased fluorescence is
indicated. Data shown represents the mean of 2 –3 experiments.
Cell line Type FITC alone (%) Anti-(siglec-10) Ig (%)
Ramos B-cell (lymphoma) 0.5 89.9
THP-1 Monocyte (lymphoma) 1.7 39.1

Jurkat T-cell (lymphoma) 0.4 76.6
U973 Monocyte (leukemia) 2.0 33.6
HL60 Monocyte (leukemia) 0.6 93.3
K562 Monocyte (leukemia) 0.5 96.2
COS7 Naive 2.4 15.4
COS7 Siglec-10 Transfected 4.7 63.3
Blood population PE1 (%) Siglec-FITC1 (%) FITC1 and PE1 (%)
PBMC 19.9
CD201 8.3 2.4
CD141 12.1 12.0
CD4lo 1 11.7 12.0
CD4hi 1 63.0 0
CD31 65.0 0
CD281 47.5 2.0
Granulocytes 88.6
Fig. 3. Expression of siglec-10 mRNA. Northern
blots of various tissues were probed with labeled
oligos that corresponded to a common region (L3
probe ¼ Ig domains 3–5) or the two spliced
regions (S1 ¼ Ig domain 2 and S2 ¼ Ig domain 5)
of siglec-10. b-Actin was also probed as a control
for mRNA integrity and loading.
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6089
are shown in Fig. 1B. The predicted secondary structure for
siglec-10 is shown in Fig. 1C. This structure was based on
comparisons to other known siglec family members as well
as results from the
GCG SEQWEB PEPTIDE STRUCTURE
program that calculates Chou–Fasman and Garnier–
Osguthorpe –Robson predictions. The siglec-10 cDNA

encodes a 697 amino-acid type I transmembrane protein
composed of a signal peptide, five Ig-like domains (one
V-set domain followed by four C2-set domains), a
transmembrane domain and a cytoplasmic tail. Compared
to other siglec family members, siglec-10 appears to be most
homologous to siglec-5, particularly in the first and fifth
Ig-like domains as determined using the
GCG SEQWEB
PILEUP
program (Fig. 2). In contrast, siglec-8, which is
also expressed in eosinophils, does not appear to be very
homologous when compared by
PILEUP (Fig. 2).
To determine chromosomal location, siglec-10 primers
were used to screen all 83 hybrids of the Stanford G3 set.
The resulting pattern of positives and negatives was
submitted to the Stanford Human Genome Center Radiation
Hybrid Mapping Server, where it was subjected to a two-
point statistical analysis against 15632 reference markers.
This yielded a linkage to two markers, D19S425 and
D19S418, at a distance of 32 centiRay (cR) [log of odds
(LOD) score ¼ 6.47] and 29 cR (LOD score ¼ 6.28),
respectively, and corresponded to an approximate physical
distance of 960 and 870 kb, respectively, in this panel
(1 cR ¼ 30 kb). Reference to the Stanford Radiation
Hybrid Map of this region of chromosome 19 gives the
most likely order of D19S418/siglec-10/D19S425 with a
cytogenetic location of 19q13. The marker D19S418 is
positive with YAC 790A05 of the Whitehead genetic map of
chromosome 19 [17].

This chromosomal location was reinforced recently
when a genomic clone of chromosome 19 was submitted
to the GenBank database (accession no.AC008750, clone
CTD-2616J11) that contains the siglec-10 sequence. The
gene structure was deduced by comparing the siglec-10
cDNA sequence to the genomic sequence. The siglec-10
gene is contained within a 44 187 bp contig (in reverse
order from 89 063 to 81 495) and is composed of at least 11
exons spanning at least 7568 bases. This is similar to the two
other known siglec genes, siglec-1 (sialoadhesin) [12] and
siglec-9 [22] The genomic sequence was compared to the
sequences of the four siglec-10 splice variants and intron/
exon junctions verified that the clones represent splice
variants and not cloning artifacts.
Fig. 5. Binding properties of the extracellular domain of siglec-10.
The binding of polyacrylamide glycoconjugates to siglec-10–hIg
fusion protein that was immobilized on an Immulon plate (first panel) or
to COS7 cells transfected with full-length siglec-10 (second panel).
Results shown are a mean ^ SD of two experiments, n ¼ 4–6 per
treatment per experiment.Results from the solid support binding assay
are shown. A 96-well Immulon plate was coated with siglec-10-hIg
fusion protein (200 ng
:
well
21
) overnight. The plate was then blocked
for 1 h with DMEM containing 1% BSA. Blood cells and cell lines
were labeled with calcein-AM (5 mL per 108 cells) for 30 min at 41 8C.
Cells were then washed twice in HBSS and added to the blocked plate
(4 Â 105 per well in 200 mL) at 37 8C for 30 min. The plate was then

gently washed with HBSS and 100 mL HBSS was added to each well.
Fluorescence was read at 485 excitation/530 emission. Sialidase
pretreatment of the cells (0.1 U
:
mL
21
for 30 min at 37 8C) did not
significantly affect binding of any of the adherent cell types. Results
shown are means ^ SD of two experiments, n ¼ 3 per treatment per
experiment.Results for COS7 binding experiments are shown. COS7
cells were transiently transfected with or without (sham) a pcDNA3
plasmid containing a full-length siglec-10. Twenty-four hours after
transfection, COS7 cells were lifted from the plates with 0.02% EDTA
and re-plated in 6 well plates containing DMEM with 10% fetal bovine
serum at a density of 2 Â 105 per well. Blood cells and cell lines were
labeled with calcein-AM (5 mL per 108 cells) for 30 min at 37 8C and
some cells were also pretreated with sialidase (0.1 U
:
mL
21
for 30 min
at 37 8C). One milliliter of blood-cell or cell-line suspension was added
to each well and incubated at 37 8C for 30 min. The plates were then
washed and fixed with 0.25% glutaraldehyde. For better contrast, the
cells were stained lightly with Wrights and Geimsa stains. Results were
expressed as the percentage of COS7 cell binding and all binding to
transfected cells was compared to sham transfected controls. Results
shown are means ^ SD of two experiments, n ¼ 2 per treatment per
experiment. *, Statistically different from sham transfected controls,
P , 0.05.

Fig. 4. Western blot of cell lysates probed with anti-(siglec-10) Ig.
Proteins (10 mg per lane) were separated by SDS/PAGE. The
electrophoresed proteins were transferred to nitrocellulose and stained
with anti-(siglec-10) Ig followed by HRP.
6090 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
Expression of siglec-10
Northern blot. Northern blot analysis did not show such a
distinct difference in distribution and suggested that perhaps
the full-length, unspliced transcript was most prominent in
those tissues with high expression (Fig. 3). Blots were
probed with three labeled oligonucleotides spanning a
common region (L3) and the two spliced regions (S1 and
S2). The tissue distribution of the three probes was similar
(Fig. 3) and did not suggest a large difference in splicing
between tissues. In addition, the S2 probe hybridized to two
unknown smaller bands that may be explained by homology
of exon 8 with other known siglec family members (e.g.
siglec-5 which is 87% homologous in that region at the
nucleotide level). Because siglec-10 splice variants would
be similar in size, it may be hard to discern differences in
expression by Northern blot. The S1 and S2 probe hybrid-
ization indicated that the two predicted splice variants were
not the major form of siglec-10 in those tissues studied.
Siglec protein expression. FACS analysis of peripheral blood
cell populations and cell lines was performed to determine
surface protein expression (Table 2). Anti-(siglec-10) Ig
bound to isolated granulocytes (eosinophils and neutrophils)
and CD141 monocytes with large shifts in fluorescence
intensity. The antibody did not bind to other blood cells
including CD281 cells and CD31 cells (data not shown).

The antibody was also used to probe Western blots of
extracts from several cell lines and purified peripheral blood
cells. The anti-(siglec-10) Ig recognized a single band
approximately at the expected M
r
of between 90 and
120 kDa. There were no other visible bands, implying
that the antibody is specific for siglec-10 (only the less
homologous siglec-4 has the same predicted M
r
as siglec-10,
while siglec-5 has a predicted M
r
of 60 kDa). Granulocytes
and several blood cell lines appear to express siglec-10
(Fig. 4).
Binding studies of the extracellular domain
PAA-glycoconjugates. The binding preference of siglec-10
for 2,3
0
-sialyllactose (2,3
0
-PAA) and 2,6
0
sialyllactose
(2,6
0
-PAA) was determined by immobilizing siglec-10–
hIg on an Immulon plate and determining the binding of the
polyacrylamide biotinylated glycoconjugates (Fig. 5A). In

the first panel, the 2,6
0
-PAA conjugate bound significantly
greater than either the unsialylated lactose (negative control)
or 2,3
0
-PAA. In the second panel, a cell-based experiment
was carried out to confirm this observation. Full-length
siglec-10 (995–2 in pcDNA3) was transfected into COS7
cells by DEAE-dextran method and PAA binding to
transfected cells was determined. There was significantly
greater binding of the 2,6
0
-PAA conjugate to transfected
COS7 cells following sialidase pretreatment. The need
for sialidase treatment suggested that cis-binding of the
siglec-10 could inhibit interaction with the added PAA.
Table 3. FACS analysis of siglec-10–hIg binding. Mixed white blood cell populations and hemapoietic cell lines were incubated with siglec-10–hIg
fusion protein then stained with fluorescein-conjugated anti-hIg Ig and/or phycoerythrin-conjugated anti-CD20 Ig, anti-CD3 Ig, anti-CD14 Ig, and
anti-CD4 Ig. mALCAM–hIg fusion protein (hIg Rg control) and CD5–hIg fusion protein (hIg Eg control) were analyzed in parallel as controls. Anti-
(rabbit IgG) Ig was also added to prevent nonspecific binding of the Ig tail on the fusion proteins to Fc receptors. The percentage of cells staining
positive for fluoresceine isothiocyanate (FITC) compared to background with mALCAM, CD5 and siglec-10 hIg is shown. One color FACS was used
for cell lines and two color FACS was used for primary peripheral blood mononuclear cells (PBMC).
Cell line Type
FITC Staining (%)
mALCAM–hIg CD5–hIg Siglec-10–hIg
MB B-cell (EBV) 0 0 0
PM B-cell (EBV) 0 0 0
TJ B-cell (EBV) 0 0 0
Ramos B-cell (lymphoma) 0 4 57

HSB-2 B-cell (lymphoma) 0 0 40
Raji B-cell (lymphoma) 1 2 36
Daudi B-cell (lymphoma) 0 0 20
Jurkat T-cell (lymphoma) 38 2 0
HEL RBC (leukemia) 0 0 0
U973 Monocyte (leukemia) 0 4 0
HL60 Monocyte (leukemia) 0 0 0
Blood population
FITC staining(%)
PE1 (%) ALCAM–hIg CD5–hIg Siglec-10–hIg
PBMC
CD201 70 0 4
CD141 11 0 0 8
CD4lo1 12 0 0 11
CD4hi1 63 8 0 0
CD31 65 4 0 0
Granulocytes 0 0 0
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6091
Binding assays on a solid support. Results from the ELISA
plate binding assays are shown in Fig. 5B. T-cells, mixed
granulocytes, purified B-cells, purified monocytes, Ramos
and Daudi (B-cell lines) significantly adhered to the
immobilized siglec-10 fusion protein. Red blood cells,
Jurkats (T-cell line) and K652 (monocytic cell line) did
not adhere to the protein-coated plate. Sialidase pretreat-
ment of the cells (0.1 U
:
mL
21
for 30 min at 37 8C) did not

significantly affect binding of any of the adherent cell types.
Binding assays with siglec-expressing COS7 cells. Results
for COS7 binding experiments are shown in Fig. 5C. There
was a significant increase in binding of mixed white blood
cells and Ramos (B-cell line) to the transfected COS7 cells
when compared to the untransfected controls. This binding
was not significantly affected by sialidase pretreatment of
the blood cells. Red blood cells, Jurkats (T-cell line), HL60
and K562 (monocytic cell lines) did not appear to bind more
to siglec-10-expressing COS7 cells. HUVEC cells, either
untreated or treated with sialidase, did not adhere to COS7
cells that were transiently transfected with full-length
siglec-10 (data not shown).
FACS analysis. Results of the FACS analyses using the
siglec-10–hIg fusion protein are shown in Table 3. When
mixed blood cell populations were incubated with siglec-10
fusion protein, only a small population of lymphocyte-sized
cells and monocyte-sized cells stained positive for siglec-10.
Double staining with either anti-CD20 Ig (for B-cells), anti-
(CD-14) Ig for monocytes, anti-CD4 Ig or anti-CD3 Ig
(for T-cells) determined that B-cells and monocytes were
binding the fusion protein but T-cells were not. Possible
binding of the fusion protein to the Fc receptors on B-cells
and monocytes was ruled out by comparison to two fusion
protein controls, one with a similar R-g hIg tail with a point
mutation that prevents Fc receptor binding and one with an
E-g hIg tail that does bind Fc receptor (data not shown).
Additional FACS analyses were carried out with cell lines to
confirm the observations with whole blood. Neither HEL, an
erythroblastic leukemia cell line, nor Jurkat, a T-cell line,

stained positive for siglec-10– hIg. EBV-transformed B-cell
lines MB, PM and TJ did not stain positive for siglec-10–
hIg fusion protein but B-cell lines Ramos, Raji, Daudi
and HSB2 did stain positive. Although, there was some
monocyte binding in whole blood, we did not observe any
binding of siglec-10–hIg to either U973 or HL60 monocytic
cell lines. Furthermore, the anti-(siglec-10) Ig could block
the binding of the siglec-10 –hIg fusion protein to Daudi
cells (data not shown).
Cytoplasmic signaling
Kinase assay results. The kinase assays indicate that the
cytoplasmic domain of the siglec protein can be phos-
phorylated by representatives of at least three of four major
families of kinases: Jak3, Lck, Emt but not ZAP-70
(Fig. 6A). By titering the kinase concentration, it was
determined that siglec-10 could be phosphorylated equally
well by Lck and Jak3, moderate phosphorylation was
observed with Emt and little to no phosphorylation occurred
with ZAP-70. Wild-type GST– siglec-10-cyto was phos-
phorylated by Lck (100%) . Jak3 (92%) Emt
(65%) ZAP-70 (20%). When compared to wild-type,
some of the mutations in the cytoplasmic domain resulted in
significant decreases in phosphorylation (Fig. 6B,C). These
results suggest that the tyrosines at positions 597 and 667,
contained within ITIM-like motifs, are likely targets of
Fig. 6. Kinase assays with the siglec-10 cytoplasmic GST fusion
constructs. Tyrosine phosphorylation (minus GST alone) of GST–
cyto-wt with individual tyrosine kinases in a cell-free format.Tyrosine
phosphorylation of GST–cyto-wt and GST–cytoY !F mutants with a
mix of Lck, Jak3, Emt and ZAP-70 tyrosine kinases (starting at a

concentration of each at 125 ng
:
mL
21
). Tyrosine phosphorylation
(minus GST alone) of GST–cyto-wt and GST–cytoY !F mutants with
individual tyrosine kinases at 62.5 ng
:
mL
21
. Results shown are
mean ^ SD of two experiments, n ¼ 3 per treatment per experiment.
6092 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
phosphorylation by several classes of signaling molecules,
including Lck, Jak3, and Emt. The tyrosine located at
position Y691 was also contributing to the phosphorylation
of the wild-type siglec tail by Lck and Jak3 kinases. By
comparison, the mutation of the tyrosine at position 641 did
not significantly affect the degree of phosphorylation by any
of the kinases that were tested. In addition, a construct
containing Y641 alone was not phosphorylated by any of the
kinases, confirming that Y641 is probably not a site for
phosphorylation (data not shown).
SHP-1 and SHP-2 association with the cytoplasmic tail of
siglec-10. The results indicate that both SHP-1 and SHP-2
from either Jurkat (Fig. 7A) or primary eosinophil cell
lysates (data not shown) are capable of binding to the
cytoplasmic domain of the siglec-10 protein. The binding of
SHP-1, however, was missing from the Y667F mutant
indicating this to be the preferred tyrosine for interaction

with SHP-1. SHP-2 binding, however, was only diminished
by < 50% in the Y667F mutant, indicating that SHP-2 may
be binding both to the tyrosine at position 667 and to other
tyrosines in the cytoplasmic tail of siglec-10. In eosinophil
lysates we did not detect any SHP-2 binding to the Y667F
mutant, maybe due to low expression of SHP-2 as
determined by Western blotting (data not shown).
Confirmation of the cell lysate data was found in a cell-
free experiment where it was determined that SHP-1 and
SHP-2 could both bind with high affinity to a phosphory-
lated peptide containing the Y667 domain (Fig. 7B).
DISCUSSION
There is mounting evidence that inflammatory cell infiltrates
play a significant role in driving the pathogenesis of asthma
and other allergic diseases by damaging tissue and releasing
pro-inflammatory agents. Activated eosinophils, neutro-
phils, macrophages and lymphocytes increase in number at
sites of inflammation and each are capable of modifying the
overall inflammatory response [23]. Eosinophils, are of
particular interest in asthma and allergy due to their con-
spicuous appearance at sites of allergen-driven inflam-
mation [24 –26]. In recent years the concept of ‘inhibitory
receptors’ that function to moderate immune responses has
grown in prominence [27,28]. Targeting these types of
receptors on inflammatory cells in diseases such as
asthma may offer new and effective approaches to
immunomodulatory therapy. In an attempt to identify such
new targets on inflammatory cells at sites of inflammation,
we searched the Incyte EST database and found that
Fig. 7. Phosphatases SHP-1 and SHP-2 in cell

lysates bind to a GST fusion of the cytoplasmic
domain of siglec-10. Ten micrograms of GST
alone, GST– cyto-wt and GST– cyto-Y667F were
tyrosine phosphorylated (-P) with a mixture of
Lck, Jak3, Emt and ZAP-70 kinases or left
untreated, and incubated with cell lysate. GST
proteins and associated proteins were recovered by
binding to glutathione–Sepharose, separated by
SDS/PAGE, and analyzed by immunoblotting with
anti-(SHP-1) Ig or anti-(SHP-2) Ig. SHP-1 and
SHP-2 bind directly to Y667 ITIM. Biotinylated
tyrosine phosphorylated siglec-10 Y667 ITIM
peptide was bound to a streptavidin coated plate,
GST–SHP-1 and GST–SHP-2 were bound
starting at 250 ng
:
mL
21
and detected with rabbit
anti-GST Ig followed by HRP-conjugated
anti-(rabbit IgG) Ig. Results shown are the
mean ^ SD of two experiments, n ¼ 3 per
treatment per experiment.
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6093
siglec-10 mRNA was highly upregulated in the eosinophils
from asthmatics.
Our expression data confirmed that siglec-10 is expressed
in eosinophils, neutrophils and moncytes and that siglec-10
expression is immune-restricted. We also have preliminary
data to suggest upregulated expression in leukocytes derived

from asthmatics (data not shown). Interestingly, siglec-10
was also highly expressed on many transformed blood cell
lines that we examined, perhaps suggesting a role for this
molecule in some types of leukemia. Similar to CD33,
siglec-10 may prove to be a useful diagnostic marker for
leukemia.
The pattern of expression of siglec-10 seems to be
somewhat parallel to siglec-5, the closest related siglec, with
69% sequence identity at the nucleic acid level, and 87%
sequence identity in the membrane proximal Ig-D5 domain.
The similarity in sequence and expression suggests that
functional similarities might exist between the two siglecs as
well. Although, little work has been published on the func-
tion of siglec-5, work with siglec-2 and siglec-3 suggests
that this family of related proteins may function as
inhibitory receptors.
Siglec-10 appears to be genetically linked to siglec-3, -5,
-6, -7, -8, and -9 within a cluster of leukocyte-associated
genes, including many inhibitory receptors, on chromosome
19q13.3-13.4 [16,29]. Interestingly, this region contains
several genes that may be associated with immune disease
such as stem cell growth factor (SCGF), markers associated
with airways hyperreactivity in asthmatics and platelet
activating factor acetylhydrolase [29]. Ober et al. [30]
recently reported strong linkage of 19q markers with asthma
and atopy in a Hutterite population of European origin.
The purported ligands for the siglec proteins are modified
glycoproteins or glycolipids on other cells, or in some
instances on the same cell. There are < 40 naturally
occurring sialic acids (SIA), the most common are Neu5Ac,

Neu9Ac2 and Neu5Gc, occurring in terminal positions
linked to other sugars such as Gal, GalNAc, GlcNAc and
SIA itself on glycoproteins and glycolipids. The siglec
family of proteins may recognize not only the terminal sialic
acids but also the context of these moieties based on
preterminal sugars to which they are attached [1,31,32].
Results of various approaches with other siglec family
members, including truncated mutants [33], site-directed
mutagenesis [2,34], X-ray crystallography and NMR [37]
have demonstrated that the GFCC
0
C
0
face of the first
N-proximal Ig domain interacts with SIA. It is thought that
this interaction with SIA is responsible for cell to cell
adhesion as sialidase often interrupts binding. In particular,
an arginine residue within the first Ig domain is a key amino-
acid residue for binding to SIA [2]. Comparison of the
N-terminal Ig-like domain (Ig-D1) of siglec-5 and siglec-10
suggests that siglec-10 have similar structural and binding
properties. There is conservation of the three cysteines in the
domain that give rise to an intrasheet disulfide bond [6].
Also conserved are Arg at position 119 and two aromatic
residues (Phe and Tyr) at positions 21 and 128 that are
thought to be necessary for SIA binding [6]. Although the
sequence strongly suggests that the binding pattern should
be the same between siglec-5 and siglec-10, we noted
differences between the two. Whereas siglec-5 bound 2,3-
and 2,6-sialated lactose equally well in a cell-free system,

siglec-10 preferred 2,6-sialated lactose.
Siglecs are also postulated to be involved in cis-
interaction in which a siglec protein recognizes glycoconju-
gates on the same cell. Such cis-interaction may regulate
intercellular adhesion for CD22 [35,36], CD33 [5] and
MAG [5]. This may explain our lack of direct binding of
fusion protein to some cells in vitro. Although we did
attempt to eliminate possible cis interactions by pretreat-
ment with sialidase, we cannot be completely sure that all
cis interactions were interrupted. Furthermore, the cells
used in the binding studies were not activated, which may
enhance the masking of potential binding. Razi & Varki [37]
suggest that as leukocytes become activated siglecs become
unmasked, although they do not provide a molecular
mechanism for such a process. Perhaps the siglec does not
bind SIA very well except in the context of coligation with
an activating type of receptor. Our data did demonstrate
an enhancement of 2,6
0
-sialolactose binding to siglec-
10-expressing COS7 following sialidase treatment. Future
studies will focus more effort to determine if siglec-10
binding can be further enhanced with activation of the cells
prior to testing.
The amino-acid sequences of the cytoplasmic tails of
several siglec proteins strongly suggest that they participate
in intracellular signaling. For example, siglec-2/CD22 has
six tyrosines in the cytoplasmic domain, two of which reside
within ITAM motifs (mediating activation), and four within
ITIM motifs (mediating inhibition) [13]. Phosphorylation of

the ITAM motif tyrosines would allow recruitment of Src
family kinases whereas phosphorylation of ITIM motif
tyrosines would allow for recruitment of phosphatases SHIP,
SHP-1 or SHP-2. Siglec-3/CD33 contains two tyrosines that
recruit SHP-1 and SHP-2 upon phosphorylation [13]. One
tyrosine, Y340, is located in an ITIM consensus sequence
((S/I/L/V)XYXX(L/V)), LHYASL, and the other,Y358, is in
a SLAM-like motif, TEYSEV. SLAM is an acronym for
signaling lymphocyte activation molecule [38]. A SLAM-
like motif [(T/N)EYSE(I/V)(K/R)] is thought to allow for
the docking of SAP (SLAM-associated protein) which acts
as a negative regulator of SHP-2 by blocking its binding site
[10]. SAP binding sites are usually within 25 residues of an
upstream ITIM motif. Siglec-3/CD33, siglec-5, siglec-6,
siglec-7, and siglec-9 all contain this ITIM/SLAM-like
motif arrangement. Similarly, siglec-10 has a motif
surrounding the tyrosine at position 691 that resembles the
SAP binding site (TQADYAEVK) and is 24 residues
downstream from the ITIM at Y667. Because of the
difference in sequence, however, it is not clear whether
Y691 is capable of binding SAP or a similar protein. Future
studies will attempt to elucidate the signaling activities
associated with Y691.
It is still not clear what cellular function is served by
siglec-10. It is likely in the class of ‘inhibitory receptors’
simply based on its cytoplasmic domain structure. Several
recent studies have begun to suggest that ‘inhibitory
receptors’ containing ITIM motifs in their cytoplasmic
region may interact with other cell surface receptors bearing
ITAM motifs, attenuating an activation signal through the

recruitment of phosphatases. We demonstrate that siglec-10
can be phosphorylated by the Src family kinase Lck as well
as Jak3 and Emt, kinases representing three major kinase
families. Phosphorylation of the tyrosine located at position
667 in an ITIM motif appears to be necessary for the
recruitment of SHP-1 and partial recruitment of SHP-2. The
6094 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001
recruitment of SHPs suggests that the function of siglec-10
is similar to that of other siglecs such as siglec-3/CD33 and
siglec-7 that demonstrate recruitment of phosphatases upon
phosphorylation [13,15,39]. The inhibitory action of siglecs,
however, probably requires a complex colocalization of
siglecs with partnered activating receptors. To date, we have
been unable to observe inhibition of eosinophil function
(with either IL-5 or IgE stimulation) by simple crosslinking
of siglec-10 with an antibody (data not shown). The
challenge of future mechanistic studies will be to determine
if siglec-10 associates with any known activating receptors
upon cell stimulation.
Here, we have described a new siglec protein family
member, identified through a genomics database mining
effort. Siglec-10 is expressed in leukocytes and appears to
bind B-cells and monocytes via its extracellular domain.
Future studies will be focused on determining the activating
receptor or receptors that are likely associating with
siglec-10 upon activation. In addition, it will be of
importance to determine the cognate binding partner of
siglec-10 so that further studies as to its functional
significance can be pursued. It is anticipated that iden-
tification of novel inhibitory receptors on inflammatory cells

and their signaling potential will offer opportunities to target
and inhibit inflammatory cells at sites of inflammation.
ACKNOWLEDGEMENTS
We would like to acknowledge the excellent technical support of Bill
Fenderson, Vanessa Haluska, Joanne Olivieri, Patty Davis, Derek
Hewgill, Michael Bowen, Dawn Stesko, Mark Stebbins, Farah Kondri,
Kathy Maruk, Bob Dunn, Christine Burke, Tiziano DiPaolo, Joe Cook,
and Molly Rodgers. We would also like to acknowledge the helpful
advice of Juan Jo Perez, Steve Kanner and Diane Hollenbaugh.
REFERENCES
1. Kelm, S., Schauer, R. & Crocker, P.R. (1996) The sialoadhesins – a
family of sialic acid-dependent cellular recognition molecules
within the immunoglobulin superfamily. Glycoconj. J. 13,
913–926.
2. Vinson, M., van der Merwe, P.A., Kelm, S., May, A., Jones, E.Y. &
Crocker, P.R. (1996) Characterization of the sialic acid-binding site
in sialoadhesin by site-directed mutagenesis. J. Biol. Chem. 271,
9267–9272.
3. Crocker, P.R., Mucklow, S., Bouckson, V., McWilliam, A., Willis,
A.C., Gordon, S., Milon, G., Kelm, S. & Bradfield, P. (1994)
Sialoadhesin, a macrophage sialic acid binding receptor for
haemopoietic cells with 17 immunoglobulin-like domains. EMBO
J. 13, 4490–4503.
4. Kelm, S., Brossmer, R., Isecke, R., Gross, H.J., Strenge, K. &
Schauer, R. (1998) Functional groups of sialic acids involved in
binding to siglecs (sialoadhesins) deduced from interactions with
synthetic analogues. Eur. J. Biochem. 255, 663–672.
5. Freeman, S.D., Kelm, S., Barber, E.K. & Crocker, P.R. (1995)
Characterization of CD33 as a new member of the sialoadhesin
family of cellular interaction molecules. Blood 85, 2005– 2012.

6. Kelm, S., Pelz, A., Schauer, R., Filbin, M.T., Tang, S., de Bellard,
M.E., Schnaar, R.L., Mahoney, J.A., Hartnell, A., Bradfield, P. &
Crocker, P.R. (1994) Sialoadhesin, myelin-associated glycoprotein
and CD22 define a newfamily of sialic acid-dependent adhesion
molecules of the immunoglobulin superfamily. Curr. Biol. 4,
965–972.
7. Cornish, A.L., Freeman, S., Forbes, G., Ni, J., Zhang, M., Cepeda,
M., Gentz, R., Augustus, M., Carter, K.C. & Crocker, P.R. (1998)
Characterization of siglec-5, a novel glycoprotein expressed on
myeloid cells related to CD33. Blood 92, 2123–2132.
8. Takei, Y., Sasaki, S., Fugiwara, T., Takahashi, E., Muto, T. &
Nakamura, Y. (1997) Molecular cloning of a novel gene similar to
myeloid antigen CD33 and its specific expression in placenta.
Cytogenet. Cell. Genet. 78, 295–300.
9. Nicoll, G., Ni, J., Liu, D., Klenerman, P., Munday, J., Dubock, S.,
Mattei, M.G. & Crocker, P.R. (1999) Identification and
characterization of a novel siglec, siglec-7, expressed by human
natural killer cells and monocytes. J. Biol. Chem. 274,
34089–34095.
10. Floyd, H., Ni, J., Cornish, A., Cornish, A.L., Zeng, Z., Liu, D.,
Carter, K.C., Steel, J. & Crocker, P.R. (2000) Siglec-8. A novel
eosinophil-specific member of the immunoglobulin superfamily.
J. Biol. Chem. 275, 861 –866.
11. Angata, T. & Varki, A. (2000) Cloning, characterization, and
phylogenetic analysis of siglec-9, a new member of the
CD33-related group of siglecs. Evidence for co-evolution with
sialic acid synthesis pathways. J. Biol. Chem. 275, 22127–22135.
12. Zhang, J.Q., Nicoll, G., Jones, C. & Crocker, P.R. (2000) Siglec-9, a
novel sialic acid binding member of the immunoglobulin
superfamily expressed broadly on human blood leukocytes.

J. Biol. Chem. 275, 22121–22126.
13. Elghetany, M.T. (1998) Surface marker abnormalities in myelo-
dysplastic syndromes. Haematologica 83, 1104 –1115.
14. Taylor, V.C., Buckley, C.D., Douglas, M., Cody, A.J., Simmons,
D.L. & Freeman, S.D. (1999) The myeloid-specific sialic acid-
binding receptor, CD33, associates with the protein-tyrosine
phosphatases, SHP-1 and SHP-2. J. Biol. Chem. 274,
11505–11512.
15. Aruffo, A., Kanner, S.B., Sgroi, D., Ledbetter, J.A. & Stamenkovic,
I. (1992) CD22-mediated stimulation of T cells regulates T-cell
receptor/CD3-induced signaling. Proc. Natl Acad. Sci. USA 89,
10242–10246.
16. Falco, M., Biassoni, R., Bottino, C., Vitale, M., Sivori, S.,
Augugliaro, R., Moretta, L. & Moretta, A. (1999) Identification and
molecular cloning of p75/AIRM1, a novel member of the
sialoadhesin family that functions as an inhibitory receptor in
human natural killer cells. J. Exp. Med. 190, 793–802.
17. Wende, H., Colonna, M., Ziegler, A. & Volz, A. (1999)
Organization of the leukocyte receptor cluster (LRC) on human
chromosome 19q13.4. Mammal. Gen. 10, 154–160.
18. Ewing, B., Hillier, L., Wendl, M. & Green, P. (1998a) Base-calling
of automated sequencer traces using phred. I. Accuracy assessment.
Genome Res. 8, 175–185.
19. Ewing, B. & Green, P. (1998b) Base-calling of automated
sequencer traces using phred. II. Error probabilities. Genome Res.
8, 86–194.
20. Gordon, D.C., Abajian, P. & Green, P. (1998) Consed: a graphical
tool for sequence finishing. Genome Res. 8, 195– 202.
21. Smith, D.B. & Johnson, K.S. (1988) Single-step purification of
polypeptides expressed in Escherichia coli as fusions with

glutathione S-transferase. Gene 67, 31–40.
22. Mucklow, S., Gordon, S. & Crocker, P.R. (1997) Characterization
of the mouse sialoadhesin gene Sn. Mamm. Gen 8, 934–937.
23. Busse, W.W. (1998) Inflammation in asthma: the cornerstone of the
disease and target of therapy. J. Allergy Clin. Immunol. 102,
S17–S22.
24. Kroegel, C., Virchow, J.C. Jr, Luttmannn, W., Walker, C. & Warner,
J.A. (1994) Pulmonary immune cells in health and disease: the
eosinophil leukocyte (Part I). Eur. Repir. J. 7, 519–543.
25. Haczku, A. (1998) T cells and eosinophils in asthma. Acta
Microbiol. Immunol. Hung. 45, 19 –29.
26. Boyce, J.A. (1997) The pathobiology of eosinophilic inflammation.
Allergy Asthma Proc. 18, 293–300.
27. Bolland, S. & Ravetch, J.V. (1999) Inhibitory pathways triggered
by ITIM-containing receptors. Adv. Immunol. 72, 149 –177.
q FEBS 2001 Immune-restricted siglec-10 (Eur. J. Biochem. 268) 6095
28. Tamir, I., Dal Porto, J.M. & Cambier, J.C. (2000) Cytoplasmic
protein tyrosine phosphatases SHP-1 and SHP-2: regulators of B
cell signal transduction. Current Opinion Immunol. 12, 307–315.
29. Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A.,
Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M.,
Gyapay, G., . Morissette, J. & Weissenbach, J. (1996) A
comprehensive genetic map of the human genome based on
5,264 microsatellites. Nature 380, 152–154.
30. Ober, C.A. & Tselenko, & Cox, N.J. (2000) Searching for asthma
and atopy genes in the Hutterites: genome-wide studies using
linkage and association. Am. J. Respir. Crit. Care Med. 161, A600.
31. Powell, L.D. & Varkin, A.J. (1994) The oligosaccharide binding
specificities of CD22 beta, a sialic acid-specific lectin of B cells.
J. Biol. Chem. 269, 10628 –10636.

32. Collins, B.E., Kiso, M., Haseqawa, A., Hasegawa, A., Tropak,
M.B., Roder, J.C., Crocker, P.R. & Schnaar, R.L. (1997) Binding
specificities of the sialoadhesin family of I-type lectins. Sialic acid
linkage and substructure requirements for binding of myelin-
associated glycoprotein, Schwann cell myelin protein, and
sialoadhesin. J. Biol. Chem. 272, 16889–16895.
33. Nath, D., van der Merwe, P.A., Kelm, S., Bradfield, P. & Crocker,
P.R. (1995) The amino-terminal immunoglobulin-like domain of
sialoadhesin contains the sialic acid binding site. Comparison with
CD22. J. Biol. Chem. 270, 26184–26191.
34. Van der Merwe, P.A., Crocker, P.R., Vinson, M., Vinson, M.,
Barclay, A.N., Schauer, R. & Kelm, S. (1996) Localization of
the putative sialic acid-binding site on the immunoglobulin
superfamily cell-surface molecule CD22. J. Biol. Chem. 271,
9273–9280.
35. Braesch-Andersen, S. & Stamendovic, I. (1994) Sialylation of the
B lymphocyte molecule CD22 by alpha-2,6-sialyltransferase is
implicated in the regulation of CD22-mediated adhesion. J. Biol.
Chem. 269 (16), 11783–11786.
36. Hanasaki, K., Varki, A., Stamenkovic, I. & Bevilacqua, M.P. (1994)
Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in
human endothelial cells mediates alpha-2,6-sialylation of
adhesion molecules and CD22 ligands. J. Biol. Chem. 269,
10637–10643.
37. Razi, N. & Varki, A. (1998) Masking and unmasking of the sialic
acid-binding lectin activity of CD22 (siglec-2) on B lymphocytes.
Proc. Natl Acad. Sci. USA 95, 7469–7474.
38. Patel, N., Brinkman-Van der Linden, E.C.M., Altmannn, S.W.,
Gish, K., Balasubramanian, S., Timans, J.C., Peterson, D., Bell,
M.P., Bazan, J.F., Varki, A. & Kastelein, R.A. (1999) OB-BP1/

Siglec-6. a leptin- and sialic acid-binding protein of the
immunoglobulin superfamily. J. Biol. Chem. 274, 22729–22738.
39. Ulyanova, T., Blasioli, J., Woodford-Thomas, T.A. & Tomas, M.L.
(1999) The sialoadhesin CD33 is a myeloid-specific inhibitory
receptor. Eur. J. Immunol. 19, 3440–3449.
40. Foussias, G., Yousef, G.M. & Diamandis, E.P. (2000) Identification
and molecular characterization of a novel member of the siglec
family (SIGLEC9). Genomics 7, 171–178.
41. Crocker, P.R., Hartnell, A., Munday, J. & Nath, D. (1997) The
potential role of sialoadhesin as a macrophage recognition
molecule in health and disease. Glycoconj. J. 14, 601–609.
6096 G. Whitney et al. (Eur. J. Biochem. 268) q FEBS 2001