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 trans
Trang 1A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33
Gena Whitney1, Shulin Wang1, Han Chang2, Ke-Yi Cheng1, Pin Lu1, Xia D Zhou1, Wen-Pin Yang2,
Murray McKinnon1and Malinda Longphre1
1 Inflammation and Pulmonary Drug Discovery Department, and2Applied 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.
Trang 2Described 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
E X P E R I M E N T A L P R O C E D U R E S
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 thePHRAPalgorithm
[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 withSEQWEB
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 inGCG(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 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 [32P]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 NaCl/Cit containing 0.05% SDS followed by a wash with 0.1 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
Resulting probe length (bp)
1436 – 1445 30-TGCACGGAGAGGCTGAGAGA-50
693 – 710 30-GAGAAGTGGGAGGTCGTT-50
1676 – 1695 30-GACGTTCCAGGCCTCACAG-50
Trang 3followed by 72 8C for 10 min The primer sequences for G3
PCR were: TMD (1720 – 1738), 50-tgcagctgccagataaga-30
and (2065 – 2083) 30-GGCTTGAGTGGATGATTT-50; 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:mL21 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 NaCl/Pito induce red blood cell lysis
for 1 min and then 1.8 NaCl/Pi 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/Pi 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 30 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 50 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 glutathioneS-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:mL21 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/Pi 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:min21 The fusion protein was then eluted with 0.1M acetic acid (pH 4.5) and immediately neutralized with 2MTris/base to
a final pH of 8.0 Siglec-10 – hIg was then dialysed against NaCl/Pi
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
Trang 5siglec-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 106cells 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 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 usingCELL
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:mL21) of a
polyacrylamide polymer containing biotin and carbohydrate
(lactose, 30-sialyllactose or 60-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:well21) and incubated with 20 mg:mL21 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, 3,30,5,50-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:well21) 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 108cells, 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 105per 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 105per 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
108cells) 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:mL21for 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/Piplus 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.
Trang 6treatment and observed at 100 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:mL21 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:mL21in 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 mM Hepes pH 7.0, 6.25 mM MnCl2,
6.25 mM MgCl2, 0.5 mM sodium vanadate, 7.5 mM ATP
and twofold dilutions of the tyrosine kinases starting at
0.25 mg:mL21 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
5 107 unstimulated cells) at 4 8C overnight The GST
fusion protein complexes were captured with 50 mL of
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:mL21) 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.
Trang 7washed and then twofold dilutions of the GST fusion
proteins, GST alone, GST – 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)
R E S U L T S 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.
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.
Trang 8are 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
also expressed in eosinophils, does not appear to be very
homologous when compared byPILEUP(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:well21) 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 (4 105 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 2 105 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.
Trang 9Expression 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 Mr 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 Mras siglec-10, while siglec-5 has a predicted Mrof 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 2,30-sialyllactose (2,30-PAA) and 2,60 sialyllactose (2,60-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 2,60-PAA conjugate bound significantly greater than either the unsialylated lactose (negative control)
or 2,30-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 2,60-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).
FITC Staining (%)
Blood population
FITC staining(%)
PBMC
Trang 10Binding 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:mL21for 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 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:mL21) 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.