Eur J Biochem 268, 6083–6096 (2001) q FEBS 2001 A 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 McKinnon1 and Malinda Longphre1 Inflammation and Pulmonary Drug Discovery Department, and 2Applied 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 cytoplasmic tail containing two ITIM-signaling motifs Siglec10 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 systemrestricted 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 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 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 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 Likewise, 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 Correspondence to G Whitney, Bristol–Myers Squibb Pharmaceutical Research Institute, Mail Stop K24-03 PO Box4000 Princeton, NJ 08543–4000, USA Abbreviations: SHP, Src homology region 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; 2,30 -PAA, 2,30 -sialyllactose; 2,60 -PAA, 2,60 sialyllactose; SCGF, stem cell growth factor; SIA, sialic acid; GST, glutathione S-transferase; SMP, Schwann cell myelin protein Note: G Whitney and S Wang share first authorship (Received April 2001, revised July 2001, accepted 25 September 2001) Keywords: sialoadhesin; CD33; inhibitory receptor; phosphatase; siglec 6084 G Whitney et al (Eur J Biochem 268) 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 E X P E R I M E N TA 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 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 siglec10d from nucleotide position 545– 710 The S2 probe includes splice variant sequences common among siglec10b 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 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 h at 68 8C The blot was then washed under stringent conditions using  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 min, followed by 30 cycles of 95 8C/56 8C/72 8C, for 30 s each Table PCR primers used to make probes for Northern blots Primer pair Corresponding nucleotides Sequence Resulting probe length (bp) L3 713–733 1436–1445 50 -TGCTCAGCTTCACGCCCAGAC-30 30 -TGCACGGAGAGGCTGAGAGA-50 732 S1 545–563 693–710 50 -CTCAGAAGCCTGATGTCTA-30 30 -GAGAAGTGGGAGGTCGTT-50 166 S2 1458–1476 1676–1695 50 2CTGCTGGGCCCCTCCTGC-30 30 -GACGTTCCAGGCCTCACAG-50 237 q FEBS 2001 followed by 72 8C for 10 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 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 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 The supernatant was harvested and centrifuged for at 500 g The cells were then resuspended in 0.2  NaCl/Pi to induce red blood cell lysis for and then 1.8  NaCl/Pi is added If eosinophils were required, the cells were then centrifuged for at 500 g and resuspended in buffer containing CD16 microbeads (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) Immune-restricted siglec-10 (Eur J Biochem 268) 6085 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 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 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 mL:min21 The fusion protein was then eluted with 0.1 M acetic acid (pH 4.5) and immediately neutralized with M Tris/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] Full-length protein expression Antibody generation 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 Balb/c mice were immunized with an intraperitoneal injection of siglec-10–hIg fusion protein in Ribi Adjuvant (Corixa, Hamilton, MT, USA) once every 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 : 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 6086 G Whitney et al (Eur J Biochem 268) q FEBS 2001 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 transferred to nitrocellulose by standard Western blotting techniques The blots were then stained with monoclonal anti-(siglec-1) Ig followed by a secondary goat HRPconjugated 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 mg protein per  106 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 h followed by washing twice with binding buffer Cells were centrifuged at 500 g for between each wash Fluorescein-conjugated anti-hIg Ig (Jackson Immunoresearch, West Grove, PA, USA) and/or phycoerythrin-conjugated anti-CD20 Ig, antiCD14 Ig, and anti-CD4 Ig (Beckton – Dickinson, San Jose,CA, USA) were added on ice for 30 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 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 Immune-restricted siglec-10 (Eur J Biochem 268) 6087 (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 h, plates were washed and treated with streptavidin – horse-radish peroxidase (HRP) (Vector Laboratories, Burlingame, CA, USA) in DMEM for 30 After a final wash, 3,30 ,5,50 -tetramethylbenzidine peroxidase substrate (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 h with DMEM containing 1% BSA Blood cells and cell lines were labeled with calcein-AM (5 mL per 108 cells, Molecular Probes, Eugene, OR, USA) for 30 at 37 8C Cells were then washed twice in Hanks buffered salt solution (HBSS) and added to the blocked plate (4  105 per well in 200 mL) at 37 8C for 30 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  105 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 108 cells) for 30 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:mL21 for 30 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 with gentle rocking The plates were then washed gently three times with NaCl/Pi 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 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 6088 G Whitney et al (Eur J Biochem 268) treatment 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 shamtransfected 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 at 37 8C) COS7 cells, suspended in binding buffer, were then added to HUVEC cells and incubated for h at 37 8C After several washes with HBSS, fluorescence in each well was measured q FEBS 2001 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 HRPconjugated 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 blocking reagent (Hitachi Genetics Systems) was bound to a strepavidin-coated ELISA plate (Pierce) Plates were 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 Baculovirus 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 mg:mL21 in sodium carbonate pH 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 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 : 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 tyrosine phosphorylation were incubated with 300 mL of cell lysate (Triton X-100-soluble fraction  107 unstimulated cells) at 8C overnight The GST fusion protein complexes were captured with 50 mL of glutathione –Sepharose beads (Amersham –Pharmacia Biotech) for h at 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 Fig 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 q FEBS 2001 Immune-restricted siglec-10 (Eur J Biochem 268) 6089 Fig 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 –5) or the two spliced regions (S1 ¼ Ig domain and S2 ¼ Ig domain 5) of siglec-10 b-Actin was also probed as a control for mRNA integrity and loading washed and then twofold dilutions of the GST fusion proteins, GST alone, GST– SHP-1SH2SH2 or GST–SHP2SH2SH2 or GST – ZAP-70SH2SH2 were added and incubated for h at room temperature Polyclonal antiGST Ig (prepared in-house by procedures similar to those detailed for anti-siglec Ig production) was added at : 1000, HRP-conjugated anti-(rabbit IgG) Ig (Biosource) at : 2000, and signal detected with peroxidase substrate (KPL) R E S U LT 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 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 –3 experiments Cell line Type FITC alone (%) Anti-(siglec-10) Ig (%) Ramos THP-1 Jurkat U973 HL60 K562 COS7 COS7 B-cell (lymphoma) Monocyte (lymphoma) T-cell (lymphoma) Monocyte (leukemia) Monocyte (leukemia) Monocyte (leukemia) Naive Siglec-10 Transfected 0.5 1.7 0.4 2.0 0.6 0.5 2.4 4.7 89.9 39.1 76.6 33.6 93.3 96.2 15.4 63.3 Blood population PE1 (%) Siglec-FITC1 (%) FITC1 and PE1 (%) PBMC CD201 CD141 CD4lo CD4hi CD31 CD281 Granulocytes 8.3 12.1 11.7 63.0 65.0 47.5 19.9 2.4 12.0 12.0 0 2.0 88.6 6090 G Whitney et al (Eur J Biochem 268) q FEBS 2001 Fig 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 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 twopoint 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 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 h with DMEM containing 1% BSA Blood cells and cell lines were labeled with calcein-AM (5 mL per 108 cells) for 30 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 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:mL21 for 30 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 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 well plates containing DMEM with 10% fetal bovine serum at a density of  105 per well Blood cells and cell lines were labeled with calcein-AM (5 mL per 108 cells) for 30 at 37 8C and some cells were also pretreated with sialidase (0.1 U:mL21 for 30 at 37 8C) One milliliter of blood-cell or cell-line suspension was added to each well and incubated at 37 8C for 30 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 per treatment per experiment *, Statistically different from sham transfected controls, P , 0.05 q FEBS 2001 Immune-restricted siglec-10 (Eur J Biochem 268) 6091 Table 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 (%) Cell line Type mALCAM–hIg CD5– hIg Siglec-10–hIg MB PM TJ Ramos HSB-2 Raji Daudi Jurkat HEL U973 HL60 B-cell (EBV) B-cell (EBV) B-cell (EBV) B-cell (lymphoma) B-cell (lymphoma) B-cell (lymphoma) B-cell (lymphoma) T-cell (lymphoma) RBC (leukemia) Monocyte (leukemia) Monocyte (leukemia) 0 0 38 0 0 0 2 0 0 57 40 36 20 0 0 FITC staining(%) Blood population PE1 (%) ALCAM–hIg CD5 –hIg Siglec-10–hIg PBMC CD201 CD141 CD4lo1 CD4hi1 CD31 Granulocytes 11 12 63 65 0 0 0 0 11 0 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 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 hybridization 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 Mr as siglec-10, while siglec-5 has a predicted Mr 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 2,30 -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 6092 G Whitney et al (Eur J Biochem 268) q FEBS 2001 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 pretreatment of the cells (0.1 U:mL21 for 30 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 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 Fig 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:mL21 Results shown are mean ^ SD of two experiments, n ¼ per treatment per experiment 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 Kinase assay results The kinase assays indicate that the cytoplasmic domain of the siglec protein can be phosphorylated 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 phosphorylated 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 q FEBS 2001 Immune-restricted siglec-10 (Eur J Biochem 268) 6093 Fig 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:mL21 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 per treatment per experiment 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 cellfree experiment where it was determined that SHP-1 and SHP-2 could both bind with high affinity to a phosphorylated 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, neutrophils, 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 conspicuous appearance at sites of allergen-driven inflammation [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 6094 G Whitney et al (Eur J Biochem 268) 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 function 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 GFCC0 C0 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 aminoacid 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 2,3and 2,6-sialated lactose equally well in a cell-free system, siglec-10 preferred 2,6-sialated lactose q FEBS 2001 Siglecs are also postulated to be involved in cisinteraction in which a siglec protein recognizes glycoconjugates 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 pretreatment 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 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 2,60 -sialolactose binding to siglec10-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 SLAMlike 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 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 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 ... mounting evidence that in? ??ammatory cell in? ??ltrates play a significant role in driving the pathogenesis of asthma and other allergic diseases by damaging tissue and releasing pro -in? ??ammatory agents... was only diminished by < 50% in the Y6 67F 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... -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)