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1.
The sialic acid binding immunglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within the Siglec family, there exists a subgroup, which bears a high degree of homology with the molecule CD33 (Siglec-3), and has thus been designated the CD33-like subgroup of Siglecs. Members of this subgroup have been localized to chromosome 19q13.4. Through the positional candidate approach, we identified a novel potential member of this subgroup of Siglecs. We have characterized the complete genomic structure of this gene, determined its chromosomal localization, its homology to other members of the Siglec family, and its tissue expression profile. This new Siglec-like gene is comprised of 11 exons, with 10 intervening introns, and is localized 278 kb telomeric to Siglec-9 and 35 kb centromeric to Siglec-8 and on chromosome 19q13.4. The coding region consists of 2094 base pairs, and encodes for a putative 76.6 kDa protein. All Siglec-conserved structural features, including V-set domain, three C-set domains, transmembrane domain, ITIM and SLAM motifs, were found in this Siglec-like gene. Also, it has the conserved amino acids essential for sialic acid binding. The Siglec-like gene has 40-66% homology with members of the CD33-like subgroup, including Siglecs 5-9. Through RT-PCR we have examined the expression profile of this new gene in a panel of human tissues and found it to be primarily expressed in the bone marrow, spleen, brain, small intestine, colon, and spinal cord. We were also able to identify three different splice variants of the new gene. This gene may represent the latest novel member of the CD33-like subgroup of Siglecs, and, given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec-7.  相似文献   

2.
3.
The Siglecs (sialic acid-binding Ig-like lectins) are a subfamily of I-type lectins, which specifically recognize sialic acids. Nine members of the family have been identified thus far. We have obtained a novel cDNA clone from a human dendritic cell cDNA library encoding a protein with sequence and structural features of the Siglec family, hence designated as Siglec-10. The full-length Siglec-10 cDNA encodes a type 1 transmembrane protein containing four extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs. The N-terminal V-set Ig domain has most of the amino acid residues typical of the Siglecs. Siglec-10 shows the closest homology to Siglec-5 and Siglec-3/CD33. Various cells and cell lines including monocytes and dendritic cells express Siglec-10. High levels of mRNA expression were seen in peripheral blood leukocytes, spleen, and liver. When expressed on COS-7 cells, Siglec-10 was able to bind human red blood cells and soluble sialoglycoconjugates in a sialic acid-dependent manner. The identification of Siglec-10 as a new Siglec family member and its expression profile, together with its sialic acid-dependent binding capacity, suggest that it may be involved in cell-cell recognition by interacting with sialylated ligands expressed on specific cell populations.  相似文献   

4.
Through efforts to investigate the CD33-like subgroup of sialic acid binding immunoglobulin-like lectins (Siglecs), which are believed to be located on chromosome 19q13.4, we have identified the precise genomic region containing the Siglec8 gene. It is located on chromosome 19q13.4, approximately 330 kb downstream of the Siglec9 gene. Further, we have identified a novel Siglec8 variant, named Siglec8-Long (Siglec8-L), which differs in its last two exons from the previously published mRNA sequence of Siglec8 (GenBank Accession No. AF195092). Both Siglec8 and Siglec8-L are comprised of seven exons, of which the first five are identical, followed by marked differences in exon usage and mRNA splicing. The 499 amino acid protein encoded by the Siglec8-L open reading frame has a molecular weight of 54 kDa. Like the other members of the CD33-like subgroup of Siglecs, except for the previously published Siglec8, Siglec8-L also contains the two tyrosine-based motifs that have been found to recruit both SH2 domain-containing tyrosine and inositol phosphatases.  相似文献   

5.
Siglecs are sialic acid-recognizing animal lectins of the immunoglobulin superfamily. We have cloned and characterized a novel human molecule, Siglec-11, that belongs to the subgroup of CD33/Siglec-3-related Siglecs. As with others in this subgroup, the cytosolic domain of Siglec-11 is phosphorylated at tyrosine residue(s) upon pervanadate treatment of cells and then recruits the protein-tyrosine phosphatases SHP-1 and SHP-2. However, Siglec-11 has several novel features relative to the other CD33/Siglec-3-related Siglecs. First, it binds specifically to alpha2-8-linked sialic acids. Second, unlike other CD33/Siglec-3-related Siglecs, Siglec-11 was not found on peripheral blood leukocytes. Instead, we observed its expression on macrophages in various tissues, such as liver Kupffer cells. Third, it was also expressed on brain microglia, thus becoming the second Siglec to be found in the nervous system. Fourth, whereas the Siglec-11 gene is on human chromosome 19, it lies outside the previously described CD33/Siglec-3-related Siglec cluster on this chromosome. Fifth, analyses of genome data bases indicate that Siglec-11 has no mouse ortholog and that it is likely to be the last canonical human Siglec to be reported. Finally, although Siglec-11 shows marked sequence similarity to human Siglec-10 in its extracellular domain, the cytosolic tail appears only distantly related. Analysis of genomic regions surrounding the Siglec-11 gene suggests that it is actually a chimeric molecule that arose from relatively recent gene duplication and recombination events, involving the extracellular domain of a closely related ancestral Siglec gene (which subsequently became a pseudogene) and a transmembrane and cytosolic tail derived from another ancestral Siglec.  相似文献   

6.
Using the positional cloning approach, we have identified siglec-9 (HGMW-approved symbol SIGLEC9) a novel member of the sialic acid-binding Ig-like lectin (Siglec) family, which belongs to the immunoglobulin superfamily (IgSF). We characterized the genomic structure of this gene and determined its chromosomal localization, its homology to other members of the siglec family, and its tissue expression profile. The siglec-9 gene is composed of seven exons, with six intervening introns. The coding region consists of 1392 nucleotides and produces a 463-amino-acid protein. Furthermore, we have localized this gene to 19q13.4, 43.19 kb more telomeric than KLK14 (a member of the kallikrein gene family) through genomic sequencing data and restriction mapping with EcoRI. This novel siglec shows a high degree of homology to many members of the siglec family, including siglec-7 (80%), siglec-8 (72%), siglec-5 (65%), and CD33 (64%). This high degree of homology is also conserved in the extracellular Ig-like domains. Through RT-PCR, we have examined the expression of siglec-9 in a large number of tissues and have found relatively high-level expression in bone marrow, placenta, spleen, and fetal liver. Based on its homology to CD33, we speculate that this gene may also have some utility as a target for immunological antineoplastic therapy.  相似文献   

7.
The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to alpha2-3 and alpha2-6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galbeta1-4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3-13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway.  相似文献   

8.
A novel mouse Siglec (mSiglec-F) belonging to the subfamily of Siglec-3-related Siglecs has been cloned and characterized. Unlike most human Siglec-3 (hSiglec-3)-related Siglecs with promiscuous linkage specificity, mSiglec-F shows a strong preference for alpha2-3-linked sialic acids. It is predominantly expressed in immature cells of the myelomonocytic lineage and in a subset of CD11b (Mac-1)-positive cells in some tissues. As with previously cloned Siglec-3-related mSiglecs, the lack of strong sequence similarity to a singular hSiglec made identification of the human ortholog difficult. We therefore conducted a comprehensive comparison of Siglecs between the human and mouse genomes. The mouse genome contains eight Siglec genes, whereas the human genome contains 11 Siglec genes and a Siglec-like gene. Although a one-to-one orthologous correspondence between human and mouse Siglecs 1, 2, and 4 is confirmed, the Siglec-3-related Siglecs showed marked differences between human and mouse. We found only four Siglec genes and two pseudogenes in the mouse chromosome 7 region syntenic to the Siglec-3-related gene cluster on human chromosome 19, which, in contrast, contains seven Siglec genes, a Siglec-like gene, and thirteen pseudogenes. Although analysis of gene maps and exon structures allows tentative assignments of mouse-human Siglec ortholog pairs, the possibility of unequal genetic recombination makes the assignments inconclusive. We therefore support a temporary lettered nomenclature for additional mouse Siglecs. Current information suggests that mSiglec-F is likely a hSiglec-5 ortholog. The previously reported mSiglec-3/CD33 and mSiglec-E/MIS are likely orthologs of hSiglec-3 and hSiglec-9, respectively. The other Siglec-3-like gene in the cluster (mSiglec-G) is probably a hSiglec-10 ortholog. Another mouse gene (mSiglec-H), without an apparent human ortholog, lies outside of the cluster. Thus, although some duplications of Siglec-3-related genes predated separation of the primate and rodent lineages (about 80-100 million years ago), this gene cluster underwent extensive duplications in the primate lineage thereafter.  相似文献   

9.
Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily   总被引:5,自引:0,他引:5  
Angata T  Varki A 《Glycobiology》2000,10(4):431-438
The Siglecs are a recently discovered family of sialic acid-binding lectins of the immunoglobulin (Ig) superfamily. We report a molecule showing homology to the six first reported Siglecs, with the closest relationship to Siglec-3(CD33), Siglec-5, and Siglec-6(OBBP-1). The extracellular portion has two Ig-like domains, with the amino-terminal V-set Ig domain including amino acid residues known to be involved in sialic acid recognition by other Siglecs. The cytoplasmic domain has putative sites of tyrosine phosphorylation shared with some Siglecs, including an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM). Expression of the full-length cDNA induces sialic acid-dependent binding to human erythrocytes. A recombinant chimeric form containing the extracellular Ig domains selectively recognizes the sequence Neu5Acalpha2-6Galbeta1-4Glc, and binding requires the side chain of sialic acid. Mutation of an arginine residue predicted to be critical for sialic acid binding abolishes both interactions. Taken together, our findings justify designation of the molecule as Siglec-7. Analysis of bacterial artificial chromosome (BAC) clones spanning the known human genomic location of Siglec-3 indicates that the Siglec-7 gene is also located on chromosome 19q13.3-13.4. Human tissues show strong expression of Siglec-7 mRNA in spleen, peripheral blood leukocytes, and liver. The combination of an extracellular sialic acid binding site and an intracellular ITIM motif suggests that this molecule is involved in trans-membrane regulatory signaling reactions.  相似文献   

10.
Sialic acid-binding immunoglobulin-like lectins (Siglecs) recognize sialylated glycoconjugates and play a role in cell-cell recognition. Siglec-7 is expressed on natural killer cells and displays unique ligand binding properties different from other members of the Siglec family. Here we describe the high resolution structures of the N-terminal V-set Ig-like domain of Siglec-7 in two crystal forms, at 1.75 and 1.9 A. The latter crystal form reveals the full structure of this domain and allows us to speculate on the differential ligand binding properties displayed by members of the Siglec family. A fully ordered N-linked glycan is observed, tethered by tight interactions with symmetry-related protein molecules in the crystal. Comparison of the structure with that of sialoadhesin and a model of Siglec-9 shows that the unique preference of Siglec-7 for alpha(2,8)-linked disialic acid is likely to reside in the C-C' loop, which is variable in the Siglec family. In the Siglec-7 structure, the ligand-binding pocket is occupied by a loop of a symmetry-related molecule, mimicking the interactions with sialic acid.  相似文献   

11.
Siglecs--the major subfamily of I-type lectins   总被引:6,自引:0,他引:6  
Varki A  Angata T 《Glycobiology》2006,16(1):1R-27R
Animal glycan-recognizing proteins can be broadly classified into two groups-lectins (which typically contain an evolutionarily conserved carbohydrate-recognition domain [CRD]) and sulfated glycosaminoglycan (SGAG)-binding proteins (which appear to have evolved by convergent evolution). Proteins other than antibodies and T-cell receptors that mediate glycan recognition via immunoglobulin (Ig)-like domains are called "I-type lectins." The major homologous subfamily of I-type lectins with sialic acid (Sia)-binding properties and characteristic amino-terminal structural features are called the "Siglecs" (Sia-recognizing Ig-superfamily lectins). The Siglecs can be divided into two groups: an evolutionarily conserved subgroup (Siglecs-1, -2, and -4) and a CD33/Siglec-3-related subgroup (Siglecs-3 and -5-13 in primates), which appear to be rapidly evolving. This article provides an overview of historical and current information about the Siglecs.  相似文献   

12.
We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1 versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K(d) 91 nM), the other two showed weak binding with K(d) values in the 1-2 microM range. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acalpha2-6GalNAcalpha (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.  相似文献   

13.
Here we characterize the properties and expression pattern of Siglec-9 (sialic acid-binding Ig-like lectin-9), a new member of the Siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding Siglec-9 was isolated from a dibutyryl cAMP-treated HL-60 cell cDNA library. Siglec-9 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two putative tyrosine-based signaling motifs. Overall, Siglec-9 is approximately 80% identical in amino acid sequence to Siglec-7, suggesting that the genes encoding these two proteins arose relatively recently by gene duplication. Binding assays showed that, similar to Siglec-7, Siglec-9 recognized sialic acid in either the alpha2,3- or alpha2, 6-glycosidic linkage to galactose. Using a specific mAb, Siglec-9 was found to be expressed at high or intermediate levels by monocytes, neutrophils, and a minor population of CD16(+), CD56(-) cells. Weaker expression was observed on approximately 50% of B cells and NK cells and minor subsets of CD8(+) T cells and CD4(+) T cells. These results show that despite their high degree of sequence similarity, Siglec-7 and Siglec-9 have distinct expression profiles.  相似文献   

14.
Siglecs are vertebrate cell-surface receptors that recognize sialylated glycans. Here we have identified and characterized a novel Siglec, named Siglec-15. Siglec-15 is a type-I transmembrane protein consisting of: (i) two immunoglobulin (Ig)-like domains, (ii) a transmembrane domain containing a lysine residue, and (iii) a short cytoplasmic tail. Siglec-15 is expressed on macrophages and/or dendritic cells of human spleen and lymph nodes. We show that the extracellular domain of Siglec-15 preferentially recognizes the Neu5Acalpha2-6GalNAcalpha- structure. Siglec-15 associates with the activating adaptor proteins DNAX activation protein (DAP)12 and DAP10 via its lysine residue in the transmembrane domain, implying that it functions as an activating signaling molecule. Siglec-15 is the second human Siglec identified to have an activating signaling potential; unlike Siglec-14, however, it does not have an inhibitory counterpart. Orthologs of Siglec-15 are present not only in mammals but also in other branches of vertebrates; in contrast, no other known Siglec expressed in the immune system has been conserved throughout vertebrate evolution. Thus, Siglec-15 probably plays a conserved, regulatory role in the immune system of vertebrates.  相似文献   

15.
Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.  相似文献   

16.
Structural implications of Siglec-5-mediated sialoglycan recognition   总被引:1,自引:0,他引:1  
Sialic acid (Sia) Ig-like binding lectins are important mediators of recognition and signaling events among myeloid cells. To investigate the molecular mechanism underlying sialic acid Ig-like lectin (Siglec) functions, we determined the crystal structure of the two N-terminal extracellular domains of human myeloid cell inhibitory receptor Siglec-5 (CD170) and its complexes with two sialylated carbohydrates. The native structure revealed an unusual conformation of the CC′ ligand specificity loop and a unique interdomain disulfide bond. The α(2,3)- and α(2,6)-sialyllactose complexed structures showed a conserved Sia recognition motif that involves both Arg124 and a portion of the G-strand in the V-set domain forming β-sheet-like hydrogen bonds with the glycerol side chain of the Sia. Only few protein contacts to the subterminal sugars are observed and mediated by the highly variable GG′ linker and CC′ loop. These structural observations, in conjunction with surface plasmon resonance binding assays, provide mechanistic insights into linkage-dependent Siglec carbohydrate recognition and suggest that Siglec-5 and other CD33-related Siglec receptors are more promiscuous in sialoglycan recognition than previously understood.  相似文献   

17.
We recently identified a novel human sialic acid binding immunoglobulin-like lectin, Siglec-8, using mRNA from human eosinophils. To search for a mouse Siglec (mSiglec) ortholog of Siglec-8 and other mouse Siglec paralogs, we conducted public database searches with cDNA sequences of human Siglec-5 to -10 and identified two novel mSiglecs. One has significant sequence identity to human Siglec-5 and is a splice variant of mSiglec-F. The other has greatest sequence identity to human Siglec-10 (mSiglec-G). Both mSiglecs have extracellular Ig-like domains and intracellular tyrosine-based motifs. To determine whether these mSiglecs were relevant to mouse eosinophils, RT-PCR and Northern blot analysis were performed. We detected expression of mSiglec-5 (or -F), -10, and -E mRNA in purified mouse eosinophils, but Northern blot data comparing expression in tissues from normal, IL-5 transgenic, and allergen-sensitized and -challenged mice suggest that mSiglec-10 is probably most relevant to mouse eosinophils.  相似文献   

18.
Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.  相似文献   

19.
While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activation and systemic depletion of CD4+ T cells, simian immunodeficiency virus (SIV) infection in sooty mangabeys or chimpanzees does not exhibit these hallmarks. Control of immune activation is thought to be one of the major components that govern species-dependent differences in the disease pathogenesis. A previous study introduced the idea that the resistance of chimpanzees to SIVcpz infection-induced hyperimmune activation could be the result of the expression of select sialic acid-recognizing immunoglobulin (Ig)-like lectin (Siglec) superfamily members by chimpanzee T cells. Siglecs, which are absent on human T cells, were thought to control levels of T-cell activation in chimpanzees and were thus suggested as a cause for the pathogenic differences in the course of SIVcpz or HIV-1 infection. As in human models of T-cell activation, stimulation had been attempted using an anti-CD3 monoclonal antibody (MAb) (UCHT1; isotype IgG1), but despite efficient binding, UCHT1 failed to activate chimpanzee T cells, an activation block that could be partially overcome by MAb-induced Siglec-5 internalization. We herein demonstrate that anti-CD3 MAb-mediated chimpanzee T-cell activation is a function of the anti-CD3 MAb isotype and is not governed by Siglec expression. While IgG1 anti-CD3 MAbs fail to stimulate chimpanzee T cells, IgG2a anti-CD3 MAbs activate chimpanzee T cells in the absence of Siglec manipulations. Our results thus imply that prior to studying possible differences between human and chimpanzee T-cell activation, a relevant model of chimpanzee T cell activation needs to be established.  相似文献   

20.
Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation.  相似文献   

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