Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways

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.     

Cloning and characterization of a novel mouse Siglec, mSiglec-F: differential evolution of the mouse and human (CD33) Siglec-3-related gene clusters

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.     

Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants.

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.     

Cloning and molecular characterization of two splice variants of a new putative member of the Siglec-3-like subgroup of Siglecs.

The sialic acid binding immunoglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within this Siglec family there exists a subgroup of molecules which bear a very high degree of homology with the molecule Siglec-3 (CD33), and has thus been designated the Siglec-3-like subgroup of Siglecs. The members of this subgroup have been localized to chromosome 19q13.4, through both in situ hybridization and precise genomic mapping at the nucleotide level. Through the positional cloning approach we have identified and characterized a Siglec-like gene (SLG), a putative novel member of the Siglec-3-like subgroup of Siglecs. We have characterized the complete genomic structure of SLG, as well as two alternative splice variants, and determined its chromosomal localization. The short isoform, SLG-S, consists of seven exons, with six intervening introns, while the longer isoform, SLG-L, consists of eight exons and seven intervening introns. The SLG gene is localized 32.9 kb downstream of Siglec-8 on chromosome 19q13.4. The putative SLG-S and SLG-L proteins, of 477 and 595 amino acid residues, respectively, show extensive homology to many members of the Siglec-3-like subgroup. This high degree of homology is conserved in the extracellular Ig-like domains, as well as in the cytoplasmic tyrosine-based motifs. Interestingly, the SLG-L protein contains two N-terminal V-set Ig-like domains, as opposed to SLG-S and other Siglec-3-like subgroup members which contain only one such domain. Through RT-PCR we have examined the expression profile of both SLG splice variants in a panel of human tissues and have found that SLG-S is highly expressed in spleen, small intestine and adrenal gland, while SLG-L exhibits high levels of expression in spleen, small intestine, and bone marrow. This gene is quite likely 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 Siglec7.     

Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells

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.     

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

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.     

The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by Siglecs-7 and -9, CD33-related Siglecs expressed on human monocytes and NK cells

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.     

Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation

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.     

Molecular characterization of a Siglec8 variant containing cytoplasmic tyrosine-based motifs, and mapping of the Siglec8 gene

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.     

SOCS3 targets Siglec 7 for proteasomal degradation and blocks Siglec 7-mediated responses

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.     

Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily

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.     

Siglecs--the major subfamily of I-type lectins

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.     

The myeloid-specific sialic acid-binding receptor, CD33, associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2

The myeloid restricted membrane glycoprotein, CD33, is a member of the recently characterized "sialic acid-binding immunoglobulin-related lectin" family. Although CD33 can mediate sialic acid-dependent cell interactions as a recombinant protein, its function in myeloid cells has yet to be determined. Since CD33 contains two potential immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail, we investigated whether it might act as a signaling receptor in myeloid cells. Tyrosine phosphorylation of CD33 in myeloid cell lines was stimulated by cell surface cross-linking or by pervanadate, and inhibited by PP2, a specific inhibitor of Src family tyrosine kinases. Phosphorylated CD33 recruited both the protein-tyrosine phosphatases, SHP-1 and SHP-2. CD33 was dephosphorylated in vitro by the co-immunoprecipitated tyrosine phosphatases, suggesting that it might also be an in vivo substrate. The first CD33 phosphotyrosine motif is dominant in CD33-SHP-1/SHP-2 interactions, since mutating tyrosine 340 in a CD33-cytoplasmic tail fusion protein significantly reduced binding to SHP-1 and SHP-2 in THP-1 lysates, while mutation of tyrosine 358 had no effect. Furthermore, the NH2-terminal Src homology 2 domain of SHP-1 and SHP-2, believed to be essential for phosphatase activation, selectively bound a CD33 phosphopeptide containing tyrosine 340 but not one containing tyrosine 358. Finally, mutation of tyrosine 340 increased red blood cell binding by CD33 expressed in COS cells. Hence, CD33 signaling through selective recruitment of SHP-1/SHP-2 may modulate its ligand(s) binding activity.     

CD33/Siglec-3 binding specificity,expression pattern,and consequences of gene deletion in mice

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to alpha2-3- or alpha2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.     

Siglec-15: an immune system Siglec conserved throughout vertebrate evolution

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.     

Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes

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.     

A uniquely human consequence of domain-specific functional adaptation in a sialic acid-binding receptor

Most mammalian cell surfaces display two major sialic acids (Sias), N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans lack Neu5Gc due to a mutation in CMP-Neu5Ac hydroxylase, which occurred after evolutionary divergence from great apes. We describe an apparent consequence of human Neu5Gc loss: domain-specific functional adaptation of Siglec-9, a member of the family of sialic acid-binding receptors of innate immune cells designated the CD33-related Siglecs (CD33rSiglecs). Binding studies on recombinant human Siglec-9 show recognition of both Neu5Ac and Neu5Gc. In striking contrast, chimpanzee and gorilla Siglec-9 strongly prefer binding Neu5Gc. Simultaneous probing of multiple endogenous CD33rSiglecs on circulating blood cells of human, chimp, or gorilla suggests that the binding differences observed for Siglec-9 are representative of multiple CD33rSiglecs. We conclude that Neu5Ac-binding ability of at least some human CD33rSiglecs is a derived state selected for following loss of Neu5Gc in the hominid lineage. These data also indicate that endogenous Sias (rather than surface Sias of bacterial pathogens) are the functional ligands of CD33rSiglecs and suggest that the endogenous Sia landscape is the major factor directing evolution of CD33rSiglec binding specificity. Exon-1-encoded Sia-recognizing domains of human and ape Siglec-9 share only approximately 93-95% amino acid identity. In contrast, the immediately adjacent intron and exon 2 have the approximately 98-100% identity typically observed among these species. Together, our findings suggest ongoing adaptive evolution specific to the Sia-binding domain, possibly of an episodic nature. Such domain-specific divergences should also be considered in upcoming comparisons of human and chimpanzee genomes.     

Cloning of two new splice variants of Siglec-10 and mapping of the interaction between Siglec-10 and SHP-1

Using a three-hybrid strategy in yeast, we have cloned a new splice variant of Siglec-10, called Siglec-10 Sv3. This splice variant lacks part of exon 3, but keeps the reading frame, as well as the crucial regions for interaction with Sias and the motifs for intracellular signaling. The expression of Siglec-10 Sv3 in T- and B-cells was detected by RT-PCR. Moreover, cDNA of another new splicing form of Siglec-10, named Siglec-10 Sv4, was identified by RT-PCR. One common characteristic of all Siglec-10 splice forms (except for Siglec-10 Sv2) is their cytoplasmic tail with two ITIMs and one CD150-like sequence. We confirmed the recruitment of SHP-1 to the Siglec-10 cytoplasmic tail by Western blot analysis and demonstrated that this interaction depends on tyrosine phosphorylation. Mutational analyses showed that ITIM Y609 of Siglec-10 and the N-terminal SH2 domain of SHP-1 play a pivotal role in the interaction between Siglec-10 and SHP-1. Finally, we demonstrated that Siglec-10 was not able to bind SAP/SH2d1A, indicating that the so-called CD150-like motif in Siglec-10 might be a docking site for other signal transduction mediators.