Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Distribution and molecular evolution of rhamnose-binding lectins in Salmonidae: isolation and characterization of two lectins from white-spotted Charr (Salvelinus leucomaenis) eggs

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.     

蓖麻蚕两种凝集素在蚕体内的出现与分布

蓖麻蚕Philosamia cynthia ricni血淋巴含两种凝集素,一种凝集兔新鲜红血球,凝血活力被L-鼠李糖和D-半乳糖抑制;另一种凝集戊二醛固定的人和鸡的红血球,凝血活力被岩藻糖抑制.它们在蚕的不同生长阶段及在蚕体各组织中的分布和凝血活力显著不同.血淋巴中这两种凝集素的凝血活力明显比其他组织中高.卵中测不到这两种凝集素活力.本文对这两种凝集素在蚕体中可能的生理功能进行了讨论.     

Calcium release at fertilization of Xenopus eggs requires type I IP(3) receptors, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.     

Identification and characterization of a novel human collectin CL-K1

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.     

The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases

Human mucus proteinase inhibitor (MPI) consists of 107 amino acids arranged in two domains showing high homology to each other. This protein is an inhibitor of different serine proteinases including trypsin, chymotrypsin, leukocyte elastase and cathepsin G. On the basis of sequence comparisons it has been suggested that the first domain inhibits trypsin, whereas the second one was thought to be active against chymotrypsin and elastase. To prove the location of the different inhibitory activities gene fragments for both domains have been cloned separately and expressed in Escherichia coli. Inhibition assays with the isolated recombinant domains showed that the second domain is active against chymotrypsin, neutrophil elastase and trypsin, whereas for the first domain only a weak activity against trypsin could be detected. These results suggest that the inhibitory activities of the native molecule towards these three proteinases are all located in the second domain.     

基于rhaSR嵌合操纵子的构建及其在大肠杆菌中表达的研究

通过PCR等重组DNA技术,构建了含rhaSR启动子表达调控元件、RhaR基因、报告基因gst(谷胱甘肽-S-转移酶)的两个嵌合操纵子,并插入大肠杆菌表达载体pALEX中构成pALEX-PR1和pALEX-PR2。其中pALEX-PR2的RhaR基因上游为原有的SD序列,而pALEX-PR1的RhaR基因上游则插入了增强的SD序列。把这两个重组表达质粒分别转入大肠杆菌BL21(DE3)中,报告基因gst能够在L-鼠李糖诱导下表达,其表达量是非诱导条件下的4～5倍,且pALEX-PR1的表达量是pALEX-PR2的3.14倍。以上结果表明,gst的表达既受L-鼠李糖诱导,同时又受RhaR的正调控。SDS-PAGE结果显示,GST占大肠杆菌培养物总可溶蛋白的5.41%(W/W),平均1L培养物可获得3.0mg纯化的GST。酶活性分析表明,所构建的嵌合操纵子表达的GST保持了正确的构型且具有很高的活性。     

Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulate human T cell function

Carcinoembryonic Ag-related cellular adhesion molecule 1 (CEACAM1) represents a group of transmembrane protein isoforms that consist of variable numbers of extracellular Ig-like domains together with either a long cytoplasmic (cyt) tail containing two immunoreceptor tyrosine-based inhibitory motifs or a unique short cyt tail. Although CEACAM1 has been reported to be expressed on the surface of T lymphocytes upon activation, its roles in T cell regulation are controversial due to the lack of functional characterization of each individual CEACAM1 isoform. We thus cotransfected Jurkat T cells with CEACAM1 isoform-encoding constructs and an IL-2 promoter-bearing plasmid or a small interference RNA targeting src homology domain 2 containing phosphatase 1. In a luciferase reporter assay and through measurements of cytokine secretion (IL-2, IL-4, and IFN-gamma), CEACAM1 containing either a long or a short cyt tail inhibited or costimulated, respectively, TCR/CD3 complex plus CD28 mediated activation with the inhibitory functions of the long cyt tail dominating. The inhibitory function of CEACAM1, was dependent upon src homology domain 2 containing phosphatase 1 activity, required both tyrosine residues within the immunoreceptor tyrosine-based inhibitory motif domains of the cyt tail and was mediated through the mitogen-activated protein kinase pathway. CEACAM1-mediated inhibition could be functionally reconstituted by incubation of PBMC with either a CEACAM1-specific mAb or CEACAM1-Fc fusion protein in the presence of an allogeneic or mitogenic stimulus, respectively. These studies indicate that the long and short cyt tails of CEACAM1 serve as inhibitory and costimulatory receptors, respectively, in T cell regulation.     

Molecular cloning of MIS, a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and SHP-2

We describe the molecular cloning and characterization of a novel myeloid inhibitory siglec, MIS, that belongs to the family of sialic acid-binding immunoglobulin-like lectins. A full-length MIS cDNA was obtained from murine bone marrow cells. MIS is predicted to contain an extracellular region comprising three immunoglobulin-like domains (V-set amino-terminal domain followed by two C-set domains), a transmembrane domain and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. The closest relative of MIS in the siglec family is human siglec 8. Extracellular regions of these two siglecs share 47% identity at the amino acid level. Southern blot analysis suggests the presence of one MIS gene. MIS is expressed in the spleen, liver, heart, kidney, lung and testis tissues. Several isoforms of MIS protein exist due to the alternative splicing. In a human promonocyte cell line, MIS was able to bind Src homology 2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2. This binding was mediated by the membrane-proximal ITIM of MIS. Moreover, MIS exerted an inhibitory effect on FcgammaRI receptor-induced calcium mobilization. These data suggest that MIS can play an inhibitory role through its ITIM sequences.     

Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.     

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid

The interaction between bacteria and three L-rham-nose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.     

Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8A were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55 degrees C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.     

Identification and expression analysis of three c-type lysozymes in Oreochromis aureus

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.     

Role of the proline-rich domain of dynamin-2 and its interactions with Src homology 3 domains during endocytosis of the AT1 angiotensin receptor

In nonneural tissues, the dynamin-2 isoform participates in the formation of clathrin-coated vesicles during receptor endocytosis. In this study, the mechanism of dynamin-2 action was explored during endocytosis of the G protein-coupled AT1A angiotensin receptor expressed in Chinese hamster ovary cells. Dynamin-2 molecules with mutant pleckstrin homology domains or deleted proline-rich domains (PRD) exerted dominant negative inhibition on the endocytosis of radiolabeled angiotensin II. However, only the PRD mutation interfered with the localization of the dynamin-2 molecule to clathrin-coated pits and reduced the inhibitory effect of the GTPase-deficient K44A mutant dynamin-2. Green fluorescent protein-tagged Src homology 3 (SH3) domains of endophilin I and amphiphysin II, two major binding partners of dynamins, also inhibited AT1A receptor-mediated endocytosis of angiotensin II. These effects were partially or fully, respectively, restored by the overexpression of dynamin-2. Transient overexpression of these SH3 domains also reduced the localization of dynamin-2 to clathrin-coated pits. These data indicate that, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles, interaction of the dynamin-2 PRD with SH3 domains of proteins such as the amphiphysins and endophilins is essential for AT1A receptor endocytosis. This mechanism could be of general importance in dynamin-dependent endocytosis of other G protein-coupled receptors in nonneural tissues.     

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.     

Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg

Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a beta-sheet secondary structure with a melting temperature of 59 degrees C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.     

Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor identified as a strong inhibitor of hepatocyte growth factor (HGF) activator and matriptase. HAI-1 is first produced in a membrane-integrated form with two Kunitz domains in its extracellular region, and subsequent ectodomain shedding releases two major secreted forms, one with a single Kunitz domain and one with two Kunitz domains. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against serine proteases, we constructed various HAI-1 mutant proteins and examined their inhibitory activity against HGF activator and trypsin. The N-terminal Kunitz domain (Kunitz I) had potent inhibitory activity against both HGF activator and trypsin, whereas the C-terminal Kunitz domain (Kunitz II) had only very weak inhibitory activity against HGF activator, although its potency against trypsin was equivalent to that of Kunitz I. These results indicate that Kunitz I is the functional domain of HAI-1 for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator, and it showed a similar, but not additive, level of inhibitory activity against trypsin when compared with that of the individual Kunitz domains. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other and that proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1.     

N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains

We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-ΔC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-ΔN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-ΔC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-ΔN and Sos1-ΔC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.     

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.