| Dendritic cells (DC) are the most potent professional antigen-presenting cells (APC), required for the initiation of immune response. Bone marrow stromal cells (BMSC) are important component of hematopoietic microenviroment, providing the structural and physiological support for hematopoietic cells. For all that the mechanisms of DC and BMSC functions are not fully elucidated. Our studies are aiming at obtaining information about important molecules involved in DC and BMSC functions by focusing on the cloning of novel genes expressed in DC and BMSC so as to understand molecular mechanisms underlying the unique capacity of DC and BMSC. Part I. Cloning and characterization of Siglec-10, novel member of Siglec superfamily, from human dendritic cellsThe Siglec (sialic acid-binding Ig-like lectin) family is a subset of structurally related Ig superfamily (IgSF) proteins, specifically recognizing sialic acid in oligosaccharide chains of glycoproteins and glycolipids, which mediate protein-carbohydrate interactions.We detailed here a novel member of Siglec family, Sigelc-10, isolated from the DC cDNA library by large-scale random sequencing. Full length of Sigelc-10 contained 1453 bp, with a methionine codon situated in a consensus Kozak sequence as start codon and a TGA as stop codon. The full ORF encoded a 602-amino acid protein. At the primary sequence level, it shared high homology with Siglec family members, especially most closely resembled Siglec-5 (overall sequence 51% similarity and 38% identity). The gene had submitted to GenBank/EMBL (GenBank Accession Number AF311905) and applied for national invention patent (Patent Application No. 01105501.4). The full-length Siglec-10 cDNA encodes a type I transmembrane protein, containing four extracellular immunoglobulin (Ig) -like domains, a 28-amino acid transmembrane region and a 123-amino acid cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs (ITIM). The amino-terminal V-set Ig domain has most of the amino acid residues typical of Siglecs. Preciously demonstrated conservation of the unusual pattern of cysteines was found in Siglec-10, which are thought to result in intrasheet and interdomain disulfide bonds. Conversation of other key amino acids included a typically placed arginine (R119). Also two aromatic amino acids (F21 and F130) on the A and G strands of the V-set domain were present, which are known to be involved in sialic acid binding. Within the putative cytoplasmic tail there were four tyrosine residues, of which the first (Y502) and the third (Y572) have been identified in the immunoreceptor tyrosine-based inhibitory motif (ITIM: S/I/L/VxYxxL/V). Siglec-10 gene was mapped in Chromosome 19q13.3, along with other Siglec member (Siglec-3,-5,-6,-7,-8 and -9) genes mapped in 19q13.3-13.4. Phylogenetic analysis of entire Siglec family revealed a close association between Siglec-10, Siglec-3/CD33, Siglec-6 and Siglec-5, indicating that there have been multiple gene duplications of the Siglec family genes during evolution from a common ancestral gene.By RT-PCR, we have demonstrated the mRNA expression of Siglec-10 in a large number of cells, including HL-60, U-937, THP-1, MOLT-4, Raji, MCF7, PC-3, A172, SMMC 7721, HeLa and A549. PBMC-derived DC expressed Siglec-10 at relatively high levels, and the expression decreased after the stimulation with LPS, reaching a minimum at 24hr, and increased again to a high level after 48hr of stimulation. Expression of Siglec-10 decreased in HL-60 and U-937 cells after PMA stimulation, but increased first and decreased later in K-562 cells after LPS or PMA stimulation. The mRNA expression pattern of Siglec-10 in human tissues was examined by Northern blot analysis. Two major forms of transcripts were observed. The larger one was about 3.3 kb in length and was found in PBL, ovary and spleen; The other transcript was estimated to be about 1.8 kb in length and was present in liver, spleen, thymus and lung, etc. GFP-Siglec-10 fusion proteinwas transiently express...
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