A novel member of mitochondrial carrier superfamily has been identified from human bone marrow stromal cells (BMSC) and designated as human BMSC-derived mitochondrial carrier protein (HuBMSC-MCP). It encodes a 321 amino-acid protein with three tandem related domains of about 100 amino acids. Each domain contains two hydrophobic stretches, which are thought to span the membrane as a-helices. Distant relationship analysis indicates that the protein is highly conserved between species from Caenorhabditis elegans to human. HuBMSC-MCP gene is mapped to chromosome 11p11. HuBMSC-MCP mRNA expression is detectable in various human tissues and cell lines. By confocal imaging, HuBMSC-MCP is localized to mitochondria and also detected in the pseudopodial protrusion of human breast adenocarcinoma MCF-7 cells. When transfected into dendritic cells (DC), HuBMSC-MCP could enhance DCs endocytotic capacity. Thus, HuBMSC-MCP is a phylogenetically conserved and widely expressed mitochondrial carrier protein which perhaps associates with mitochondrial oxidative phosphorylation.

Trypsin-like serine proteases are involved in diverse biological processes such as complement activation, tissue remodeling, cellular migration, tumor invasion, and metastasis. Here we report a novel human C1r-like serine protease analog, CLSPa, derived from dendritic cells (DC). The 487-residue CLSPa protein contains a CUB domain and a serine protease domain, possessing characteristic catalytic triad but lacking typical activation/cleavage sequence. It shares great homology with complement C1r/C1s and mannoseassociated serine proteases. CLSPa mRNA is widely expressed, especially abundant in placenta, liver, kidney, pancreas, and myeloid cells, which are a major resources of serine proteases. Upon stimulation by agonistic anti-CD40 Ab, TNF-a, or LPS, CLSPa mRNA expression was significantly up-regulated in monocytic cells and monocyte-derived immature DC. When overexpressed in 293T cells, CLSPa protein was synthesized into the culture supernatants as a secretory protein, which had an inhibitory effect on complementmediated cytotoxicity to antibody-sensitized erythrocytes. However, CLSPa itself possesses little protease activity, but it plays an inhibitory role in other active protease catalytic processes. The identification of human CLSPa as a novel C1r-like protein might facilitate future investigation of the regulatory mechanism of CLSPa in complement pathways during inflammation.

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2ab, hence is designated as MIP-2g. Mouse MIP-2g was identified by electrocloning and is highly homologous to human MIP-2g. Northern blotting revealed that MIP-2g was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2g was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2g expression by monocyte-derived DC. Recombinant MIP-2g from 293 cells is about 9.5kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2g is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2g does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251 bp with an open reading frame (ORF) of 636 bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.

Purpose: To identify the characteristics and function of a cadherin-like molecule, cloned from a human dendritic cell (DC) cDNA library and designated DC-derived cadherin-like molecule (DC-CLM). Methods: The mRNA expression of DC-CLM in tissues and cells was analyzed by Northern blot and RT-PCR, respectively. In order to express DC-CLM in target cells, we constructed a pcDNA3.1/DC-CLM expression vector and transfected it into MCF-7 human breast cancer cells. Tumor growth was demonstrated by cell proliferation and colony formation. Results: DC-CLM cDNA encoded a protein of 260 amino acids and the gene was localized to chromosome 5q31. The predicted protein possessed a definitive cadherin-specific sequence motif and shared homology with classical cadherin. However, no transmembrane segment was observed in DC-CLM. Northern blot revealed the ubiquitous nature of DCCLM transcripts in human tissues, with high expression in heart, brain, prostate, testis and ovary. RT-PCR demonstrated that DC-CLM was widely expressed in hematopoietic and epithelial tumor cell lines, but was not expressed in MCF-7. Interestingly, DC-CLM expression was upregulated in DC activated by lipopolysaccharides. DC-CLM expression in the stable transfectant (MCF-7/DC-CLM) was confirmed by RTPCR and Western blot. DC-CLM protein was found to be secreted by MCF-7/DC-CLM but not expressed on the membrane of MCF-7/DC-CLM. DC-CLM transfection resulted in significant inhibition of in vitro growth and colony formation of MCF-7 cells. Conclusions: A cadherin-like molecule DC-CLM was cloned from human DC and it may be a soluble cadherin-like molecule for tumor suppression. DC-CLM was upregulated in activated DC and may be involved in the effector function of activated DC.

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit antitumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1β, tumor necrosis factor-α(TNF-α), and the chemokines IP-10, macrophage inflammatory protein-1α(MIP-1α), MIP-1β, and normal Tcell expressed and secreted (RANTES). The induction of interferon-γ-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbu min(OVA)257-264 induces an OVA257-264-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVAtumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

The cystatins are physiological cysteine proteinase inhibitors. Here we report the cloning of a novel human cystatin-like molecule (CLM) from human bone marrow stromal cell (BMSC) cDNA library. The putative CLM protein contained 159 residues with a 29-residue signal peptide. CLM protein was highly homologous to family 2 cystatins, especially mouse and human testatin. The CLM gene spanned two exons and was mapped on chromosome 20p11.2, among cystatin superfamily gene clusters. CLM mRNA was barely detected in most tumor cell lines except for breast adenocarcinoma MCF-7 cells and glioblastoma U251 cells, but after LPS or PMA stimulation, CLM expression was increased in myelogenous leukemia cell lines HL-60 and U-937. Northern blot analysis revealed CLM was ubiquitously expressed in normal tissues, which was clearly different from the testis-specific expression pattern of most family 2 cystatins. When overexpressed in 293 cells, GFP-fused CLM targeted extracellularly through secretory pathway by Golgi apparatus. The results indicated that the secreted CLM protein might play roles in hematopoietic differentiation or inflammation.

Several ubiquitin-like proteins recently discovered have been confirmed to modify proteins akin to ubiquitinization for fineregulation of intracellular proteins. In the present study, we report a novel ubiquitin-like protein from human dendritic cells (DC), named as dendritic cell-derived ubiquitin-like protein (DC-UbP). The full-length of DC-UbP cDNA is 565 bp and encodes 106 amino acids. Ubiquitin domain (UBQ) in DC-UbP shares 28.6% identity and 55% similarity to ubiquitin, but does not possess the conserved C-terminus Gly-Gly of ubiquitin required for ubiquitinization. DC-UbP localized in cytoplasm, especially in mitochondrion, indicating that it may play a role in mitochondrial biology. DC-UbP mRNA was expressed in various tumor cells, but not in adult human normal tissues, suggesting that DC-UbP might be related to tumor genesis. In addition, DC-UbP mRNA expression decreased in the HL60 cells undergoing apoptosis after being stimulated with TRAIL and in the differentiated HL60 cells induced by ARTA. Taken together, DC-UbP might be downregulated during cellular differentiation and apoptosis.

Septins are a family of cytoskeletal proteins with GTPase activity involved in various cellular biological processes. Here, we describe the identification of septin 10, a novel septin family member from human dendritic cells. The 3018-bp full-length cDNA potentially encodes a 517-residue peptide, sharing closest homology to human septin 8 and septin 6. With a conserved GTP-binding motif at its N-terminus, septin 10 protein can bind to GTP and exert GTPase activity. Human septin 10 gene is electronically mapped to 8q11.2-12. It is ubiquitously expressed in normal tissues, with the abundant expression in heart and kidney, lacenta, skeletal muscles, liver and lung, as well as various tumor cell lines. Interestingly, dendritic cells express upregulated septin 10 upon LPS-induced maturation. Based on its GFP-fusion protein, septin 10 is found to localize in cytoplasm and nucleus, with a subcellularpatter n independent of the filamentous state of actin.

Ubiquitin is one of phylogenetically well-conserved proteins in all eukaryotes. Ubiquitin-dependent modification of protein contributes to fine regulation of cellular biological processes. Using large-scale screening of human bone marrow stromal cell (BMSC) cDNA library, we isolated a full-length cDNA of 1352 bp encoding 380 amino acids with a ubiquitin domain (UBQ), which was designed as bone marrow stromal cell-derived ubiquitin-like protein (BMSC-UbP). In addition to UBQ domain at its Nterminus, BMSC-UbP also possesses a ubiquitin-associated domain at its C-terminus, sharing moderate homology to some ubiquitin-like proteins such as UBIN, Chap1, and ubiquilin. BMSC-UbP localizes at chromosome 15q22.3-q23 as confirmed by blast search in human genome. BMSC-UbP mRNA is widely expressed in human multiple tissues and various tumor cell lines. Moreover, BMSC-UbP mRNA decreased in BMSC stimulated with PMA and increased in HL60 cells stimulated with LPS, suggesting that BMSC-UbP might play roles in regulation of BMSC function or cell differentiation through an evocator- and cellspecific pattern.