Tmtsp, a Novel Gene Encoding a Cell Surface Protein Highly Specific to Hematopoietic Stem and Endothelial Cells.

In the adult mouse bone marrow, hematopoietic stem cells (HSC) are highly enriched in the CD34 (−/low), c-Kit (+), Sca-1 (+), and lineage marker (−) (CD34 (−) KSL cell population. Using in silico data base search against NCBI EST database, we identified a novel cell surface molecules specific to CD34 (−) KSL HSCs. We cloned its full-length cDNAs of both mouse and human counterparts from total embryo (E15) and human spleen cDNAs, respectively. They were 2815-bp and 2953-bp long and the deduced amino acid sequences consisted of 851 and 852 amino acids, respectively. They contain a signal sequence, three Ig-like domains, a Thrombospondin type 1 (TSP1) domain, and a transmembrane region. Its domain composition shows high similarity to that of Unc5h3, a receptor for netrin, which plays a key role in neuronal axon guidance. Therefore, we designated this gene as Transmembrane Molecule with Thrombospondin Module (Tmtsp). BLAST search against the mouse genome database displayed that the mouse Tmtsp is composed of four exons and spanning over 26kbp on the chromosome 8qA2. Tmtsp was translated into a 170 kDa transmembrane protein. An alternative splicing which splices out the 3rd exon was detected in all major organs tested, leading to in-frame translation of a variant protein without TSP1 domain. RT-PCR analysis of hematopoietic cells demonstrated that Tmtsp is confined to KSL cells in expression and shows even higher expression in CD34 (−) KSL HSCs. On the other hand, Northern blot analysis detected its expression in all major organs tested and the highest expression in the lung. To specify the cells that express Tmtsp, we generated monoclonal antibodies against the extracellular domain of Tmtsp. Immunohistochemical analysis using anti-Tmtsp monoclonal antibodies revealed that its expression is tightly restricted to endothelial cells in tissues. These findings indicate that Tmtsp is a novel cell surface transmembrane molecule specific to both hematopoietic stem/progenitor cells and endothelial cells. Among a list of molecules shared by HSC and endothelial cells, Tmtsp is the first one that possesses the TSP1 domain implicated in cell adhesion. In this context, structure and expression of Tmtsp strongly suggest important roles in the development and maintenance of HSCs and endothelial cells including HSC homing and formation of vascular network.