Molecular and cellular characterization of SEL‐OB/SVEP1 in osteogenic cells in vivo and in vitro

Abstract

We describe a novel human gene, named SEL-OB/SVEP1, expressed by skeletal tissues in vivo and by cultured osteogenic cells. The mRNA expression was analyzed on frozen tissues retrieved by laser-capture microscope dissection (LCM) and was detected in osteogenic tissues (periosteum and bone) but not in cartilage or skeletal muscle. The SEL-OB/SVEP1 cDNA of 11,139 bp was in silico translated into a 3574AA protein with expected molecular weight of 370 kDa. The protein is composed of multiple domains including complement control protein (CCP) modules with selectin superfamily signature; sushi and other domains, such as vWA, EGF, PTX, and HYR. Stromal osteogenic cells were analyzed for the protein expression using anti-SEL-OB/SVEP1 for immuno-precipitation and Western blot application confirm the presence of high molecular weight protein. Immuno-histochemistry and fluorescence-activated cell sorting (FACS) were applied to detect SEL-OB/SVEP1 on the surface of stromal cells. ELISA quantified the dependence of protein expression on cell density. Bioinformatic analysis of SEL-OB/SVEP1 revealed domains compositions recognized in cell surface molecules and suggested its role in cell adhesion. Analysis of mesechymal osteogenic cells' adhesion in presence of anti-SEL-OB/SVEP1 antibody demonstrated its interference with initial adhesion stages. In summary, present study describes novel SEL-OB/SVEP1 protein with a unique composition of functional domains, restricted pattern of expression in skeletal cells and demonstrated involvement in attachment of mesenchymal cells. The unusual composition of functional domains puts SEL-OB/SVEP1 in the discrete new group of membrane proteins involved in cell adhesion processes. All together makes SEL-OB/SVEP1 an attractive marker for studying the role of stromal osteogenic cells and their interactions within the bone marrow microenvironment creating a network that regulates the skeletal homeostasis.

The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6−/− mice

“…However, 9 of these genes are potential candidates because of their documented or implied association with thrombosis or their expression by cells of the vascular bed. These are the genes for: the ␣ chain of type XV collagen (Cofa1; 47.2 Mb) 22 ; transforming growth factor ␤ receptor I (Tgfbr1; 47.4 Mb) 23 ; ATP-binding cassette subfamily A member 1 (Abca1; 53.1 Mb) 24 ; Klf4 (55.5 Mb), which encodes Kruppel-like factor 4 (Klf4), also known as gut-enriched Kruppel-like factor (GKLF) or epithelial zinc-finger protein (EZF) [25][26][27][28] ; ␣-catulin (␣-catenin-related protein) (Ctnnal1; 56.9 Mb) 29 ; a sushi-, von Willebrand factor type A-, and EGF-domain bearing protein (Svep1; 58.2 Mb) 30 ; the ␣ chain of type XXVII collagen (Col27a1; 62.7 Mb) 31 ; tenascin C (Tnc; 63.5 Mb) 32 ; and toll-like receptor 4 (Tlr4; 66.3 Mb). 33 To further refine the Mh locus, we are pursuing additional selected intercrosses to identify meaningful recombinant offspring.…”

Polydom/SVEP1 Is a Ligand for Integrin α9β1

“…Polydom comprises multiple domains characteristic of ECM proteins (i.e. a von Willebrand factor A domain, a pentraxin domain, 10 EGF-like domains, and 34 CCP (also called "sushi") domains) (19,20) (Fig. 1A).…”

“…We extended this protocol to the Ensembl mouse and human genome databases to identify more ECM proteins by focusing on transcripts encoding proteins with Ͼ1500 amino acid residues. One of the obtained candidates was polydom (also named SVEP1), a putative secreted protein harboring EGF domains with strong similarities to those in the Notch family proteins (19,20). Polydom comprises multiple domains characteristic of ECM proteins (i.e.…”

“…Polydom was originally identified in murine bone marrow stromal cells as a protein containing EGFlike domains that share strong similarities with those in the Notch family proteins (19). The human orthologue of polydom, named SVEP1/SEL-OB, was reported as a protein expressed on the surface of osteogenic cells (20). We encountered polydom in our in silico screening for functionally unknown ECM proteins (21).…”

Biochemical Characterization of Major Bone-Matrix Proteins Using Nanoscale-Size Bone Samples and Proteomics Methodology

“…Originally, LCM was developed for dissection of soft biological materials (21) such as a specific group of cultured osteobalsts (22), cells from mice intervertebral discs (23), odontoblasts from biodegradable polymer scaffolds (24), cranial neural crest mesenchyme (25), dental follicle, and surrounding mandibular bone of newborn rats (26). Notably, a skeletal tissue of newborn mice (27) was also dissected using LCM. However, skeletal tissue of newborn mice is known to be not fully mineralized up to 3-4 weeks after birth (28,29), and thus, cannot be compared with laser microdissection of a cortical bone of an adult human.…”