Domain and Functional Analysis of a Novel Platelet-Endothelial Cell Surface Protein, SCUBE1

Abstract

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1 ⌬cub/⌬cub ) to investigate the domain function and biological significance. A number of cellbased assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGFlike repeats 7-9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1 ⌬cub/⌬cub embryos.Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is the founding member of an evolutionarily conserved SCUBE gene family (1, 2). To date, three distinct isoforms have been cloned and named SCUBE1 to SCUBE3 according to their order of discovery in mammals (1-4). These genes coding for polypeptide molecules of about 1000 amino acids share an organized protein domain structure of at least 5 recognizable motifs: an N-terminal signal peptide sequence, 9 tandem repeats of epidermal growth factor (EGF) 3 -like modules, a large N-glycosylated spacer region followed by three repeated stretches of 6-cysteine residues with unique and regular spacing, and one CUB domain at the C terminus (see Fig. 1A). Our previous study demonstrated that the signal peptide sequence is sufficient to direct the expressed SCUBE1 protein into the secretory pathway and results in a secreted and surface-associated protein (1). However, little is known about the functional significance of the other four domains in SCUBE1.SCUBE genes have been shown to be expressed predominantly in a variety of developing tissues, including gonads, the central nervous system, dermomyotome, digital mesenchyme, and limb buds during mouse embryogenesis (2, 3, 5), which implies that the proteins may play important roles in development. However, direct functional studies of the roles of the SCUBE genes during mammalian development are still lacking. In addition to its embryonic expression, SCUBE1 was found to be expressed in the endothelium and platelets (1, 6). Our recent study showed that SCUBE1 is stored in the platelet ␣-granules and exposed to the cell surface upon platel...

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…Neto2 is a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein abundantly expressed in neurons (12), which is important for proper neurological function (13). CUB domains are evolutionarily conserved protein domains (14) that participate in protein-protein interactions (15,16). In the present study, we performed an unbiased proteomic screen and discovered that Neto2 interacts with KCC2.…”

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s, Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

“…Because of the large and complex genomic structure of the mouse Scube1 gene, designing a targeting strategy for generating a null allele is difficult. Indeed, our previous targeting study disrupted only the COOH-terminal CUB domain of mouse Scube1 (mutant scube1 ⌬cub allele), which resulted in brain malformation and perinatal lethality (34). Although this gene-targeting study clearly demonstrated the role of Scube1 during early stages of central nervous system development, its function in primitive hematopoiesis was not obvious and not thoroughly examined (34).…”

“…Indeed, our previous targeting study disrupted only the COOH-terminal CUB domain of mouse Scube1 (mutant scube1 ⌬cub allele), which resulted in brain malformation and perinatal lethality (34). Although this gene-targeting study clearly demonstrated the role of Scube1 during early stages of central nervous system development, its function in primitive hematopoiesis was not obvious and not thoroughly examined (34). Given the essential role of Scube1 in primitive hematopoiesis in zebrafish, we are interested in producing a conditional null allele to further explore the biological functions of Scube1 in mammalian hematopoiesis or other developmental processes.…”

“…The FLAG or Myc epitope-tagged version of zebrafish Scube1 expression plasmid was constructed in the pFLAG-CMV1 (Sigma) or pSecTag2 (Invitrogen) as described (4). The expression plasmids coding for Myctagged BMP2 or BMP4 were prepared as described (34).…”

“…Morphants-Because human SCUBE1 or SCUBE2 have been implicated in regulating the long range signaling of BMP (3,34) and because BMP signaling is important for mesoderm patterning and specifying the blood fate (19,21,38), we then investigated the genetic relationship between scube1 and bmp genes by co-injection of bmp2b or bmp4 mRNA with scube1 tMO1 in embryos. Overexpression of bmp2b or bmp4 restored scl expression in scube1 morphants (Fig.…”