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

Cheng-Fen Tu,Yu-Ting Yan,Szu-Yao Wu,Bambang Djoko,Ming-Tzu Tsai,Chien-Jui Cheng,Ruey-Bing Yang

doi:10.1074/jbc.m705872200

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(Delta cub/Delta cub)) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like 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(Delta cub/Delta cub) embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.

Highlights

SCUBE1 is the founding member of an evolutionarily conserved SCUBE gene family [1, 2]
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
Molecular Dissection of the SCUBE1 Domain Responsible for Secretion and Cell Surface Association—We previously showed that overexpression of SCUBE1 in human embryonic kidney (HEK)-293T cells resulted in a secreted glycoprotein tethered on the cell surface [1]

Summary

Introduction

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is the founding member of an evolutionarily conserved SCUBE gene family [1, 2]. To delineate the domain required for the secretion and cell surface anchorage, we generated a comprehensive panel of mutated SCUBE1 expression constructs (D1–D7) with specific deletions of the EGF-like repeats, spacer region, cysteine-rich repeats, or CUB domain (Fig. 1A).

Results

Conclusion