title: Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.
abstract: SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish.
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Wei-Ju
authors.initials: WJ
authors.lastname: Liao
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan Molecular Medicine Graduate Program, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Ku-Chi
authors.initials: KC
authors.lastname: Tsao
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan Molecular Medicine Graduate Program, Academia Sinica, Taipei 115, Taiwan Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan rbyang@ibms.sinica.edu.tw.
authors.forename: Ruey-Bing
authors.initials: RB
authors.lastname: Yang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006410:Hematopoiesis; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D055672:Static Electricity; D015027:Zebrafish / Q000097:blood
chemical_list: D019485:Bone Morphogenetic Proteins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009844:Oligosaccharides; C469998:SCUBE1 protein, human
keywords: N-glycosylation; SCUBE1; bone morphogenetic protein; electrostatic interaction; peripheral membrane protein; signalling