Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

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. * ALM, : anterior lateral mesoderm; BMP, : bone morphogenetic protein; CR, : cysteine-rich; CUB, : complement protein C1r/C1s, Uegf and Bmp1; EGF, : epidermal growth factor; FL, : full-length; HEK, : human embryonic kidney; ICM, : intermediate cell mass; ISH, : in situ hybridization; MO, : morpholino oligonucleotide; PLM, : posterior lateral mesoderm; PNGase F, : peptide N -glycosidase F; S1, : SCUBE1 (signal peptide/CUB/EGF domain-containing protein 1); tMO, : translation-blocking morpholino; WT, : wild-type