Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form

Abstract

Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action.

Emilin3 is required for notochord sheath integrity and interacts with Scube2 to regulate notochord-derived Hedgehog signals

“…Scube2 is a multi-domain protein, with a signal peptide sequence at the N-terminal end followed by nine EGF repeats, one spacer region, three cysteine-rich motifs and one CUB domain at the Cterminal end. The CUB domain was found to interact with Shh and its presence is required for the activity of Scube2 on Shh release (Creanga et al, 2012;Tukachinsky et al, 2012). Our finding that Emilin3 is able to interact with the EGF repeats of Scube2 supports the hypothesis that this region of the protein may be important for targeting Scube2 to the ECM and fine-tuning its localization and activity in the extracellular milieu.…”

“…We show that Emilin3 interacts with Scube2 in the extracellular environment. Recently, Scube2 has been identified as a secreted, permissive factor, acting non-cell autonomously in the release of lipidated Shh from producing cells (Creanga et al, 2012;Tukachinsky et al, 2012). Scube2 is a multi-domain protein, with a signal peptide sequence at the N-terminal end followed by nine EGF repeats, one spacer region, three cysteine-rich motifs and one CUB domain at the Cterminal end.…”

“…Recent studies demonstrated a role for the secreted protein Scube2 in Shh release (Creanga et al, 2012;Tukachinsky et al, 2012). We therefore investigated whether Emilin3 could interact with Scube2.…”

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans

“…6A, left, asterisk). Another soluble protein fraction represented by an upper medium band also lacked the radiolabel, demonstrating that this fraction does not simply represent cellular (lipidated) material that was leaked into the medium or released by some other means (Creanga et al, 2012;Palm et al, 2013). Instead, most of these proteins represent Shh that escaped N-palmitoylation during biosynthesis and were therefore released in an N-terminally unprocessed (but C-terminally processed) form.…”

“…Media were harvested after another 24 h and processed. Based on the established role of Scube2 glycoproteins (signal sequence, cubulin domain, epidermal growth factor 2) in Hh release from 293 T cells (Tukachinsky et al, 2012), HEK293S cells (Creanga et al, 2012), and Bosc23 cells (Jakobs et al, 2014), Scube2-conditioned medium was used in all Shh release experiments.…”