Scube2 enhances proteolytic Shh processing from the surface of Shh-producing cells

Abstract

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N-and Cterminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.

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

“…Cell-surfaceassociated HA-tagged Shh C25A was converted into a C-terminally processed, but N-terminally unprocessed soluble form (yellow band), showing that the N-terminal processing of unpalmitoylated morphogens was impaired (Ohlig et al, 2012). Consistent with our previous observation that Shh palmitoylation in vitro is incomplete (Jakobs et al, 2014), Shh processing in CHO-K1 cells was strongly enhanced after stable hhat transfection (CHO-K1 tg cells, Fig. 2B, asterisk).…”

“…One attractive idea is that not only do Gpcs serve as platforms for Hh assembly and storage, but they also associate with Hh release factors, such as Hh sheddases (Dierker et al, 2009) or the sheddase activator Scube2 (Jakobs et al, 2014) (Fig. 8).…”

“…We and others have shown that proteolytic removal of N-and C-terminal lipidated peptides (called shedding) releases bioactive Shh morphogens from the surface of producing cells in vitro (Damhofer et al, 2015;Dierker et al, 2009;Jakobs et al, 2014;Ohlig et al, 2011). We further showed that because of the lack of the N-terminal Hhat acceptor cysteine (C25S or C25A in mouse Shh) (Hardy and Resh, 2012) non-palmitoylated Hh proteins require only C-terminal shedding for their solubilization, whereas most Shh N-termini are left intact (Jakobs et al, 2014). Here, we confirm this observation by SDS-PAGE and immunoblotting of N-palmitoylated Shh and .…”

“…2A), a HEK293-derived cell line commonly used to analyze Shh production and release. To monitor C-terminal Shh processing, we tagged both proteins with hemagglutinin (HA), resulting in the extended C-terminal membrane anchor N 190 SVAAKSG-YPYDVPDYA-G 198 (G198 represents the cholesterol-modified glycine; underlined italicized letters represent the tag) (Jakobs et al, 2014). To confirm N-terminal processing during release, we used anti-Cardin-Weintraub antibodies raised against the CardinWeintraub peptide K 33 RRHPKK 39 located adjacent to the palmitoylated cysteine residue (Ohlig et al, 2012).…”

“…However, because redundancy in enzymes and substrates is common (Kheradmand and Werb, 2002), activities and colocalization of sheddases with their substrate(s) requires tight regulation. One recently described Hh shedding regulator is the glycoprotein Scube2 (signal peptide, cubulin domain, epidermal growth factor-like protein 2) (Jakobs et al, 2014). Another group of molecules that regulate Hh signaling is the glycosylphosphatidylinositol (GPI)-linked glypican (Gpc) heparan sulfate proteoglycans (HSPGs, Gpc1-Gpc6 in vertebrates) (Chatterjee and Mayor, 2001;Mayor and Riezman, 2004;Rietveld et al, 1999), called Division abnormally delayed (Dally) (Nakato et al, 1995) and Dally-like protein (Dlp) (Khare and Baumgartner, 2000) in the fly.…”