Disp and Scube2 enhance shedding of cell surface-associated dual-lipidated Shh into delipidated soluble forms.

Media containing 10% serum were exchanged for serum-free DMEM 36 h post-transfection and proteins solubilized for 6 h. Cells were not washed between media changes to leave residual serum traces in the assay. We refer to this experimental condition as “serum-depleted” throughout this work. A) Cap-dependent Shh translation and cap-independent Hhat translation from one bicistronic mRNA generated dual-lipidated, plasma membrane-associated proteins (asterisk in the cellular (c) fraction) in all transfected cells. Disp and Scube2 synergistically and specifically enhance dual-lipidated Shh precursor conversion into truncated soluble Shh (arrowhead). m: media, c: cell lysate. A’) Quantification of relative Shh release from nt ctrl and Disp-/- cells in the presence of Scube2. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh solubilization from nt ctrl cells (set to 100%, black bar). A’’) Reverse-phase HPLC analyses by using a C4 column revealed that specifically solubilized Shh (the same fraction indicated by the arrowhead in A, black line) was less hydrophobic than its cell surface-associated precursor (gray line, the asterisk denotes analysis of the same cellular fraction as shown in A). C4 column calibration and color-coding of fractions is explained in Fig. S2. B-D) Solubilization of non-palmitoylated C25SShh, non-cholesteroylated ShhN and lipid-free control C25SShhN under the same serum-depleted conditions. Arrowheads denote the Disp- and Scube2-solubilized material. B’-D’) Quantifications of B-D, again from nt ctrl and Disp-/- cells in the presence of Scube2. B’’-D’’) Reverse-phase HPLC demonstrates similar elution of C25SShh, ShhN, and non-lipidated C25SShhN. This suggests that lipidated peptide termini were removed during C25SShh and ShhN (as well as Shh, A’’) release. Unpaired t-test, two-tailed. ****: p<0.0001, *: p<0.016, n.t.: 0.79. n=20 datasets (A’), n=13 datasets (B’), n=10 datasets (C’), and n=17 datasets (D’). See Table S1 for detailed statistical information.

Shh shedding depends on Disp activation by furin and the presence of serum traces.

A) nt ctrl cells were transfected with Shh and Scube2, and Shh solubilization was monitored in the presence or absence of 50 μM CMK furin inhibitor. B) Quantification of CMK-inhibited Shh shedding. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh solubilization in the absence of inhibitor (set to 100%, black bar). Unpaired t-test, two-tailed. ****: p<0.0001, n=9. See Table S1 for additional statistical information. C) Immunoblotted cellular (c) and medium (m) fractions of Shh expressing nt ctrl and Disp-/- cells in the complete absence of serum (referred to as “serum-free” conditions throughout this work). Note that Shh solubilization is completely abolished and that cellular precursors accumulate in the cells.

Dual-lipidated cell-surface Shh converts into delipidated soluble forms under low-serum and high-serum conditions.

Media were changed for serum-free DMEM 36 h post-transfection (cells were not washed) or DMEM containing the indicated amounts of serum, and proteins were solubilized for 6 h (serum-depleted) or 24h (with 0.05%, 5% and 10% serum). A) Under serum-depleted conditions, Disp and Scube2 increase dual-lipidated Shh shedding into truncated soluble forms (arrowhead). m: media, c: cell lysate. A’) RP-HPLC confirmed loss of both terminal lipidated Shh peptides during Disp- and Scube2-regulated shedding. B-D) The appearance of truncated Shh in serum-containing media remained dependent on Disp and Scube2 (arrowheads). B’-D’) RP-HPLC revealed that increased serum amounts shift dual Shh shedding (low serum amounts, [1]) toward N-terminally restricted shedding and release of a cholesteroylated Shh form [2]. Low levels of dual-lipidated Shh are also detected [3]. See Fig. S4 for experimental details.

Activities and SEC of dual-lipidated Shh/Hh and depalmitoylated Shh variants solubilized into serum-containing media. A) Immunoblotted proteins (inset) were used for quantification and visualization of dually lipidated R&D 8908-SH, Shh, C25AShh (this artificial variant has the cysteine palmitate acceptor changed for a non-accepting alanine) and unlipidated C25SShhN. Data were normalized to mock-treated C3H10T1/2 cells. Shh, C25AShh and C25SShhN were solubilized from Scube2- and Disp-expressing cells into media containing 10% FCS. 0-4 ng of R&D 8908-SH induced C3H10T1/2 reporter differentiation in a concentration-dependent manner, as determined by Alp activity measured at 405nm (blue line). Similar amounts of solubilized Shh, as determined by immunoblotting, increased C3H10T1/2 osteogenesis over that of the dual-lipidated R&D 8908-SH standard (black line). Notably, C25AShh was also highly bioactive if released into serum-containing medium and tested in this experimental system (green line). Inset: Immunoblot of proteins used in this assay. The arrow indicates proteolytic processing of the palmitoylated N-terminal peptide from Shh and C25AShh, but not from R&D 8908-SH. Artificial unlipidated soluble C25SShhN showed only little activity 63 (gray line). B) Similar induction of transcription of Hh target genes Ptc1 and Gli1 in C3H10T1/2 reporter cells. Cells were stimulated with 4ng R&D 8908-SH or similar amounts of Shh and C25AShh. Shh and C25AShh were both solubilized from Bosc23 cells into serum-containing media in the presence of Scube2 (100μl Shh/C25AShh+Scube2). C) SEC reveals substantial amounts of increased molecular weight Shh in media that contain 10% serum (black line). The increased molecular weight Shh eluted together with ApoA1 (orange line). In contrast, Scube2 was largely monomeric in solution (blue line). Levels of Shh-induced alkaline phosphatase activity in C3H10T1/2 cells were again measured as absorption at 405 nm, demonstrating the strongest C3H10T1/2 differentiation by eluted fractions containing large Shh assemblies. D) SEC of Drosophila Hh (black line) and of a variant lacking its HS binding site (HhHS, black dotted line). Both proteins were expressed under actin-Gal4/UAS-control from S2 cells and solubilized into media containing 10% FCS.

HDL increases N-processed Shh solubilization by Disp.

Media were changed for serum-free DMEM 36 h post-transfection (and cells washed three times) or serum-free DMEM supplemented with 40 mg/mL HDL before proteins were solubilized for 6 h. A) Immunoblotted cellular (c) and medium (m) fractions of Shh expressing nt ctrl and Disp-/- cells in the complete absence of serum. Note that Shh solubilization is strongly reduced [1]. A’) RP-HPLC of the material labeled [1] revealed complete delipidation during release. A’’) SEC of the same delipidated material demonstrates that it is readily soluble and not associated with Scube2. B) Immunoblotted cellular (c) and medium (m) fractions of Shh expressing nt ctrl and Disp-/- cells in the presence of 40 μg/mL HDL. Shh shedding and solubilization are strongly increased by Disp [2] but not by Scube2. B’) RP-HPLC of the material labeled [2] revealed that HDL shifts Shh shedding from dual processing (A’, [1]) toward the release of cholesteroylated Shh. B’’) SEC of the same material [2] (black line) shows a molecular weight increase that matches the molecular weight range of HDL (orange line). Again, the soluble Shh elution profile does not overlap with that of Scube2 (blue line). C) Quantification of HDL-induced Shh solubilization from nt ctrl cells and Disp-/- cells. Unpaired t-test, two-tailed. ****: p<0.0001, n=6. See Table S1 for additional statistical information.

Cholesteroylated C-terminal peptides are required and sufficient for Disp-mediated protein transfer to HDL.

A) Immunoblotted cellular (c) and medium (m) fractions of ShhN-expressing nt ctrl and Disp-/- cells. Shown is unspecific ShhN solubilization into serum-free media (top blot, labeled [1]) or into serum-free DMEM supplemented with HDL (bottom blot, labeled [2]). A’) ShhN [1] expressed under serum-free conditions is solubilized in a monomeric state. A’’) ShhN [2] expressed in the presence of HDL remained monomeric. B) Cholesteroylated C25SShh solubilization into serum-free medium is strongly impaired (top blot, labeled [3]), but increases Disp-dependently in the presence of HDL (bottom blot, labeled [4]). Asterisks denote C25SShh solubilized independent of Disp function. B’) Most C25SShh [3] in serum-free media is monomeric, but a small fraction also assembles into larger units. B’’) C25SShh [4] expressed in the presence of HDL increases in molecular weight to match the molecular weight range of HDL (orange line). C) SEC of C25SShh solubilized from Disp-expressing cells (solid green line) or from Disp-/- cells (dotted line). D) SEC of cholesteroylated mCherry solubilized from nt ctrl cells (solid lines) or from Disp-/- cells (dotted lines) under the same conditions. Dashed lines indicate proteins solubilized under serum-free conditions. Note that most mCherry associates with HDL in a Disp-regulated manner. E) C25SShh (green line) disassembles from HDL in 50% ethanol (bright green line) or in 0.1% Triton X-100 (bright green dashed line). HDL (orange line) disassembly under the same conditions is confirmed by ApoA1 size shift towards the monomeric 32 kDa protein (bright orange line).

Model of two-way Disp-mediated Shh solubilization.

A) Dual lipidation protects Shh from unregulated cell-surface shedding by tight plasma membrane association of both lipids (blocked shedding is indicated by an x in [1]). In contrast, monolipidated ShhN [2] and C25SShh [3] are prone to unregulated membrane-proximal shedding (indicated by the dashed line) or non-enzymatic desorption. B) Representation of surface hydrophobicity of pdb:7RPH 45 (red) suggests an extended hydrophobic surface channel that may function as a “slide” for lipophiles extending from the plasma membrane (dashed lines) to a cavity of the second extracellular domain (blue arrows). An upward lifted sterol (green stick representation) at the start point of the hydrophobic track may represent an intermediate state of sterol extraction from the membrane, and a lipidic group modeled as the amphiphilic detergent lauryl maltose neopentyl glycol (violet stick structure) may represent the end point of the transfer 45 prior to hand-over to HDL. C) We suggest two sequences of Shh transfer events. In the first event [1], plasma membrane sterol is transferred through the Disp hydrophobic surface channel to HDL acceptors. This process resembles established reverse cholesterol transport. In the second event, if present, C-terminal cholesterol moieties of Shh can also be transferred [2]. This partial Shh extraction exposes the N-terminal cleavage site [3] and makes it prone to proteolytic processing (as is the case for ShhN in A). N-terminal Shh shedding may then release the protein from the plasma membrane [4] to complete the transfer [5]. In addition to, or competing with this process, cholesterol depletion of the plasma membrane (representing the first event, [1]) may trigger shedding of both terminal Shh peptides and the solubilization of monomeric proteins in an indirect manner [6]. See discussion for details.