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 ensured the generation of 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 variants during release (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 revealed that Shh solubilized by Disp and Scube2 (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). RP-HPLC calibration and color coding of Shh fractions is shown in Fig. S2 F-K. B-D) Solubilization of non-palmitoylated C25SShh (in this artificial variant, the N-terminal palmitate acceptor cysteine is changed for a non-accepting serine; functionally equivalent constructs with the cysteine exchanged for a non-accepting alanine (C25AShh) were also used in our study), non-cholesteroylated yet palmitoylated ShhN and lipid-free control C25SShhN under the same serum-depleted conditions. Arrowheads denote Shh variants that were solubilized by Disp- and Scube2. 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 demonstrates that terminal lipids 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 CMK furin inhibitor. CMK impaired proteolytic processing and release of truncated (arrowheads) soluble Shh in a concentration-dependent manner. Right: Shh release in the presence or absence of 50 μM CMK furin inhibitor. B) Quantification of CMK-inhibited Shh shedding. Ratios of solubilized versus cellular Shh in the presence of 50 μM CMK inhibitor were determined and expressed relative to Shh solubilization in the absence of inhibitor (set to 100%, black bar). Unpaired t-test, twotailed. ****: p<0.0001, n=9. See Fig. S3A for loading controls and 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 under serum-free conditions is abolished.

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 for 24 h (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 – to a lesser degree – on 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) Shh, C25AShh (this artificial variant has the cysteine palmitate acceptor changed for a non-accepting alanine), and non-lipidated C25AShhN were expressed into media containing 10% FCS; their protein amounts determined by immunoblotting and normalized; and conditioned media added to C3H10T1/2 reporter cells to induce their Hh-dependent differentiation into alkaline phosphatase (Alp)-producing osteoblasts. Mock-treated C3H10T1/2 cells served as non-differentiating controls. At lower concentrations (1x), Shh and C25AShh induced C3H10T1/2 differentiation in a similar manner, as determined by Alp activity measured at 405 nm. At higher concentrations (2x), the bioactivity of C25AShh was increased over that of Shh. C25AShhN was inactive. One-way ANOVA, Sidaks multiple comparisons test. ****: p<0.0001, n.s.=0.99, n=3-9. See Table S1 for additional statistical information and Fig. S5E,F, which demonstrates protein activities similar to, or exceeding, that of a dual-lipidated Shh control. B) Similar transcription of Hh target genes Ptch1 and Gli1 by Shh and C25AShh in two independent experiments. C3H10T1/2 reporter cells were stimulated with similar amounts of Shh, C25AShh, and C25AShhN at high (2x) and lower (1x) concentrations. 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 Alp activity in C3H10T1/2 cells were 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 (HhβHS, 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 μg/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, as indicated by marker apolipoproteins ApoA1 (orange line) and mobile ApoE4. The former lends structural stability to the particle and stimulates cholesterol efflux to HDL; the latter facilitates cholesterol storage and core expansion and is therefore a marker of larger mature HDL particles (brown dashed 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.

Activities of HDL-associated Shh and non-palmitoylated variants. A) Shh, non-palmitoylated C25AShh, and non-lipidated C25AShhN were released into media containing 80 μg/mL HDL; their protein amounts determined by immunoblotting and normalized; and conditioned media added to C3H10T1/2 reporter cells to induce their differentiation into Alp-producing osteoblasts. Mock-treated C3H10T1/2 cells served as non-differentiating controls. At low (1x) and high (2x) concentrations, Shh and C25AShh induced C3H10T1/2 differentiation in a similar manner, as determined by Alp activity measured at 405 nm. Again, C25AShhN was completely inactive, in contrast to bioactive HDL-associated non-palmitoylated C25SShh. One-way ANOVA, Sidaks multiple comparisons test. ****: p<0.0001, ***: p<0.001, n.s. >0.1, n=4. See Table S1 for additional statistical information. B) Similar transcription of Hh target genes Ptch1 and Gli1 by HDL-associated Shh and C25AShh in two independent experiments. C3H10T1/2 reporter cells were stimulated with similar amounts of Shh, C25AShh, and C25AShhN at high (2x) and half (1x) concentrations.

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 (e.g. is not HDL associated). B) Cholesteroylated C25SShh solubilization into serum-free medium is strongly impaired (top blot, labeled [3]), but increases Dispdependently 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. 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) indicates Disp-independent physical desorption and unregulated HDL association of the monolipidated protein. 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 Dispmediated 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 toward 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 Disp (pdb:7RPH 47) suggests an extended hydrophobic surface channel (hydrophobic residues are shown in red) 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 47 prior to handover 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 (similar to ShhN as shown 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.