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

Media containing 10% serum was changed to serum-free DMEM 36 h post-transfection and proteins were 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 paper. A) Cap-dependent Shh translation and cap-independent Hhat translation from bicistronic mRNA ensured the generation of dual-lipidated, plasma membrane-associated proteins (asterisk in the cellular fraction) in all transfected cells. Disp and Scube2 synergistically and specifically enhance the conversion of dual-lipidated Shh precursors 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 or absence of Scube2. Amounts of solubilized Shh with higher electrophoretic mobility (the lower bands) were quantified and expressed as % relative to the respective cellular Shh, which was always set to 100%. One-way ANOVA, Dunnett’s multiple comparisons test. ****: p<0.0001. See Table S1 for detailed statistical information. 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 indicates analysis of the same cellular fraction as shown in A). RP-HPLC calibration and color coding of Shh fractions are shown in Fig. S2 I-N. B-D) Solubilization of non-palmitoylated C25SShh (in this artificial variant, the N-terminal palmitate acceptor cysteine is replaced by 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 but palmitoylated ShhN and lipid-free control C25SShhN under the same serum-depleted conditions. Arrowheads indicate Shh variants that were solubilized in Disp- and Scube2 presence. B’-D’) Processed protein quantifications from B-D, again from nt ctrl and Disp-/- cells in the presence or absence of Scube2. One-way ANOVA, Dunnett’s multiple comparisons test. ns: p>0.05. See Table S1 for detailed statistical information. B’’-D’’) Reverse phase HPLC shows similar elution of C25SShh, ShhN, and non-lipidated C25SShhN. This indicates that terminal lipids were removed during the release of C25SShh and ShhN (as well as Shh, A’’).

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 the furin inhibitor CMK. CMK impaired proteolytic processing and release of truncated (arrowheads) soluble Shh in a concentration-dependent manner. B) Truncated (arrowhead) Shh release in the presence or absence of 50 μM CMK furin inhibitor. C) Quantification of CMK-inhibited Shh shedding. Ratios of solubilized versus cellular Shh (set to 100%) in the presence of 50 μM CMK inhibitor were determined and expressed relative to Shh solubilization in the absence of inhibitor (DMSO). Unpaired t-test, two-tailed. **: p=0.0021, n=6. See Fig. S3A for loading controls and Table S1 for additional statistical information. D) 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 paper). Note that Shh solubilization is greatly reduced under serum-free conditions. E) Processed Shh quantifications after secretion from nt ctrl and Disp-/- cells in the presence or absence of Scube2 into serum-free medium. One-way ANOVA, Dunnett’s multiple comparisons test. **: p=0.0059, *: p=0.02, ns: p=0.54. See Table S1 for detailed statistical information.

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

Media were changed to serum-free DMEM (cells were not washed) or DMEM containing the indicated amounts of serum 36 h post-transfection, 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 the conversion of dual-lipidated Shh into truncated soluble forms (arrowhead). m: media, c: cell lysate. A’’) RP-HPLC confirmed the loss of both terminal lipidated Shh peptides during Disp- and Scube2-regulated shedding, as shown previously (Fig. 1A’’). B-D) The appearance of truncated Shh in serum-containing media remained dependent on Disp and, to a lesser extent, Scube2 (arrowheads). B’-D’) Quantifications of B-D. One-way ANOVA, Dunnett’s multiple comparisons test. ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05. See Table S1 for detailed statistical information. B’’-D’’) RP-HPLC revealed that increased serum levels shift dual Shh shedding (low serum levels, [1]) towards N-terminally restricted shedding and release of a cholesteroylated Shh form [2]. Low levels of dually lipidated Shh are also detected [3]. E) Quantification of unprocessed Shh release in the presence of 10% FCS (top band in D). One-way ANOVA, Dunnett’s multiple comparisons test. ns: p>0.05. See Fig. S4 for additional information.

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 to a non-accepting alanine), and non-lipidated C25AShhN were expressed in media containing 10% FCS; their protein levels were determined by immunoblotting and normalized; and the conditioned media were 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, ns=0.99, n=3-9. See Table S1 for additional statistical information and Fig. S5E,F, which shows protein activities similar to or exceeding those of a dual-lipidated Shh control. B) Similar transcription of the Hh target genes Ptch1 and Gli1 by Shh and C25AShh in three independent experiments. C3H10T1/2 reporter cells were stimulated with similar amounts of Shh, C25AShh, and C25AShhN at high (2x) and low (1x) concentrations. C) SEC shows significant amounts of Shh of increased molecular weight in media containing 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). The level of Shh-induced Alp activity in C3H10T1/2 cells was measured as absorbance at 405 nm, showing the strongest C3H10T1/2 differentiation by eluted fractions containing large Shh aggregates. D) SEC of Drosophila Hh (black line) and of a variant lacking its HS binding site (HhΔHS, black dotted line). Both proteins were expressed from S2 cells under actin-Gal4/UAS-control and solubilized into media containing 10% FCS.

HDL enhancess N-processed Shh solubilization by Disp.

Media were changed to serum-free DMEM (and cells washed three times) or serum-free DMEM supplemented with 40 µg/mL HDL 36 h post-transfection before protein solubilization 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 greatly reduced [1]. A’) RP-HPLC of the material labeled [1] in A showed complete delipidation during release. A’’) SEC of the same delipidated material shows 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] in B showed that HDL shifts Shh shedding from dual processing (A’, [1]) to release of cholesteroylated Shh. B’’) SEC of the same material [2] (black line) shows an increase in molecular weight corresponding to the molecular weight range of HDL, as indicated by the marker apolipoproteins ApoA1 (orange line) and mobile ApoE4. The former provides 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. One-way ANOVA, Dunnett’s multiple comparisons test. ***: p=0.0008, **: p=0.0023, ns: p=0.77, n=7. Additional statistical information is provided in Table S1.

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 levels were determined by immunoblotting and normalized, and conditioned media were 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 the bioactive HDL-associated non-palmitoylated C25SShh. One-way ANOVA, Sidaks multiple comparisons test. ****: p<0.0001, ***: p<0.001, ns >0.1, n=4. Additional statistical information is provided in Table S1. B) Similar transcription of Hh target genes Ptch1 and Gli1 by HDL-associated Shh and C25AShh in C3H10T1/2 cells three independent experiments. Experiment 4 confirms similar Shh and C25AShh activities in NIH3T3 cells. Reporter cells were stimulated with similar amounts of Shh, C25AShh, and C25AShhN at high (2x) and low (1x) concentrations as determined by immunoblotting.

Cholesterylated C-terminal peptides are necessary 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 (upper blot, labeled [1]) or into serum-free DMEM supplemented with HDL (lower 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 (i.e. not HDL-associated). B) Solubilization of cholesterylated C25SShh into serum-free medium is strongly impaired (upper blot, labeled [3]), but increases in a Disp-dependent manner in the presence of HDL (lower blot, labeled [4]). Asterisks denote C25SShh solubilized independently 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) shows 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 of the mCherry associates with HDL in a Disp-mediated manner. E) C25SShh (green line) dissociates from HDL in 50% ethanol (bright green line) or in 0.1% Triton X-100 (bright green dashed line). The disassembly of HDL (orange line) under the same conditions is confirmed by the size shift of ApoA1 towards the monomeric 32 kDa protein (light 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) Depiction of the 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). A sterol lifted upward (green stick representation) at the starting 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 delivery to HDL. C) We propose two sequences of Shh transfer events. In the first event [1], plasma membrane sterol is transferred through the hydrophobic Disp surface channel to HDL acceptors. This process is similar to the 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 susceptible to proteolytic processing (similar to ShhN as shown in A). N-terminal Shh shedding can 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 indirectly trigger shedding of both terminal Shh peptides and the solubilization of monomeric proteins [6]. See Discussion for details.