Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly

Abstract

Holoprosencephaly (HPE), a human developmental brain defect, usually is also associated with varying degrees of midline facial dysmorphism. Heterozygous mutations in the Sonic hedgehog (SHH) gene are the most common genetic lesions associated with HPE, and loss of Shh function in the mouse produces cyclopia and alobar forebrain development. The N-terminal domain (ShhNp) of Sonic hedgehog protein, generated by cholesterol-dependent autoprocessing and modification at the C terminus and by palmitate addition at the N terminus, is the active ligand in the Shh signal transduction pathway. Here, we analyze seven reported missense mutations (G31R, D88V, Q100H, N115K, W117G, W117R, and E188Q) that alter the N-terminal signaling domain of Shh protein, and show that two of these mutations (Q100H and E188Q), which are questionably linked to HPE, produce no detectable effects on function. The remaining five alterations affect normal processing, Ptc binding, and signaling to varying degrees. These effects include introduction of a recognition site for furin-like proteases by the G31R alteration, resulting in cleavage of 11 amino acid residues from the N terminus of ShhNp and consequent reduced signaling potency. Two other alterations, W117G and W117R, cause temperature-dependent misfolding and retention in the sterol-poor endoplasmic reticulum, thus disrupting cholesterol-dependent autoprocessing.

Fig. 1.

Ptch-binding and signaling activities of HPE mutant ShhN proteins. (A) Sequences of human ShhN, mouse ShhN, rat ShhN, Xenopus Banded hedgehog (Bhh), Drosophila Hh, and mouse Desert hedgehog (Dhh) are aligned with nonidentical residues in blue. Human and mouse sequences are identical except for a Ser in place of Thr at position 67 in the human sequence; the numbering of corresponding human and mouse residues differs by one because of different signal sequence lengths. Residues altered in human HPE are boxed in yellow, and the altered residues are shown above in green. (B) Binding of altered ShhN mutant proteins to the Ptch receptor. Binding of ³²P-ShhN to EcR-293 cells stably expressing Ptch-CTD (15) was measured in the presence of unlabelled recombinant proteins; binding was normalized to the amount (100%) bound in absence of any competitor. (C) Signaling activities of altered ShhN proteins in intermediate neural plate explants from chick embryos. After incubation in presence of recombinant proteins (concentrations in nM are indicated by the numbers in each panel), explants were double stained with antibodies against Pax-7 (green) and the floor plate marker HNF3-β (red).

Fig. 2.

Temperature dependence of WG and WR protein activities. (A) Neural plate signaling of WG and WR variants. Neural plate explants were incubated at 37°C and 32°C at the indicated concentrations of ShhN (in nM) and stained to monitor expression of Pax-7 (green; control panels) and HNF3β (red). Stimulation with WT protein (4 nM) suppressed Pax-7 expression at both temperatures, whereas variant proteins at 37°C failed to suppress Pax-7 at 200 nM protein concentrations (WG and WR) or at 400 nM protein concentrations (WR). However, at 32°C, WG and WR proteins at 200 and 400 nM suppressed Pax-7 (data not shown) and induced HNF3-b expression (red). (B) Ptch-binding of WG and WR variants. Proteins were preincubated at 37°C or 32°C for 1 h, then added (5 nM) to EcR-293 cells stably expressing Ptc-CTD in the presence of ³²P-ShhN. WG and WR variants failed to compete with ³²P-ShhN when preincubated at 37°C, but showed some activity when preincubated at 32°C. (C) Immunoprecipitation of variant proteins by 5E1 monoclonal antibody. WT ShhN and surface D (SD) (15), WG, and WR variants were all immunoprecipitated by 5E1-monoclonal antibody when preincubated at 4°C (bulk of proteins found in pellet, not supernatant), but after preincubation at 37°C, WG and WR were predominantly found in the supernatant. Proteins were detected by immunoblotting with anti-ShhN polyclonal antibodies.

Fig. 3.

Effect of HPE mutations on Shh protein processing. (A and B) Western blot analysis of cell extracts from HEK-293 cells transiently transfected with full-length WT and variant Shh constructs. Anti-ShhN polyclonal antibodies detected the full-length Shh protein (45 kDa, indicated by arrow), the processed and lipid-modified N-terminal fragment (ShhNp, indicated by arrowhead), and an abnormal processed N-terminal fragment (ShhNp^*, indicated by star) generated from the GR variant (A), whereas anti-ShhC antibodies detected full-length Shh and the ShhC fragment (B). The WG and WR variants produced neither ShhNp nor ShhC fragments. (C-E) Glycosidase digestion. Extracts (C and D) or conditioned medium (E) from HEK-293 cells transfected with Shh constructs were immunoblotted after denaturation and no further treatment (N) or treatment with Endo H (E) or PNGase F (P). Proteins were separated in 10% (C) or 15% (D and E) SDS/PAGE and immunoblotted with anti-ShhN (C) or anti-ShhC (D and E) antibodies. (F) Conditioned medium from transfected HEK-293 cells was immunoblotted and analyzed with anti-ShhN antibodies. Note the greater abundance of the abnormally processed ShhN GR variant in conditioned medium.

Fig. 4.

Abnormal processing and functional characterization of ShhN from the GR variant. (A) Heparin-agarose binding. Conditioned media from HEK-293 cells transfected with ShhN or GR-ShhN-Myc were separated with heparin-agarose and immunoblotted with anti-ShhN polyclonal antibodies. The C-terminal Myc epitope can be detected in both the slower- and faster-migrating forms of GR-ShhN-Myc (data not shown), indicating that the abnormal truncation occurs at the N terminus. WT ShhN and the longer form of GR-ShhN-Myc are both retained by heparin agarose, but the truncated form of the GR variant is not. Note the comigration of slower-migrating GR-ShhN-Myc and recombinant ShhN-Myc protein purified from E. coli.(B) Determination of cleavage site. The abnormally truncated ShhN^*-Myc protein was purified by lack of binding to heparin-agarose and with the monoclonal antibody 5E1 and subjected to N-terminal sequence analysis by Edman degradation. This sequence indicated that cleavage occurred between Arg (35) and His (36) residues. (C and D) Furin cleavage of recombinant GR-ShhN protein in vitro and in vivo. (C) Purified, recombinant ShhN (1.5 μg) and GR-ShhN (2.0 μg) proteins treated with 0, 40, 80, and 200 units of furin protease (Sigma) were analyzed by SDS/PAGE and Coomassie blue staining. (D) EcR-293 cells with stably integrated construct for expression of GR-Shh full-length protein were induced with ponasterone in the presence of 0, 5, 10, 20, and 50 μM of furin inhibitor I, and cell extracts were analyzed by immunoblotting with anti-ShhN antibodies. (E and F) Ptc-binding and Shh signaling activities of recombinant ShhN^*. (E) Recombinant WT ShhN and protein lacking 11 N-terminal residues (N-11) proteins were purified (Inset) and used as competitor for ³²P-ShhN binding to EcR-293 cells expressing Ptc-CTD. (F) Signaling activities of ShhN WT and N-11 in C3H10T1/2 cells.