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FUNCTIONAL CHARACTERIZATION OF SHH MUTATIONS
ASSOCIATED WITH HOLOPROSENCEPHALY
by
Elisabeth Traiffort1†, Christèle Dubourg2, Hélène Faure1, Didier Rognan3,
Sylvie Odent4, Marie-Renée Durou2, Véronique David2 and Martial Ruat1
Running title: SHH mutants implicated in HPE
1Institut de Neurobiologie Alfred Fessard, IFR 2118 CNRSLaboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040 CNRSBâtiment 33, 1 avenue de la terrasse 91198 Gif sur Yvette, France2Génétique Humaine, UMR 6061, Faculté de Médecine, 2 avenue du Pr Léon Bernard, CS 34317,35043 Rennes, France3Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081 CNRS, 74 route duRhin, B.P. 24, 67401 Illkirch, France4Unité de Génétique Médicale, Hôpital Sud, 16 boulevard de Bulgarie, BP 90347, 35023 Rennes,France† Corresponding author: [email protected];Phone: 33 1 69 82 43 01, fax: 33 1 69 82 36 39
JBC Papers in Press. Published on July 28, 2004 as Manuscript M405161200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Mutations of the developmental gene Sonic hedgehog (SHH) and alterations of SHH
signaling have been associated with holoprosencephaly (HPE), a rare disorder characterized
by a large spectrum of brain and craniofacial anomalies. Based on the crystal structure of
mouse aminoterminal and drosophila carboxyterminal hedgehog proteins, we have developed
three-dimensional models of the corresponding human proteins (SHH-N, SHH-C) which have
allowed us to identify, within these two domains, crucial regions associated with HPE
missense mutations. We have further characterized the functional consequences linked to
eleven of these mutations. In transfected HEK293 cells, the production of the active SHH-N
fragment was dramatically impaired for eight mutants (W117R, W117G, H140P, T150R,
C183F, L271P, I354T, A383T). The supernatants from these cell cultures showed no
significant SHH signaling activity in a reporter cell-based assay. Two mutants (G31R,
D222N) were associated with a lower production of SHH-N and signaling activity. Finally,
one mutant harboring the A226T mutation displays an activity comparable to the wild-type
protein. This work demonstrates that most of the HPE-associated SHH mutations analysed
have a deleterious effect on the availability of SHH-N and its biological activity. However,
because of the lack of correlation between genotype and phenotype for SHH-associated
mutations, our study suggests that other factors intervene in the development of the spectrum
of HPE anomalies.
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INTRODUCTION
Holoprosencephaly (HPE) is the most common brain anomaly in humans, involving
abnormal formation and septation of the developing central nervous system and occurring in 1
in 16000 live births and 1 in 250 spontaneous abortion cases (1). The clinical manifestations
of the disease are variable, ranging from a single cerebral ventricle and cyclopia to minor
anomalies of midline structures. The etiology of HPE is extremely heterogeneous including
genetic factors and environmental agents. The majority of HPE cases are sporadic, although
autosomal dominant familial cases have been described. Genetic studies have shown that
more than ten chromosomal loci are implicated in HPE and seven genes have been already
identified: Sonic hedgehog (SHH) isolated from the human critical region HPE3 on
chromosome 7q36 (2, 3), ZIC2 (13q32 ; HPE5) (4), SIX3 (2p21 ; HPE2)(5), TGIF (18p11.3 ;
HPE4) (6), Patched (PTC) (9q22) (7), TDGF1 (3p21.31) (8) and GLI2 (2q14) (9).
SHH mutations including nonsense and missense mutations, but also deletions and
insertions constitute about 50 % of the known HPE mutations as reported by several genetic
screenings (10-16). If the deleterious role of nonsense or frameshift mutations is evident in
the pathogenesis of genetic diseases, the implication of missense mutations has to be proved.
Moreover, the ability to discriminate between a loss-of-function mutation and a silent
polymorphism is important for genetic testing of inherited diseases like HPE where the
opportunity for genetic counseling exists (17).
SHH is a morphogen molecule involved in embryonic development including the
induction of the floorplate and the establishment of the ventral polarity within the central
nervous system (18). SHH is synthesized as a precursor that undergoes autocatalytic cleavage
into a highly conserved N-terminal domain (SHH-N) responsible for the signaling activities of
the molecule, and a more divergent C-terminal domain (SHH-C) implicated in the
autoproteolysis reaction and the addition of a cholesterol moiety covalently attached to the C-
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terminus of SHH-N (19). This cholesterol molecule is presumably important for tethering the
protein to the plasma membrane and thus restricts the tissue localization of hedgehog
signaling. The addition of another lipid, a palmitoyl group, on the N-terminal cysteine residue
of SHH-N dramatically increases the biological activity of the protein (20, 21). Thus, these
modifications of SHH may exert a key role in targeting the protein to lipid rafts (22) that
could represent functional platforms for SHH signaling. Diffusion of a soluble cholesterol-
modified multimeric form of mouse Shh-N has been proposed to mediate long-range
signaling during embryonic development (23).
SHH mediates its action via a receptor complex associating two transmembrane
proteins: PTC displaying a transporter-like structure and Smoothened (SMO) presumably
belonging to the G protein-coupled receptor superfamily. The repression exerted by PTC on
SMO is relieved when SHH binds PTC, which leads to a complex signaling cascade involving
the transcription factors of the Gli family and to the activation of target genes including PTC
itself (18, 24). Two PTC genes have been molecularly cloned (25-27). During embryonic
development, PTC-1 is widely expressed, whereas PTC-2 RNA distribution is more restricted
(18). SHH binds also HIP (hedgehog interacting protein), a negative regulator of the pathway
which sequesters and antagonizes SHH (22, 28). In rodent, Shh-N secretion from the
synthesizing cell requires Dispatched, a PTC related protein proposed to control the release of
the morphogen (29, 30).
Animal models support the concept that mutations resulting in modulation of SHH
activity signaling pathway can cause HPE. The phenotype observed in Shh knockout mice
(31) associating cyclopia and loss of normal ventral specification in the forebrain mimics
clinical manifestations of HPE. A percentage of mice with increased PTC activity driven by
the nestin enhancer display a fusion of the lateral ventricles consistent with HPE (32). Smo
homozygous knockout mice also present cyclopia and HPE (33). Finally, homozygous null
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mutant for Gli2 displays a single central maxillary incisor constituting a microform of HPE
(34).
We have now developed a three dimensional model of human SHH-N and SHH-C
domains that were used to highlight important regions associated with HPE mutations within
both peptides. We have mutated a panel of amino acid residues associated with HPE and
distributed both in SHH-N and SHH-C. We present biochemical and functional analysis of the
mutated proteins and discuss their functionality in the development of HPE.
EXPERIMENTAL PROCEDURES
Modeling the human SHH-N and SHH-C proteins
The three-dimensional models of the human SHH-N and SHH-C domains were
constructed from the x-ray structures of mouse Shh-N (35) and drosophila carboxy terminal
hedgehog (hh-C) (36), respectively. After alignment of the amino acid sequences of the
above-cited proteins using standard parameters of the ClustalW program (37), human SHH-N
and SHH-C proteins were modeled using the BIOPOLYMER module of the SYBYL 6.9
package (TRIPOS Assoc., Inc., St.Louis, MO). Two insertions of one and three residues in
SHH-C were modeled using a classical loop search procedure as already described (38),
whereas a nineteen residue insertion could not be modeled. Standard geometries for the
mutated side chains were given by the BIOPOLYMER module of SYBYL. Whenever
possible, the side chain torsional angles were kept to the values occurring in the x-ray
templates. Otherwise, a short scanning of side chain angles was performed to remove steric
clashes between the mutated side chain and the other amino acids. After the heavy atoms were
modeled, all hydrogen atoms were added, and the protein coordinates were then minimized
with AMBER (39) using the AMBER95 force field (40). The minimizations were carried out
by 1,000 steps of steepest descent followed by conjugate gradient minimization until the root-
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mean-square gradient of the potential energy was less than 0.05 kcal/mol.Å. A twin cut-off
(10.0, 15.0 Å) was used to calculate non-bonded electrostatic interactions at every
minimization step, and the non-bonded pair-list was updated every 25 steps. A distance-
dependent (ε=4r) dielectric function was used.
Site-directed mutagenesis
To mutate SHH amino acids, the full-length cDNA encoding the human wild-type
(WT) SHH (Genbank accession number L38518), kindly provided by Dr C. Tabin (Harvard
Medical School, Boston, USA), was used. Mutations G31R, W117G, W117R, H140P,
T150R, C183F, D222N, A226T, L271P, I354T and A383T were introduced into SHH cDNA
using the QuikChange XL Site-directed Mutagenesis Kit (Stratagene, La Jolla, USA)
according to the manufacturer’s instructions with primers that are available upon request.
The mutations were confirmed by automated DNA sequence analysis using the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) and the
ABI Prism 3100 Genetic Analyzer. The WT and mutated SHH cDNAs were subcloned into
the pRK5 vector for use in transfection experiments.
Cell culture
HEK293 and C3H10T1/2 cells (ATCC, Molsheim, France) were cultured at 37°C,
under 5% CO2, in DMEM high glucose (Life Technologies, Cergy Pontoise, France)
supplemented with 10% heat inactivated FCS ( Eurobio, Les Ulis, France).
Transfection, expression in cell cultures and conditioned media
HEK293 cells (107/well) were electrotransfected (270 V, 960 µF; Gene Pulser II, Bio-
Rad, Marnes-la-Coquette, France) in buffer (K2HPO4, 50 mM; KCH3CO2, 20 mM; KOH, 20
mM; MgSO4, 26.6 mM; pH 7.4) with 1.5 µg of WT or mutated SHH cDNAs. The DNA
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amount was normalized to 10 µg with the empty vector. 48 h after transfection, the culture
media were harvested and the cells were rinsed and scraped in cold PBS, both were
centrifuged (4°C, 100g, 10 min) and processed for Western blotting. Alternatively, the culture
media were harvested, centrifuged (4°C, 500g, 10 min) and used as conditioned media to
evaluate the differentiation of C3H10T1/2 cells.
Western blotting and densitometry analysis
48 h following the transfection, cell pellets were homogenized in ice-cold 10 mM
Tris.HCl (pH 7.4), 1 mM EDTA, aprotinin (10 µg/ml), leupeptin (10 µg/ml),
phenylmethylsulfonyl fluoride (100 µg/ml) and benzamidine (60 µg/ml), and protein content
was determined (41). Media and cellular homogenates were separated on 12.5 % acrylamide
gel, blotted on nitrocellulose membrane, probed 2 h with specific polyclonal antisera 167Ab
(1/1000) directed to mouse Shh-N (42) or 1229Ab (1/1000) directed to the human SHH-C
peptide 288-302 and kindly provided by A. Galdes and K.P. Williams (Biogen, Cambridge,
USA). Immunoreactivity was revealed as described (42). The effective quantity of loaded
protein was determined by probing the blots with a 1/500 dilution of the mouse monoclonal
IgG2a α-actin antibody (AC-40) (Sigma, Saint Quentin Fallavier, France). Quantification of
the chemiluminescent signals was performed by scanning the films with a Canon scanner
(Canoscan 300, Canon, Courbevoie, France) and measuring the pixel densities in the signal
areas using SigmaGel™ (Version 1.0, SPSS Inc., USA). Significance was assayed using the
Excel 98 Student’s t Test (Microsoft®, Seattle, USA). Data were from at least three
independent experiments carried out with different preparations of DNA.
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Alkaline phosphatase (AP) assay
C3H10T1/2 cells were seeded in 96-well plates at a density of 5x103 cells per well.
24 h later, the culture medium was replaced by 200 µl of conditioned media from HEK293
cells as described above. Five days later, AP assay was performed as described (22). Data are
means ± S.E.M. of quadruplicates and experiments have been performed 3-5 times with
similar results. Significance was assayed by the Excel 98 Student’s t Test (Microsoft®,
Seattle, USA).
RESULTS
Mapping SHH-N and SHH-C mutations associated with HPE
During the last few years, genetic studies have associated non syndromic HPE with
about 40 missense SHH mutations, one nucleotide insertion, seven nucleotide deletions and
nine mutations introducing a stop codon leading to a premature SHH protein (Fig. 1 and Table
1). These genetic modifications affect residues located in the signal peptide as well as in
SHH-N and SHH-C. They are reported in Table 1 together with the expected effect at the
level of the SHH protein and the clinical signs seen in the HPE-affected cases and in the
kindred.
To further locate the residues mutated in HPE within the SHH protein, the three-
dimensional models of N- and C-terminal domains of human SHH were separately obtained
from the known x-ray structures of mouse Shh-N (35) and drosophila hh-C (36) solved at 1.7
and 1.9 Å resolution, respectively. Human SHH-N differs from mouse Shh-N by only one
residue (S versus T67) so that the human SHH-N structure could be directly obtained by
threading to the murine Shh-N x-ray structure and by modeling the mutated residue (Figs. 2A
and 3A). Human SHH-C differs more significantly from drosophila hh-C (sequence identity
of 31%, Figs. 2B and 3B). Three insertions occur in human SHH-C: one residue between
strands β4a and β5b and three residues between strands β3b and β4b were modeled using a
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classical loop search procedure as already described (38), nineteen residues between strands
β1b and β2b are indicated in dashed line in Fig. 2B. In our current models, SHH-N model
begins at K38 and ends at K194, whereas the SHH-C model encompasses amino acids C198
and A365.
The SHH-N model was used to locate both the triad residues –H140, D147 and H182–
implicated in Zn2+ coordination (35) and SHH-N residues mutated in HPE, except those
corresponding to a nonsense mutation (Fig. 1 and Table 1) and those located before residue
38. We further identified two main regions associated with HPE mutations within SHH-N.
The first one containing the highest number of SHH-N mutated residues, includes amino acids
located in two α-helices (Q100H, A110D, I111F, N115L, W117R, W117G, T150R) and in
the C-terminal end of SHH-N (E188Q). These residues lie at the surface of the protein
suggesting that they may be implicated in signaling and potentially in protein interactions.
The second region involves residues implicated in or surrounding the Zn2+ binding site. These
mutations would modify residues directly implicated in Zn2+ coordination such as H140, or
affect such residues, such as C183 which is adjacent to H182. The G31R mutation affects a
residue located in the N-terminal region that has been shown to be away from the globular
domain of Shh-N and to possibly make hydrophobic contacts with H182 of a symmetrical
SHH-N molecule (43).
In the SHH-C model, we observed that D243, T267 and H270 residues are facing
C198 residue in agreement with their implication in the internal thioester re-arrangement (Fig.
3B) as previously shown in drosophila hh (36). A first cluster of SHH-C residues mutated in
HPE comprises D222, V224 and S236 and are located in the same region of the protein (Fig.
3B). Except D222, the two other amino acids are not readily accessible for protein
interactions since they appear to be buried in the SHH-C ternary structure. A second cluster of
residues including P347 and I354 is found in the C-terminal region which is incompletely
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shown in our model (Fig. 3B). This region has been proposed to bind cholesterol and to be
responsible for its transfer during the autoprocessing reaction. R381, A383, P424 and S436
(not shown in our model) probably belong to this cluster. The other mutations do not
segregate in a particular region (Fig. 3B). However, the L271P mutation which is located next
to H270 proposed to intervene in the thioester formation (see above) probably introduces a
dramatic change in the folding of the SHH-C protein further blocking the amino acid re-
arrangement. We were not able to get more insights from the model, neither for the mutation
affecting the V332 residue that lies at the surface of SHH-C nor for the G290 residue located
in the nineteen residue insertion region.
Analysis of human WT SHH processing in transfected HEK293 cells
To examine the effect of mutations on the processing of SHH and on its biological
activity, we first characterized the expression of WT SHH transiently transfected in HEK293
cells. Synthesis and secretion of the protein were further monitored by Western blot analysis
using two polyclonal antisera recognizing specifically SHH-N (167Ab) (42) or SHH-C
(1229Ab) (Fig. 1). Both sera detected SHH precursor protein as 48-51 kDa polypeptides in
homogenates from transfected cells in agreement with the predicted molecular mass deduced
from SHH sequence (44). These polypeptides might reflect SHH protein prior and after signal
peptide cleavage as previously observed for chicken and mouse Shh proteins (45). As
expected, SHH-N and SHH-C fragments were further identified as a major (Fig. 4A) and a
faint (Fig. 4B) polypeptide migrating with a relative molecular mass of 22 or 33 kDa,
respectively, indicating that the autoprocessing reaction had occurred.
SHH-N was further released in the culture medium of these transfected cells as a
22 kDa polypeptide (Fig. 4A). A broad signal migrating with a relative molecular mass of 35-
38 kDa was also detected in the culture medium indicating that SHH-C fragment was also
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secreted (Fig. 4B). The decrease in SHH-C mobility observed in the culture medium is
consistent with glycosylation maturation of N-linked carbohydrates as previously reported for
drosophila hh, chicken and mouse Shh (45, 46). All these signals were absent in homogenates
and media from mock cell preparations thereby indicating their specificity (Fig. 4).
Biochemical and functional analysis of missense mutations located within the SHH-N
domain
We decided to model by site-directed mutagenesis 11 of these missense mutations
located within the main regions of SHH associated with HPE mutations identified in our
model. These mutants were transiently transfected in HEK293 cells and their expression
analyzed by Western blot. The expression pattern of the first mutant examined harboring the
G31R mutation, was comparable to that of WT SHH, except for the appearance of a band
above the major 22 kDa SHH-N detected with WT sample. However, SHH-N secreted
peptide was identified as a major 21 kDa peptide instead of 22 kDa for the WT protein (Fig.
5A, 6C). We further compared the in vitro biological activity of the WT and G31R mutant
SHH by measuring their ability to induce AP activity in C3H10T1/2 cells. This assay
represents a reliable measure of the differentiation of these cells to an osteoblast lineage (47).
The culture media from HEK293 cells transfected with either the WT- or G31R-SHH were
applied to C3H10T1/2 cells. The AP activity induced by the G31R mutant was significantly
reduced and represented 20 ± 12% (mean ± S.E.M., n=5, p<0.005) of that observed with the
WT protein (Fig. 7A). To determine whether the low activity of the G31R mutant was due to
proteolytic degradation during the course of the assay, we submitted to Western blot analysis
a sample of the culture medium at the start of the culture (J=0) and at the end of the
experiment 5 days later (J=5). The level of WT SHH-N peptides was only slightly decreased
whereas that of G31R mutated SHH-N peptides was dramatically reduced at the end of the
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experiment indicating that this G31R mutant is highly susceptible to proteolytic degradation
(Fig. 7B-C).
The signals corresponding to SHH-N peptides were absent from homogenates or
culture media of cells expressing SHH harboring the W117R, W117G, H140P, T150R or
C183F mutations indicating a profound modification of the SHH-N expression pattern, and to
a lesser extent of that of the SHH precursor (Fig. 5A, 6A-B). Indeed, the signal corresponding
to the precursor protein (48-51 kDa) was greatly diminished for the T150R and C183F
mutants (Fig. 6A). A faint signal corresponding to SHH-C peptide of W117R, W117G and
H140P mutants was detected in the culture medium of transfected cells (Fig. 5B) and was
more evident after a longer exposure time of the film (data not shown). This observation
suggests that at least a limited autoprocessing reaction of the mature protein had occurred in
these three mutants. In agreement with the absence of detectable released SHH-N peptides
(Fig. 5A, 6C), we did not detect a significant functional activity in the culture medium of
HEK293 cells transfected with the five mutants in the differentiation assay (Fig. 7A). These
results suggest either a susceptibility to proteolysis of W117R, W117G, H140P, T150R, and
C183F mutants or an impairment of their autoproteolysis reaction or both.
Biochemical and functional analysis of missense mutations located within the SHH-C
domain
The D222N and A226T mutations affect residues located in the Hint (Hedgehog
Intein) module which is related to self-splicing proteins (36). D222 and to a lesser extent
A226 residues are highly conserved between species (Fig. 2B) (36), but a role in
autoprocessing of SHH protein has not yet been described. We observed that the precursor
protein of mutants harboring the D222N and A226T mutations significantly increased
compared to the WT (n=3, p<0.05; Figs. 5A, 6A) suggesting an impairment of the
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autoprocessing reaction. In agreement with this hypothesis, membrane-associated SHH-N as
well as soluble SHH-N and SHH-C peptides from the D222N mutant were reduced
significantly compared to the WT protein (n=3, p<0.05) (Figs. 5A-B, 6B-C) and the
corresponding functional activity represented 53 ± 5 % (mean ± S.E.M., n=7, p<0.005) of WT
activity (Fig. 7A). However, the A226T mutant did not show any further differences
compared to the WT.
For cells transfected with the mutant harboring the L271P mutation, Western blot
analysis using the anti SHH-N serum revealed the presence of several peptides that might
correspond to proteolytic fragments of the precursor protein in both the cell homogenate and
culture medium (Fig. 5A). Interestingly, the anti SHH-C serum did not detect the fragments
corresponding to either the mature protein or the processed peptides (Fig. 5B) suggesting
again that proteolysis had occurred. Alternatively, the mutation may have modified the three-
dimensional structure of the protein preventing recognition by SHH-C antiserum. The
deleterious effect of the L271P mutation was confirmed by the very low AP activity (Fig.
7A).
The two last mutations studied belong to the cluster comprising residues mutated in
the carboxyl end of SHH-C. The SHH-N peptides corresponding to either I354T or A383T
mutants were not detected in the cell homogenates suggesting that the autoprocessing reaction
had not occurred in these proteins. The A383T mutation led to the accumulation of the
precursor protein before its secretion in the culture medium compared to the WT (n=3,
p<0.05) (Fig. 5, 6A). However, we did not detect a significant increase of AP activity when
the culture medium was assayed on C3H10T1/2 cells showing that the secreted precursor
protein observed in the culture medium was not active in this assay (Fig. 7A).
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DISCUSSION
We have modeled for the first time a large panel of SHH mutations associated with
HPE. After transfection of the mutant proteins in HEK293 cells, we have analyzed their
biological activities by using the hedgehog-induced response of the C3H10T1/2 cells. Our
results show that at least 3 different classes of inactivating SHH mutants exist. The first one
includes mutants affecting stability of the precursor protein or of SHH-N and displaying
mutations located either within SHH-N (G31R, W117R, W117G, H140P, T150R, C183F) or
SHH-C (L271P). The absence or low functional activity in the cell-based assay with these
mutants is in agreement with the absence or the very low expression level of mutated SHH-N
fragment in the supernatant of the transfected cells. The crystal structure of murine Shh-N has
revealed the presence of a Zn2+ ion coordinated by H141, D148 and H183 that respectively
correspond to H140, D147 and H182 in human SHH-N. This arrangement is similar to that
found in several Zn2+ hydrolases and has led to the hypothesis that SHH might display
hydrolase activity (48, 49). Denaturation studies conducted on the H140A and D147A SHH-N
mutants have previously shown that the loss of Zn2+ binding is accompanied by a misfolding
of the proteins, which were thus highly susceptible to proteolytic degradation and very
difficult to purify (49). One can speculate that the substitution of a histidine by a proline
residue (H140P) would also impair Zn2+ binding. In the same way, the substitution of
threonine (T150) and cysteine (C183) located next to the important D147 and H182 residues,
by an arginine and a phenylalanine, respectively, would also affect Zn2+ binding. Thus, the
H140P, T150R, C183F SHH-N mutants might be very unstable and susceptible to proteolysis
which is in agreement with our results. This may be also the case for the W117R and W117G
mutants. When expressed in vivo in the chick ventral neural tube or the forebrain, the W117R
and W117G SHH mutants failed to induce appropriate transcription factors associated with
SHH signaling (50). This study did not allow to conclude if this was due to a reduced activity
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or another alteration of the mutant protein. Our present studies conducted with the W117R
and W117G mutants indicate that the lack of activity would be primarily due to the lack of
secreted SHH-N.
The G31R mutation, is located in the aminoterminal region of SHH-N and lies next to
a phenylalanine (F30) and a proline (P26) postulated to make hydrophobic interactions with
residues delimitating the Zn2+ binding cleft of a SHH-N symmetrical molecule (43).
Therefore, the G31R mutation might destabilize these interactions leading to a protein with an
increased susceptibility to proteolysis.
Our model of SHH-N indicates that several of these mutated residues belong to a
cluster of amino acids that lie at the surface of the protein (Fig. 3A). Therefore, it is plausible
that this region may interact directly with an as yet unidentified protein to stabilize or to
prevent further degradation of SHH-N, or alternatively of SHH protein before autoproteolysis.
The L271 residue is a highly conserved residue found in most inteins (36) residing
next to H270 and T267. These two conserved residues have been demonstrated to participate
directly in an internal thioester formation with C258 in drosophila hh-C (36), allowing further
cholesterol transfer. The replacement of a leucine by a proline at position 271 would imply a
profound modification of the ternary structure of the protein not only preventing the thioester
formation but also increasing its susceptibility to proteolysis as observed in our study.
The second class of mutations includes those that affect the autoprocessing reaction.
The first one, D222N, is located in the aminoterminal tail of SHH-C and found in almost all
inteins (36). The conservative mutation replacing an aspartate by a slightly less charged
asparagine, might impair ionic interactions and modify the ternary structure of the protein,
possibly reducing the overall autoprocessing reaction leading to a reduced production of
SHH-N.
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The two other mutations, I354T and A383T, are located in the carboxy-terminal tail of
SHH-C and were characterized by a very low level of secreted SHH-N in the culture medium
of transfected cells. The crystal structure and molecular studies of the autoprocessing domain
of drosophila hh have revealed the role exerted by the carboxyl domain. A construct lacking
the last 63 amino acids at the carboxyl-terminal was able to form the thioester intermediate,
but failed to transfer cholesterol and a direct role in cholesterol binding for this region has
been proposed (36). Therefore, it is plausible that structural modifications introduced by the
I354T and A383T located in the same region, prevent further the binding and/or the transfer
of the cholesterol molecule.
A226T belongs to the last class of mutations which do not change the biological
activity of the mutated protein compared to the WT SHH. Whereas our data suggest an
impairment of the autoprocessing reaction of the mutated protein as indicated by the
accumulation of the precursor protein, further in vitro and in vivo experiments are required for
identifying functional differences between the WT and the mutated protein.
Interestingly, among 44 mutations reported in Table 1, 26 are also found in the
kindred. Nevertheless, the correlation between genotype and phenotype is elusive, as in 10
cases the SHH mutation in the kindred is not associated with evident signs of the disease.
There is also a lack of correlation between the phenotype severity on the one hand, and the
alteration of the biochemical properties and biological activity of the associated mutant
proteins on the other hand (Table 1). This phenomenon may be due to penetrance differences
or to the existence of modifier genes. HPE was first described as a "single gene" autosomal
dominant disease with a proved genetic origin associated with haploinsufficiency of SHH
(51). Then, the description of patients presenting with mutations in both SHH and a second
HPE gene (11), suggested that HPE was a multigenic disease (52).
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Animal models of HPE confirm this hypothesis as Shh-/- mice have features of HPE or
die during embryonic development, whereas the heterozygous mice appear normal (31). Other
animal models, heterozygous for mutations in genes belonging to the Shh or TGFβ pathways,
show that only double mutants show signs of HPE (53, 54), suggesting a bigenic inheritance
in mouse models. Several human genetic diseases such as Usher syndrome, insulin resistance
or Hirschprung disease, are now described as multigenic diseases (55-57). Recently Gabriel
and collaborators (58) conducted a genome scan in families with Hirschprung disease and
identified susceptibility loci at 3p21, 10q11 (RET, receptor tyrosine kinase for GDNF) and
19q12, necessary and sufficient to explain the recurrence risk and population incidence. They
propose a multiplicative effect of these three loci where the 3p21 and 19q12 loci could be
RET-dependent modifiers. This approach could serve as a model for dissecting a complex
disease such as HPE, provided a sufficient number of families is available. Besides the effect
of modifier genes, environmental or metabolic factors may contribute to variable
expressiveness and explain the absence of a strict genotype-phenotype correlation.
In summary, we have, to our knowledge, characterized for the first time the
biochemical and biological properties of a large panel of SHH mutations associated with HPE.
We have shown that most of these mutations have a deleterious effect on the availability of
the SHH-N fragment and its biological activity evaluated by the AP reporter cell-based assay,
which appears to be an appropriate functional assay, due to the correlation found between
functional and biochemical data. However, because of the lack of correlation between
genotype and phenotype for SHH-associated mutations, our study underlines the necessity for
further careful analysis to understand the complex molecular and biochemical traits linked to
HPE.
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ACKNOWLEDGEMENTS
This work was supported by the GIS “Institut des Maladies Rares” to V.D., M.R., D.R.
and S.O. We acknowledge Leïla Lazaro for helpful discussions on HPE.
ABBREVIATIONS
AP, alkaline phosphatase; HPE, holoprosencephaly; PTC, Patched; SMO,
Smoothened; SHH, Sonic hedgehog; SHH-N, Sonic hedgehog aminoterminal protein; SHH-
C, Sonic hedgehog carboxyl terminal protein; WT, wild-type.
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FIGURE AND TABLE LEGENDS
Figure 1. Schematic representation of SHH protein with the location of mutations,
deletions and insertions associated with HPE. The mutations are distributed in the signal
peptide (SP, amino acid 1-23) and both in the signaling (SHH-N, amino acids 24-197) and in
the autocatalytic (SHH-C, amino acids 198-462) domains. The black arrowhead and arrow
indicate the removing of the signal peptide and the autoprocessing reaction associating
cleavage and attachment of cholesterol to the C-terminal end of SHH-N, respectively.
Asterisks denote the nonsense mutations; ins, insertion; del, deletions. Horizontal black lines
indicate the position of the polypeptides against which the antibodies 167Ab and 1229Ab are
directed. Bottom panels illustrate post-translational maturation of SHH precursor leading to
the active SHH-N fragment modified by a cholesterol- and a fatty acid-molecules at its C- and
N-terminal ends, respectively.
Figure 2. Comparison of the amino acid sequences of drosophila, mouse and human
hedgehog proteins. (A) Amino acid sequence alignment of the human SHH-N terminal
domain (SHH_HUMAN, residues 38-194) with Shh from mouse (SHH_MOUSE, residues
39-195). (B) Amino acid sequence alignment of the human SHH-C terminal domain
(SHH_HUMAN, residues 198-365) with hh from Drosophila melanogaster (HH_DROME,
residues 258-402). Identical and homologous residues between aligned sequence pairs are
enclosed in black and gray boxes, respectively. The alpha-helices and beta strands found in
the X-ray structure of SHH_MOUSE (35) and HH_DROME (36) are depicted by rectangles
and arrows, respectively. The residue numbering for each protein is indicated.
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Figure 3. Three-dimensional model of human SHH-N terminal (A) and C-terminal
domains (B) and localization of some SHH mutations associated with HPE. The main
chain course is indicated by a yellow ribbon. Catalytically important amino acids are labeled
in red; residues whose missense mutation has already been linked to HPE signs are labeled in
white. Yellow dots indicate amino acid residues not modeled.
Figure 4. Western blot analysis of WT SHH transiently expressed in HEK293 cells. Cells
were transfected with WT SHH (SHH) or control (mock) vector. Cell homogenates (Ho,
20 µg) or media (Med, 15 µl) were analyzed using antisera directed against SHH-N (A) or
SHH-C (B) terminal fragments to verify expression. SHH-N is detected as a major 22 kDa
signal (black arrowhead) in both the homogenate (Ho) and medium (Med) from SHH
transfected cells (A). The uncleaved SHH protein is identified as 48-51 kDa signals (black
arrows) in homogenates with both sera (A, B). The white arrowhead marks the predicted
SHH-C protein (33-38 kDa) in both the homogenates and medium (B). These signals were not
detected in homogenates and culture media from mock transfected cells with any sera.
Molecular weights (kDa) are indicated on the right.
Figure 5. Analysis of the expression and the processing of some SHH mutations observed
in HPE. Cell homogenates (upper panels) and culture media (middle panels) derived from
transient transfection of HEK293 cells with the WT or mutated SHH cDNAs were analyzed
by Western blot using antisera to SHH-N (A) or SHH-C (B). Missense mutations associated
with HPE are identified at the top of each lane. The black arrowheads, white arrowheads and
black arrows indicate the localization of the signals expected for WT SHH-N, WT SHH-C
and uncleaved polypeptides, respectively. Protein loading was verified using the mouse
monoclonal actin antibody (lower panel). Molecular weights (kDa) are indicated on the left.
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Figure 6. Quantification of SHH precursors and SHH-N polypeptides from HPE
mutants. Densitometry analysis of autoradiography from Western blots analysed with anti
SHH-N serum was performed according to Experimental Procedures. Data are means ±
S.E.M. from at least 3 independent experiments. Relative intensity of the signals
corresponding to (A) the 48-51 kDa SHH precursor bands in cellular homogenates (% of WT;
smaller size for L271P and A383T), (B) the 22 kDa SHH-N polypeptide in cellular
homogenates (% of WT), (C) the 21 kDa (white bar) and 22 kDa (grey bar) polypeptides in
culture media (% of WT 22 kDa signal).*: p<0.05 compared with WT.
Figure 7. Functional analysis of SHH alleles in C3H10T1/2 cells reporter assay and
susceptibility to proteolytic degradation of the SHH-N fragments. (A), The relative
potencies of WT and mutated SHH were assessed in C3H10T1/2 cells measuring the AP
activity induced by conditioned media derived from transfected HEK293 cells. Basal (mock)
and WT SHH-induced AP activity correspond to 0.19 ± 0.01 and 1.89 ± 0.27 optical density
units, respectively. The data are expressed as % of the WT-induced AP activity and are the
mean ± S.E.M. from 3 to 7 independent experiments performed in quadruplicates.*: p<0.005
compared with WT. (B), Samples (15 µl) of conditioned media derived from HEK293 cells
transiently transfected with WT or SHH mutants were harvested at the start (J=0) and at the
end (J=5) of the AP reporter assay and analyzed by Western blotting using the anti SHH-N
antibody. Molecular weight markers (kDa) are indicated on the right. (C), Densitometry
analysis of the signals corresponding to 21-22 kDa SHH-N peptides at J=0 and J=5 shown in
(B). The profile of the A226T mutant protein was comparable to that of the WT protein. The
G31R signal was dramatically reduced at J=5 indicating proteolysis.
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Table 1. Comparison of genetic and functional traits linked to SHH mutations associated
with HPE. For cDNA numbering (c.), +1 corresponds to the A of the ATG translation
initiation codon, and for protein numbering (p.), +1 corresponds to the initiator codon.
GenBank accession number for SHH cDNA is NM_000193.2. A: alobar HPE ; SL: semilobar
HPE ; L: lobar HPE ; Min: minor signs of HPE spectrum ; AF: atypic features of HPE ; ND:
not defined in the reference article ; +: presence ; −: absence ; ▲: presence of a mutation in
another HPE gene (c.G869A : mutation in ZIC2 ; c.1132del9 : mutation in TGIF). References
for clinical data are listed in the last column. *Values for AP activity and for intensity of the
soluble 22 kDa SHH-N peptide are from the present study. Intensities: 0, not detectable; 1+,
low; 2+, moderate; 3+, strong; 4+, comparable to WT.
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Table 1 :
Mutation(+1 is the A of the
ATG initiator)
Amino acidchange
Exon HPEtype
Mutationin
kindred
Clinicalsigns inkindred
APactivity
(presentstudy)*
SHH-Npeptide(presentstudy)*
Reference
c.9_10insGCTG Frameshift 1 A − − (11)c.G17C p.R6T 1 SL ND ND (16)
c.38_45del Frameshift 1 L ND ND (11)c.T50C p.L17P 1 L ND ND (60)c.C72A p.C24X 1 A ND ND (16)c.G91A p.G31R 1 ND + + 1+ 1+ (3)
c.211delG Frameshift 1 SL + + (16)c.A263T p.D88V 1 A + − (11)c.C298T p.Q100X 1 ND + + (3)c.G300C p.Q100H 1 SL − ND (12)c.A307T p.K105X 2 ND + − (3)
c.316_321del p.L106_N107del 2 A − ND (16)c.C329A p.A110D 2 Min + +/− (16)c.A331T p.I111F 2 Min + +/− (13)c.C345A p.N115K 2 L + − (11)c.T349G p.W117G 2 ND + + 0 0 (3)c.T349C p.W117R 2 ND + + 0 0 (3)c.G383A p.W128X 2 A + + (15)c.G388T p.E130X 2 SL + + (16)c.A419C p.H140P 2 ND + +/− 0 0 (14)c.C449G p.T150R 2 SL − ND 0 0 (16)c.C475G p.Y158X 2 AF + + (12)
c.527_535del p.E176_K178del 2 Min + − (16)c.G548T p.C183F 2 ND ND ND 0 0 (14)c.G562C p.E188Q 3 L + − (12)c.C625T p.Q209X 3 SL − ND (11)c.G664A p.D222N 3 SL + +/− 2+ 2+ (12)c.T671A p.V224E 3 ND + + (10)c.G676A p.A226T 3 ND + − 4+ 4+ (10)c.C708A p.S236R 3 ND + − (11)c.G766T p.E256X 3 L − ND (10 , 11)
c.788_808del p.R263_A269del 3 ND ND ND (11)c.T812C p.L271P 3 AF + + 0 1+ (16)c.G850T p.E284X 3 ND ND ND (10)
c.G869A▲ p.G290D 3 SL ND ND (11)c.T995C p.V332A 3 Min + − (16)
c.C1040A p.P347Q 3 A + + (16)c.T1061C p.I354T 3 A − ND 0 0 (16)
c.1132_1140del▲ p.A378_F380del 3 SL + − (11)c.G1142C p.R381P 3 SL ND ND (16)c.G1147A p.A383T 3 ND ND ND 0 0 (10)
c.1210_1224del p.G404_G408del 3 ND + − (11)c.C1270G p.P424A 3 SL + − (11)c.C1307T p.S436L 3 SL ND ND (11)
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Figure 1:
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SHH mutants implicated in HPE
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Figure 2:
A
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1b 2b β3b β4b
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Figure 3:
E188
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Figure 4:
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Figure 5:
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Figure 6 :
A
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C
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50
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200
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Figure 7 :
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Marie-Renée Durou, Véronique David and Martial RuatElisabeth Traiffort, Christèle Dubourg, Hélène Faure, Didier Rognan, Sylvie Odent,
Functional characterization of SHH mutations associated with holoprosencephaly
published online July 28, 2004J. Biol. Chem.
10.1074/jbc.M405161200Access the most updated version of this article at doi:
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