Functional analysis of missense mutations in the SHH gene of ...

SHH mutants implicated in HPE

1

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 2:

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Figure 3:

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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.

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Functional analysis of missense mutations in the SHH gene of ...

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