 |
INTRODUCTION |
The ED1 gene encodes a protein, ectodysplasin-A
(EDA),1 recently recognized
to be a member of the tumor necrosis factor (TNF) superfamily of
ligands. Mutations within the ED1 gene cause an X-linked
recessive disorder, hypohidrotic or anhidrotic ectodermal dysplasia
(ED1, XLHED) (Mendelian inheritance in man 305100), involving
abnormal morphogenesis of teeth, hair, and eccrine sweat glands.
Various splice forms of the ED1 transcript have been detected, but two
isoforms differing only by two amino acids, EDA-A1 (391 aa) and EDA-A2
(389 aa), contain a TNF homology domain (1-3). EDA is a type II
transmembrane protein with a small N-terminal intracellular domain and
a larger C-terminal extracellular domain containing a
(Gly-X-Y)19 collagen-like repeat with a single
interruption and a C-terminal TNF homology domain (Fig. 1A).
The TNF homology domain is similar to other members of the TNF family,
consisting of 10 predicted anti-parallel
-sheets linked by variable
loops (Fig. 1A). TNF family ligands homotrimerize to form a
pear-shaped quaternary structure able to bind a receptor molecule at
each monomer-monomer interface (4, 5). The closest EDA homologues in
the TNF family are BAFF/BLyS, APRIL, and TWEAK, although none of them
contains collagen-like repeats (6-9). All four ligands contain
consensus sequences for proteolytic cleavage by furin within their
extracellular domain. In the case of EDA, two overlapping consensus
sites are located between the transmembrane and the collagen-like
domains (Fig. 1A). EDA-A1, but not EDA-A2, has been shown to
specifically bind to EDAR, a member of the TNF receptor superfamily
that, like most members of the TNF receptor family, activates the
NF-
B and c-Jun N-terminal kinase pathways (3, 10). Mutations in DL
(EDAR), the human homologue of the murine downless locus, produce an
identical phenotype to loss of function of EDA (11, 12). XEDAR, another
member of the TNF receptor superfamily that also activates the NF-
B
pathway, binds EDA-A2 but not EDA-A1. Although EDA-A1 and EDA-A2 are
closely related splice variants, the respective proteins appear to have
different patterns of expression in mouse skin and hair follicles (3). Intracellular signals elicited by EDA in vivo rely at least
in part on the activation of NF-
B, because a rare form of HED
associated with immunodeficiency (HED-ID) correlates with mutations in
NEMO/IKK-
, an essential component of the NF-
B pathway (13).
In order to get insight into the structure-function relationship of
EDA, we identified 44 mutations (17 of which have not been reported
previously) in unrelated families with XLHED and studied their effect
on the properties of EDA in vitro. The mutations clustered
in three functionally important domains as follows: a TNF homology
domain necessary for receptor binding, a bundle-forming collagen domain, and a cleavage site for a furin protease. This indicates that the receptor binding ability of EDA and also its oligomerization and proteolytic processing to a soluble form are critical events for its action in vivo.
 |
MATERIALS AND METHODS |
Families Analyzed--
Seventy apparently unrelated families
with hypohidrotic ectodermal dysplasia were identified by clinicians at
various centers and recruited into a research study approved by the
institutional review board of the Oregon Health Sciences University.
Consent was obtained for the use of clinical information, relevant
family history, and DNA samples. Family histories and clinical data
were provided by the clinical centers. A single individual was screened for mutations in each family. Fifty affected males had the classical findings of hypodontia, hypotrichosis and hyphidrosis (decreased amount
of teeth, hair, and sweat glands, respectively), whereas 20 carrier
females were either obligate carriers or had clear manifestations of
the disorder. 38 of the families had more than one affected individual
(multiplex families), whereas the remaining 32 cases were sporadic
(simplex families). A subset of the families had been analyzed
previously and shown to have no detectable mutations within exon 1 of
ED1 (14).
Mutation Detection--
The eight exons coding for EDA-A1 and
EDA-A2 were amplified by PCR from genomic DNA of patients and
controls using a Stratagene Robocycler with the primers and
conditions listed in Table I. Genomic DNA
samples from controls and 15 known mutations, previously detected by
complete sequencing of the ED1 gene, were run as controls under the same conditions utilizing single-stranded conformation polymorphism (SSCP) analysis (14). PCR fragments from exons 1, 5, and 9 were digested to produce restriction fragments in the 100-270-base
pair range (Table I). All samples were then denatured at 95 °C for 5 min, chilled on ice, and electrophoresed on a 0.5× MDETM
polyacrylamide gel (FMC Corp.) at room temperature for SSCP analysis. Electrophoresis of all samples, except for those from exon 1, was
performed in 10% glycerol. DNA was visualized by silver staining (14).
Samples having abnormally migrating bands were re-amplified from stock
genomic DNA, purified by use of Geneclean (Bio 101), and sequenced on
both strands by use of ABI end-terminator chemistry on either a 373A or
377 automated sequencer. A previously identified recurrent mutation,
G467
A (R156H) was not detectable by SSCP using several
sets of conditions. However, the mutation eliminates a restriction site for Fnu4H1 (gc/ngc). Therefore, PCR fragments from exon 3 with normally
migrating bands on SSCP were digested with Fnu4H1 and electrophoresed
on a 3% agarose gel. Genomic DNA from the mothers of individuals with
confirmed mutations were also sequenced. Sequence alignments were
performed using a pairwise sequence alignment program.
Production of Recombinant Proteins--
Cloning of ligands and
receptors in suitable vectors and expression of the recombinant
proteins in 293T cells were performed essentially as described
previously (15). The source of cDNAs used in this study was as
follows: mouse EDA-A1 (aa 245-391) and EDA-A2 (aa 245-389) were
amplified by nested reverse transcriptase-PCR from mouse lung and brain
cDNAs, respectively (using primer pair 5'GGA TTC CAG GAA CAA CTG
TTA TGG3' and 5'CCT ACA CAC AGC AAG CAC CTT AGA G3' for the initial
PCR). In this region, the murine and human proteins are 100%
identical. Full-length cDNAs for hEDA and hEDAR have been described
earlier (2, 12), and XEDAR cDNA was from clone 5091511 (LifeSeq®
Gold, Incyte Genomics, Palo Alto, CA). The expression construct for
XEDAR:Fc carried a human immunoglobulin µ chain signal
sequence, the region coding for amino acids 1-135 of XEDAR flanked by
AatII and SalI sites, and the Fc portion of human
IgG1, in a modified PCR-3 expression vector (Invitrogen). The
extracellular domain of hEDAR (aa 1-183) was cloned into the Fc fusion
expression vector. Expression vectors for various soluble forms of
FLAG-tagged EDA were constructed in a vector containing the signal
peptide of hemagglutinin (see Fig. 1B) as follows: EDA-A1
Glu245 (aa 245-391), EDA-A2 Glu245 (aa
245-389), EDA-A1 Ser160 (aa 160-391), and EDA-A1
Ser66 (aa 66-391). The following point mutations were
generated by PCR-based methods in both EDA-A1 and EDA-A2 E245: H252L,
Y343C (Y341C), A356D (A354D), S374R (S372R), T378M (T376M)
(mutations in parentheses refer to EDA-A2 which lacks amino
acids 307 and 308. For the sake of clarity, the EDA-A1 mutant
nomenclature will be used for both ligands.). The following mutants
were also constructed: EDA-A1 Ser66 R153C, EDA-A1
Ser66 R156C, and EDA-A1 Ser66
185-196. For
the expression vectors for EDA, FasL fusion proteins contained the
hemagglutinin signal peptide, a FLAG tag, the entire or
truncated collagen domain of EDA (aa
Ser160-Arg244, aa
Ser160-Arg244
185-196, aa
Ser160-Arg244
218-223, aa
Ser160-Arg244 G207R, aa
Gly210-Arg244), and the TNF homology domain of
FasL (aa Glu139-Leu281 of hFasL). Finally,
mutations C86R and R87P were introduced in hEDAR:Fc. All constructs
were sequenced on both strands. CHO cells were transfected with
Polyfect reagent (Qiagen), according to the manufacturer's instructions.
Cell Lines--
HEK-233T cells were maintained in Dulbecco's
modified Eagle's medium and CHO dhfr
cells in
-minimum essential medium containing ribonucleosides and
deoxyribonucleosides (Life Technologies, Inc.). Culture media were
supplemented with 10% of heat-inactivated fetal calf serum and antibiotics.
Deglycosylation--
Denatured samples of EDA were submitted to
deglycosylation with peptide N-glycanase F for 16 h at
37 °C, following the manufacturer's recommendations (New England Biolabs).
Receptor Binding ELISA--
The following steps were performed:
(a) coating with 5 µg/ml mouse anti-human IgG antibodies
(Jackson ImmunoResearch) in 50 mM carbonate buffer, pH 9.6;
(b) incubation in block buffer (PBS, 0.5% Tween 20, 4%
skimmed milk); (c) incubation with cell supernatants containing the indicated receptor:Fc fusion proteins (20 µl
supernatant in 100 µl of incubation buffer: PBS, 0.05% Tween 20, 0.4% milk); (d) incubation with cell supernatants
containing the indicated FLAG ligands (20 µl of supernatant in 100 µl of incubation buffer); (e) incubation with 0.5 µg/ml
biotinylated anti-FLAG M2 antibody (Sigma) in incubation buffer;
(f) incubation with horseradish peroxidase-coupled
streptavidin (1/4000, Jackson ImmunoResearch) in incubation buffer.
Alternatively, steps d
f were replaced by an incubation
with horseradish peroxidase-coupled goat anti-human IgG. Four washing
steps with PBS 0.05% Tween 20 were performed between incubations. 100 µl of ortho-phenylenediamine solution was added (Sigma
fast o-phenylenediamine dihydrochloride tablet sets, Sigma),
and the reaction was stopped by addition of 50 µl of 2 N
HCl, and A490 nm was taken.
Immunoprecipitations--
FLAG ligands (about 100 ng in 100-400
µl of cell supernatants) were added to 5 µl of M2-agarose affinity
matrix (Sigma). Receptors:Fc (about 500 ng in 100-400 µl of cell
supernatants) were mixed with FLAG ligands (about 100 ng) and 5 µl of
protein A-Sepharose. All samples were diluted to 1 ml with PBS and
incubated on a rotating wheel for 1 h at 4 °C. Beads were
recovered in mini-columns, washed with 2× 400 µl of PBS, and eluted
in 15 µl of 0.1 M citrate/NaOH, pH 2.7. Neutralized
eluates were prepared for Western blot analysis under reducing
conditions. Membrane were probed with anti-FLAG M2 antibody or rabbit
anti-EDA antibodies and subsequently reprobed with goat anti-human IgG antibodies.
Gel Permeation Chromatography--
200 µl of transfected cell
supernatants mixed with internal standards (40 µg of catalase and 100 µg of ovalbumin) was applied onto a Superdex-200 column and eluted in
PBS at 0.5 ml/min. Fractions of 700 µl were collected and
supplemented with 40 µg of lysozyme. Proteins were recovered by
precipitation in chloroform/methanol and analyzed by Western blot.
Cytotoxic Assay--
Cytotoxic assays in the presence or absence
of 1 µg/ml of M2 antibody were performed as described previously,
using the FasL-sensitive Jurkat cell line and measuring cell viability
after 16 h with the phenazine methosulfate/3-[4,
5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfonyl]-2H-tetrazolium test (16).
N-terminal Sequence Determination--
EDA
Ser66 and EDA Ser66 R153C were expressed
in 293T cells. Supernatants (10 ml) were immunoprecipitated with 10 µg of EDAR:Fc and 20 µl of protein A-Sepharose as described above.
Samples were reduced, blotted onto polyvinylidene difluoride membranes,
and stained with Ponceau S. Bands of interest were submitted to
automated Edman degradation using an ABI 120A gas phase sequencer
coupled to an ABI 120A analyzer equipped with a
phenylthiohydantoin C18 2.1 × 250 mm column. Data were
analyzed using ABI 610 software.
Anti-EDA Antibodies--
Anti-EDA rabbit anti-serum (AL166) was
obtained by custom rabbit immunization using purified FLAG-EDA E245 as
immunogen (Eurogentech, Seraing, Belgium). Serum was used at a dilution
of 1/500 for Western blotting.
 |
RESULTS |
Identification of Mutations in HED--
Twenty five different
mutations of the ED1 gene were detected in 44 of the 70 unrelated families analyzed (63%), and 9 were demonstrated to
have occurred de novo (footnoted in Table
II). As 17 of these mutations had
not been described previously, the number of different ED1
mutations identified to date in XLHED patients totals 53 (Table II).
Analysis of the variants identified in this study showed a
significantly non-random distribution of the mutations within the
sequence of EDA. Fifteen separate families had mutations within the
7-amino acid domain (aa 153-159) encoding two adjacent
potential furin cleavage sites. Mutations in 13 other families
specifically affected the collagen-like domain. Ten of the families had
18 or 36 nucleotide in-frame deletions eliminating 2 or 4 of the
(GlyXY) repeats, and three families had missense mutations
altering glycine residues. Nine missense mutations were detected within
the TNF-like domain (aa 245-391), which is necessary and sufficient
for receptor binding (3, 10). In the latter class of mutations,
threonine 378 was altered in 4 independent families. In addition, 6 families had nonsense or frameshift mutations altering one or more of
the domains cited above. Altogether, these data strongly suggest that
the predicted furin, collagen, and TNF-like domains are essential for
the function of EDA in vivo. Finally, a single splice site
mutation was detected in the current study (IVS8 +5G
A),
which affects the donor site of EDA-A1 but not that of EDA-A2. This
mutation is predicted to eliminate EDA-A1 only, in contrast to all
other mutations that alter both EDA-A1 and EDA-A2.
In an effort to understand the mechanism by which mutations detected in
families with XLHED affect the function of EDA, we prepared a range of
wild type and mutant recombinant EDA proteins and analyzed them in
different in vitro assays. The mutations selected in this
study are shown in bold in Table II, and the various protein constructs
are schematized in Fig.
1B.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 1.
Sequence alignment and features of EDA and
EDA constructs. A, sequence alignment of EDA and of its
closest relatives in the TNF family. The transmembrane domain, furin
recognition sequences, collagen domain, and -sheets A-H of the TNF
homology domain are indicated (bold lines). The starting
position (Ser66, Ser160, Gly210,
and Glu245) of the various recombinant proteins is shown by
an arrow, and the various mutations studied are also
indicated. Potential N-glycosylation sites are
boxed. Amino acids Val307 and Glu308
are absent in EDA-A2, but numbering of mutations in EDA-A1 and EDA-A2
is conserved for clarity. B, schematic representation of
expression constructs for soluble EDA. F = FLAG tag
plus a linker of 10 amino acids. Point mutations are indicated by an
asterisk. In FasL fusion molecules, the TNF homology domain
of EDA was replaced by that of FasL.
|
|
Binding Specificity of EDA-A1 and EDA-A2--
We have previously
shown the specific interaction of EDA-A1 with mouse EDAR (10), and we
now document the interaction of EDA-A1 with human EDAR (Fig.
2, A and C).
Recombinant EDA always migrated as a double band when analyzed by
SDS-polyacrylamide gel electrophoresis, and both bands interacted with
the receptor (Fig. 2A). Peptide N-glycanase F
digestion of EDA-A1 indicated that the lower and upper bands
corresponded to unglycosylated and N-glycosylated isoforms,
respectively (Fig. 2B). To demonstrate the specificity of
EDA/EDAR interaction, we expressed EDAR:Fc with mutations previously
detected in two families with autosomal recessive hypohidrotic
ectodermal dysplasia (online Mendelian inheritance in man
224900) (12). These point mutations either abolished (C87R) or strongly
decreased (R89H) EDAR binding to EDA-A1. In addition, EDAR C87R was
recovered in poor yield, suggesting folding or solubility defects (Fig.
2A). In agreement with recently published data (3), we found
that the splice variant EDA-A2 interacted with a distinct receptor,
XEDAR, and that XEDAR interacted only with EDA-A2 among 17 ligands of
the TNF family tested (Fig. 2C).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
Characterization of EDA-A1/EDAR and
EDA-A2/XEDAR interactions. A, soluble recombinant
EDA-A1 was immunoprecipitated (IP) with EDAR:Fc fusion
protein (wt or with the indicated mutations) and analyzed by Western
blot (WB) using anti-FLAG antibody. Precipitated EDAR:Fc is
shown in the top panel. B, deglycosylation of EDA-A1.
EDA-A1s (wt or S374R mutant) were treated ± peptide
N-glycanase F (PNGaseF) and analyzed by Western
blot with anti-FLAG antibody. C, receptor binding ELISA.
XEDAR:Fc, EDAR:Fc, and various control receptors:Fc were captured on an
ELISA plate. FLAG-EDA-A1, FLAG-EDA-A2, and various control FLAG ligands
were added as indicated. Interactions were revealed using a monoclonal
anti-FLAG antibody. Fn14 is a recently described receptor for TWEAK
(31).
|
|
Mutations in the TNF Homology Domain Affect Receptor
Binding--
Most of the selected EDA-A1 and EDA-A2 mutants affecting
the TNF homology domain could be expressed as soluble, FLAG-tagged secreted proteins, but mutant A356D was entirely retained inside the
cells, suggesting that it experienced folding or solubility problems
(Fig. 3, A and B).
Interestingly, the glycosylation pattern of the mutant S374R was
indistinguishable from that of the wild type protein, although this
mutation abolishes one of the two predicted N-glycosylation
sites within the TNF-like domain (Fig. 2B). We deduce that
N-glycosylation of EDA occurs on Asn313, whereas
Asn372 remains unglycosylated. All mutants lost their
ability to interact with EDAR and XEDAR, in sharp contrast with the
wild type proteins. The only exception was EDA-A1 S374R, which
displayed weak and variable but apparently specific binding to EDAR
(Fig. 3A). Surprisingly, only the unglycosylated isoform of
this mutant interacted with the receptor.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Expression and receptor binding of wild type
and mutated forms of EDA-A1 and EDA-A2. A, binding of
wt and mutant EDA-A1 to EDAR. Supernatants and cell extracts of
transfected cells are shown in the top two panels. Ligands
in supernatants were immunoprecipitated (IP) with EDAR:Fc
and analyzed by Western blot (WB) in the 3rd
panel. Precipitated EDAR:Fc is shown in the bottom
panel. Results of the immunoprecipitation with a control receptor
(Fas:Fc) is shown for EDA-A1 wt and S374R. B, binding of wt
and mutant EDA-A2 to XEDAR. Analysis was performed essentially as in
B, except that EDA-A2 and XEDAR were used.
|
|
In order to assess whether the mutations affected the quaternary
structure of EDA, the size of the recombinant proteins was estimated by
gel permeation chromatography (Fig. 4).
Wild type EDA and the mutant Y343C eluted with an apparent mass
of about 70-kDa, which is compatible with the predicted trimeric
structure (Fig. 4). Mutants H252L and S374R displayed dissociation of
the glycosylated and unglycosylated subunits, and the latter mutant had
the propensity to elute as high molecular weight complexes. EDA-A1
T378M was poorly expressed and eluted apparently as high molecular
weight aggregates, whereas EDA-A2 T378M was readily secreted and
migrated with an apparent molecular weight smaller than that of
the wild type protein. Taken together, the results indicate that,
although the impact of the various mutations on the structure of EDA is
different, they all affect interactions of EDA with both EDAR and
XEDAR.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 4.
Gel filtration analysis of mutated EDA-A1 and
EDA-A2. wt and mutant EDA-A1 Glu245 (left
panels) and EDA-A2 Glu245 (right panels)
were loaded onto a Superdex-200 column and eluted in phosphate-buffered
saline. Fractions (700 µl) were precipitated and analyzed by
anti-FLAG Western blot. The top panel shows Ponceau S
staining of the co-injected internal standards catalase and
ovalbumin.
|
|
The Collagen Domain Induces Multimerization of EDA
Trimers--
Soluble, FLAG-tagged recombinant EDA with and without the
collagen domain (EDA Ser160 and EDA
Glu245) and a deletion mutant lacking four of the
GlyXY repeats (EDA Ser160
185-196) all bound
EDAR equally well (Fig. 5A).
The collagen domain therefore appears not to play a direct role in
receptor binding. In related proteins with collagen domains, such as
C1q, collagen triple helices form a bundle, therefore assembling
several globular trimeric heads into a single bouquet-like structure
(17). In order to test whether the collagen region of EDA multimerizes, we generated EDA:FasL fusion proteins. We have shown previously that
the TNF family member FasL is not cytotoxic as a soluble trimer, unless
trimers are multimerized by means of cross-linking antibodies (here an
anti-FLAG tag antibody) (16) (Fig. 5C, upper left panel).
When FasL was fused to the collagen domain of EDA (amino acids
Ser160-Gly242), its cytotoxic activity was
increased by more than 100-fold, indicating that ligand multimerization
had likely occurred. A similar effect was obtained with the collagen
domain of EDA
185-196 and EDA
218-223, suggesting that these
deletions did not affect multimerization. In contrast, no such
aggregation effect was observed when the first half of the collagen
domain was deleted (EDA Gly210) or when the point mutation
G207R was introduced (Fig. 5C).Taken together, these results
indicate that the collagen triple helix of EDA forms multimers that are
apparently not affected by in frame deletions but can be disrupted by
point mutations.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of the collagen domain on the receptor
activity of EDA-A1 and on the cytotoxic activity of FasL.
A, EDA-A1, with or without wild type or mutated collagen
domain, was immunoprecipitated (IP) with EDAR:Fc and
analyzed by Western blot (WB) as indicated. B,
FasL and the various EDA:FasL fusion proteins were immunoprecipitated
with Fas:Fc and analyzed by Western blot. C, cytotoxic
activity of FasL and of the various EDA:FasL fusion proteins was
monitored on Jurkat cells, in the presence (black squares)
or in the absence (open squares) of cross-linking anti-FLAG
antibody. Cytotoxic activity in the absence of anti-FLAG reveals
oligomerization of FasL trimers by the collagen domain of EDA.
|
|
Mutations in the Furin Cleavage Site Impair Proteolytic Processing
at Arg159--
Mutation of Arg residues in the predicted
furin cleavage sites is a common cause of XLHED, strongly suggesting
that proteolytic processing of EDA at this site is necessary for its
function in vivo (Table II). A soluble form of EDA
containing the entire extracellular domain with a N-terminal FLAG tag
(EDA Ser66) was expressed in 293T cells. As expected, this
protein was processed very efficiently, and a small 12-kDa N-terminal
fragment bearing the FLAG tag was recovered in cell supernatants
instead of the full-length 40-kDa protein. In contrast to the 12-kDa
fragment, the untagged C-terminal fragment of EDA was readily recovered by affinity purification on the immobilized receptor and detected by
Western blot using anti-EDA antibodies (Fig.
6A). N-terminal sequencing of
purified processed EDA yielded the sequence SKSNEGADGPVKNKK (Ser160-Lys174), demonstrating that
proteolytic cleavage occurred after Arg159. When expressed
in 293T cells, mutation R153C did not prevent proteolytic processing of
EDA, which still occurred after Arg159. However, partial
inhibition of the processing was observed when EDA R153C was expressed
in CHO cells (Fig. 6A). Mutation R156C, which affects both
predicted furin recognition sequences, had a more drastic effect and
entirely prevented the degradation of EDA and loss of the FLAG tag in
both cell lines (Fig. 6A). Interestingly, the unprocessed
portion of wt and mutant EDA were clearly distinguishable following
SDS-polyacrylamide gel electrophoresis under non-reducing conditions,
with a proportion of the mutants R153C and R156C migrating as
disulfide-linked dimers (Fig. 6B). In summary, the results indicate that cleavage of EDA occurs at Arg159, which
corresponds to a predicted furin cleavage site, and that mutations
within the furin recognition sequence affect the cleavage to various
extents.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of mutations in the furin recognition
sequence of EDA-A1. A, the indicated EDA-A1 constructs
were transfected in both 293T and CHO cells, and cell supernatants were
collected and immunoprecipitated (IP) with anti-FLAG
antibody or with EDAR:Fc. Immunoprecipitates were analyzed by Western
blot (WB) using either anti-FLAG or anti-EDA antibodies. The
identity of the observed fragments is indicated on the right-hand
side. Proteolytic processing results in loss of the FLAG tag,
which is recovered as a small N-terminal fragment (EDA
N-term). B, supernatants of CHO cells transfected with
the indicated EDA-A1 constructs were immunoprecipitated with EDAR:Fc,
treated plus or minus dithiothreitol (DTT), and analyzed by
Western blot with anti-FLAG antibodies. Different volumes of
supernatants (indicated by +, ++, and +++) were utilized to compensate
for the loss of FLAG tag after furin-mediated processing.
|
|
 |
DISCUSSION |
In this study, mutations in the EDA gene were detected in 63% of
the families with XLHED, which is lower than the 95% rate we found
previously (2) by the direct sequencing of affected males. The lower
detection rate is the consequence of two factors. The use of a single
set of conditions lowers the sensitivity of SSCP analysis, as only 11 of the 15 known mutations run as controls could be detected under these
conditions (73%). In addition, this study included 28% of families
with "affected" females only, which was not the case in our
previous study. Indeed, the detection rate was lower in families with
female probands (45%), and this may well be due to genetic
heterogeneity for autosomal forms of HED.
A number of point mutations are located within the TNF homology domain
of EDA, but only one of them (Y343C) affects a residue which, based on
structural homology with known ligand-receptor structures (4, 5), is
predicted to interact with the receptor. Indeed, this mutation
abolished receptor binding without affecting the trimeric structure of
EDA, although we cannot exclude indirect conformational effects. All
other mutations are predicted to have indirect effect on receptor
binding, e.g. by altering the folding of EDA. It is
noteworthy that four independent mutations (G291W, G291R, A356D, R357P)
occurred in two short loops at the bottom of EDA (loops BC
and FG, see Fig. 1A). The affected amino acids are probably crucial for proper folding of the monomer, as mutation A356D resulted in insoluble EDA-A1 and EDA-A2. Another group of mutations (H252L, S374R) seems to affect the stability of the trimer,
because the resulting proteins contain a proportion of monomers. The
propensity of unglycosylated subunits to form larger aggregates support
the idea that glycosylation of some TNF family members promotes their
solubility (18). Interestingly, one of the mutations (S374R) destroys a
potential N-glycosylation site without affecting the
glycosylation of EDA, suggesting that this particular site is not
recognized by the N-glycosyltransferase. A single mutant
(S374R) retained some binding activity to EDAR. Although preferential
binding of the unglycosylated (and most probably aggregated) isoform
was observed, the interaction appeared specific, suggesting that
residual activity may be associated with this mutation in
vivo. In general, no apparent phenotype/genotype correlation was
observed, but the family with missense mutation S374R had two affected
males and an affected maternal grandfather with isolated hypodontia.
Whether residual activity of this mutant may account for the milder
phenotype, and whether there is truly a tissue-specific difference in
the function of this mutant protein remains to be determined. Finally,
one mutation (T378M) affected secretion and aggregation of EDA-A1 and
EDA-A2 in a strikingly different manner. The structural reason for this
differential behavior is unclear. In summary, all missense mutations in
the TNF homology domain result in abolished or much impaired binding of
EDA-A1 to EDAR and EDA-A2 to XEDAR.
A number of mutations occurring outside the TNF homology domain did not
affect binding to the receptor in an in vitro assay, indicating that the interaction of EDA with its receptor(s) is necessary but not sufficient for its function in vivo. In
particular, the integrity of the collagen domain appears to be
functionally essential, and we provided evidence that it may serve to
multimerize EDA trimers. This is in strong support of the hypothesis
that EDA belongs to the C1q as well as to the TNF family of proteins (19). C1q family members are characterized by the presence of a
C-terminal globular trimeric domain, with striking structural homology
to TNF (20), which is prolonged by a collagen triple helix further
assembling into an N-terminal bundle structure, giving rise to a highly
multimeric superstructure. In the TNF family, it has been shown that
the activity of soluble trimers can be dramatically increased by
antibody-mediated multimerization, thereby mimicking the membrane-bound
form of the ligand (16, 21). A highly multimeric structure of EDA would
provide a powerful means for the soluble protein to signal through high
valency receptor clustering. In line with this hypothesis, we found
that a naturally occurring point mutation (G207R) in the collagen
domain completely abolished the bundle effect. It is, however, likely
that the collagen domain also serves additional functions; a number of
families with XLHED displayed in frame deletion of 2 or 4 GlyXY repeats in the predicted bundle domain of the collagen
triple helix, i.e. before the interruption in the
GlyXY repeats. Deletions of 2 GlyXY repeats can
also be found C-terminal to the interruption. Surprisingly, the
activity of recombinant proteins containing these types of deletions
was indistinguishable from wild type, at least in the model systems
utilized. The deletions may specifically affect multimerization of the
collagen domain under in vivo conditions. Alternatively, the
collagen domain may have additional functions, e.g.
interaction with other proteins. It is well known for C1q that the
collagen domain interacts with the serine proteases C1r and C1s to form
C1, the first component of the serum complement system, and with a
number of other proteins, including membrane-bound receptors (22).
Further investigations are required to understand the molecular
mechanism underlying loss of function of EDA in these particular
deletion mutants.
15 families with XLHED displayed 5 distinct mutations in the furin
consensus recognition sequences of EDA, demonstrating an important
functional role for this 7-aa sequence. The release of soluble EDA upon
proteolytic processing is an expected event, as mRNAs for EDA and
EDAR are not expressed in adjacent cells but rather in spatially
distinct tissues, at least in the developing tooth (10). EDA contains
two overlapping furin recognition sequences (RVRR and RNKR spanning aa
153-156 and 156-159, respectively). Because Arg156 is
part of both sequences, it is hardly surprising that mutation R156C
completely abolished EDA processing. Mutation R153C also affected the
cleavage of EDA, but in a less dramatic manner. Although this mutation
destroys only one of the two furin sites, this must be sufficiently
disturbing to prevent efficient release of EDA from the cells naturally
expressing it. These cells may express low amounts of furin or furin
isoforms whose specificity may extend further than the canonical
tetrapeptide recognition sequence. However, as mutations affecting the
first furin domain invariably yield Cys residues, and as these Cys
residues appear to form novel disulfide bridges, it is possible that
this novel structural constraint prevents proper recognition of the
remaining intact furin site. It also clearly appears from this study
that, beside the furin cleavage sites, there are no alternative sites
for solubilization of EDA. In particular, the sequence RRER (aa 69-72)
and the basic motives KNKK and KGKK (aa 171-174 and 175-178) were not
cleaved in our expression system. The latter two motifs are encoded in the small exon 4 and are also found in the sequences of Tweak and
APRIL. However, mutations in these sequences have not been described so
far in association with XLHED.
A single splice site mutation was detected in the current study (IVS8
G+5
A), which affects the splice donor site of
exon 8 utilized to generate the 391-aa EDA-A1 isoform of the ligand.
EDA-A2 utilizes an alternate splice site 6 base pairs 5' to this site.
This mutation probably interferes with splicing of EDA-A1 but not
EDA-A2, as computer analysis by HSPL (prediction of splice sites in
human DNA sequence) demonstrates a complete loss of the A1 but not of the A2 donor site. This together with the fact that genetic defects in
EDA and in EDAR both lead to identical phenotypes indicate a crucial
role for EDA-A1/EDAR interactions during morphogenesis. The role of the
parallel EDA-A2/XEDAR interaction is less well established. If at all
involved in hair, sweat gland, and teeth formation, it is not able to
rescue a genetic deficiency in EDAR. In addition, there is no evidence
to date for mutations in XEDAR being associated with the HED phenotype.
XEDAR may play a distinct role in skin development, which does not
translate into an HED phenotype upon dysfunction. Alternatively,
inactivation of XEDAR might be lethal, but this would only be possible
if it binds another ligand beside EDA-A2 or fulfills a vital,
ligand-independent function. TROY/TAJ is a close sequence homologue of
XEDAR, which is also expressed in the developing skin (23, 24). The
precise functional roles of XEDAR and TROY and their interplay with the
EDAR pathway remain to be determined.