1 Developmental and Cell Biology, University of California Irvine, Irvine, CA
92697, USA
2 Howard Hughes Medical Institute, Department of Cellular and Molecular
Medicine, University of California, San Diego, La Jolla, CA 92093, USA
3 Department of Molecular and Cellular Biology, University of Arizona, Tucson,
AZ 85721, USA
4 Departments of Pediatrics and Genetics, Cell Biology and Development,
University of Minnesota, Minneapolis, MN 55455, USA
* Author for correspondence (e-mail: rwarrior{at}uci.edu)
Accepted 5 January 2004
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SUMMARY |
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Key words: Growth factor signaling, Heparan sulfate proteoglycan, Hedgehog, Wingless, Decapentaplegic, Tout velu, Sister of tout velu, Hereditary multiple exostoses, EXT1, EXT2
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Introduction |
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Proteoglycans can be divided into several classes based on the sugar subunits incorporated into the GAG chains. Among these, HSPGs are composed of glucuronic acid-N-acetylglucosamine (GlcA-GlcNAc) repeating disaccharide units, whereas chondroitin sulfate (CS) proteoglycans contain glucuronic acid-N-acetylgalactosamine (GalNAc-GlcA) repeats. The initial steps of both CS and HS synthesis share a common pathway: nucleotide sugar precursors are transported to the golgi where a tetrasaccharide linker is added to the core protein. Following this, the two pathways diverge. If the proteoglycan is an HSPG, the next residue added is GlcNAc, after which the sugar chain is extended by HS polymerase. If instead GalNAc is added, chondroitin synthesis ensues.
Biochemical studies suggest that human HS polymerase is a complex of two
related proteins, EXT1 and EXT2, encoded by the causative genes for the bone
overgrowth syndrome, HME (Lind et al.,
1998; McCormick et al.,
1999
). Individuals with HME develop bony nodules (exostoses) at
the ends of bones, particularly in limbs, and are at increased risk for
malignancy, suggesting that EXT1 and EXT2 are tumor
suppressors (Ahn et al., 1995
;
Stickens et al., 1996
;
Wuyts et al., 1996
).
Drosophila contains homologs for both EXT1 (ttv)
and EXT2 (sotv; Ext2 FlyBase)
(Bellaiche et al., 1998
;
The et al., 1999
). Direct
evidence that Ttv (and by extension vertebrate EXT1) is required for HS
biosynthesis in the organism comes from the finding that HS-derived GAG levels
are greatly reduced in ttv mutant larvae, while CS levels remain
essentially unchanged (Toyoda et al.,
2000a
; Toyoda et al.,
2000b
). These data suggest that HS biosynthesis in
Drosophila and vertebrates takes place through conserved
mechanisms.
Although the biochemical assays argue that Ttv plays an integral role in
GAG chain synthesis, phenotypic analysis of the ttv mutants yielded
surprising results. Loss of ttv function was reported to impair only
Hedgehog (Hh) signaling without affecting the Wingless (Wg) or the Fibroblast
Growth Factor (FGF) pathways (Bellaiche et
al., 1998; The et al.,
1999
). This result was puzzling, as mutations in
sugarless (sgl) and sulfateless (sfl), two
other genes involved in HS biosynthesis, compromise signaling by all three
ligands. Sgl is a UDP-glucose dehydrogenase that generates a nucleotide sugar
donor essential for both HS and CS synthesis. Thus, abolishing Sgl function
might be expected to cause more dramatic defects than loss of HS synthesis
alone. However, loss of Sfl should not cause more severe defects than
ttv, as Sfl is an HS-specific N-deacetylase/N-sulfotransferase:
without Sfl certain GAG modifications are lost, but without Ttv, the GAGs
themselves are not synthesized (Toyoda et
al., 2000a
). To explain this apparent inconsistency, it was
postulated that Sotv might act independently of Ttv to provide HS polymerase
activity at levels sufficient for normal Wg and FGF function but too low to
support Hh signaling (Bellaiche et al.,
1998
; The et al.,
1999
). This hypothesis predicts that the Hh pathway is more
sensitive to loss of HSPGs than are the other two pathways. Alternatively, it
has been proposed that ttv activity might be involved in generating a
specific HSPG required only for Hh signaling
(Bellaiche et al., 1998
;
The et al., 1999
).
Our data do not support these models and argue against a Hh-specific role for ttv. We have isolated mutations in sotv and have found that the loss of either sotv, ttv or both genes compromises Hh, Wg and Dpp signaling pathways in the wing imaginal disc. In each instance, there were no obvious differences in the nature or severity of the effects in the single or the double mutants, suggesting that both co-polymerases are required for the biologically relevant activity. Our biochemical data also support this conclusion, because we find that HS levels drop dramatically in ttv or sotv homozygous mutant larvae. Finally, we also observed that Hh protein levels are reduced in Hh-expressing cells that lack HS polymerase activity. This result suggests that the reduced range of Hh signaling in the absence of HSPGs may be due to increased lability of the Hh ligand. Taken together, our data argue that ttv and sotv are equally required for HS synthesis and that HS is required in at least three major signal transduction pathways.
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Materials and methods |
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Generation of germline and somatic clones
The sotv1.8.1 allele was used to generate FRT-G13
sotv1.8.1 and FRT-G13 ttv00681,
sotv1.8.1 lines by recombination. FRT-G13
ttv02055 was kindly provided by Norbert Perrimon. Embryos
lacking maternal and zygotic ttv or sotv activity were
generated essentially as described (The et
al., 1999). Mosaic mothers were mated to heterozygous males as
follows: ttv02055 crossed to
ttv02055/CyO-Ftz-lacZ;
sotv1.8.1 crossed to Df(2R)Jp8 Ftz-lacZ; and
ttv00681, sotv1.8.1 crossed to
ttv00681, sotv1.8.1/CyO Ftz-lacZ.
Imaginal disc clones were generated by crossing male flies from the FRT lines
described above to females homozygous for hs>FLP; FRT-G13
2XUbi-GFPnls. Two-to 3-day-old progeny were heat-shocked for 2 hours at
37°C and reared at room temperature until dissection 4-5 days later.
Immunohistochemistry
Antibodies were used at the concentrations listed: mAb anti-Wg
(Developmental Studies Hybridoma Bank, DSHB), 1:3 for extracellular
(Baeg et al., 2001) and 1:1000
for cytoplasmic Wg (Brook et al.,
1996
); mAb anti-Ac (DSHB)
(Skeath and Carroll, 1991
),
1:100; rabbit anti-Sal provided by Rheinhard Schuh
(Kuhnlein et al., 1994
),
1:300; rabbit anti-pMad (PS1) from Carl-Henrik Heldin
(Persson et al., 1998
),
1:1000; rabbit anti-Hh from Philip Ingham
(Taylor et al., 1993
), 1:200;
rat anti-Ci provided by Robert Holmgren
(Motzny and Holmgren, 1995
),
1:5; Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, 1:200; and
Alexa-568 secondary antibodies (Molecular Probes), 1:500. mAbs from the DSHB
were developed under the auspices of the NICHD and maintained by the
University of Iowa, Department of Biological Sciences, Iowa City, IA
52242.
Sugar chain biochemistry
Profiling of HS and CS-derived disaccharides was conducted according to
published procedures (Toyoda et al.,
2000b) with the following modifications: (1) the chromatographic
equipment included a Hitachi L-7250 autosampler and Hitachi D-7000 computer
interface, permitting disaccharide levels to be determined by comparing the
area of each peak with reference standards; and (2) HPLC profiles in
Fig. 8A and
Fig. 9A were derived from 100
third instar larvae, whereas experiments in
Fig. 8C and
Fig. 9B used 300 third instar
larvae. Standard unsaturated HS and CS disaccharides were purchased from Sigma
and Seikagaku America (Falmouth, MA), respectively.
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Results |
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The sotv transcription unit encodes a protein of 717 residues that
contains a strongly hydrophobic domain (residues 26 to 47) indicative of a
type II transmembrane protein and three aspartic acid residues (DDD)
surrounded by a stretch of hydrophobic residues in the C-terminal third of the
protein (Fig. 1). This DXD
sequence is a signature motif for UDP-sugar-dependent glycosyltransferases and
is important for their catalytic activity
(Negishi et al., 2003;
Pedersen et al., 2003
).
Comparison of Sotv with human EXT-related genes using clustal analysis
revealed that Sotv is most homologous to EXT2 (47% amino acid identity),
suggesting it is likely to be a glycosyltransferase resident in the
endoplasmic reticulum or golgi (McCormick
et al., 2000
).
|
Embryos lacking sotv activity show defects suggestive of impaired Hh and/or Wg signaling
Homozygotes for sotv die as pupae, consistent with the presence of
substantial amounts of maternally contributed sotv transcript in 0-
to 2-hour-old embryos (data not shown). To determine if sotv is
required in the early embryo, we generated females lacking sotv
function in the germline. When these females were crossed to
Df(2R)Jp6/CyO hemizygous males, embryos lacking both maternal and
zygotic sotv activity failed to hatch and showed a strong segment
polarity phenotype (Fig. 2).
The ventral surface of the wild-type larval cuticle bears rows of denticles at
the anterior of each segment interspersed with posterior naked regions.
Embryos mutant for sotv lack naked cuticle and instead display a near
contiguous lawn of denticles, a phenotype resembling, but less severe than,
that of null mutations in hh or wg
(Fig. 2B-D). Loss of both
maternal and zygotic ttv activity results in similar segmentation
defects (The et al., 1999).
The phenotype of sotv germ line clone embryos appears
indistinguishable from ttv null germline clones
(Fig. 2E). Thus, the two genes
do not have independent functions that can be identified at this level of
analysis. To examine whether sotv and ttv have additive
effects on patterning, we generated animals that lacked both gene activities.
These double mutant embryos were phenotypically equivalent to either single
mutant alone, arguing that ttv and sotv do not have
partially redundant functions (Fig.
2F).
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Dpp signaling is sensitive to the loss of HS polymerase activity
We next examined whether loss of sotv or ttv affects Dpp
signaling, as both Dpp and its human homolog BMP-2 bind heparin with high
affinity in vitro (Groppe et al.,
1998; Ruppert et al.,
1996
). Furthermore, changes in the level of Division abnormally
delayed (Dally), a GPI-anchored HSPG, affect the Dpp gradient in the wing disc
and Dpp target gene expression in many tissues
(Fujise et al., 2001
;
Fujise et al., 2003
;
Jackson et al., 1997
). To
monitor Dpp signaling, we followed phosphorylated Mothers against Dpp (pMad),
which is detected in a graded pattern that reflects Dpp activity
(Fig. 7). In wild type, the
levels of pMad are highest close to the source of Dpp at the AP boundary,
except in Dpp transcribing cells, where signaling is attenuated due to
Hh-dependent downregulation of the Dpp receptor thick veins
(Tanimoto et al., 2000
). We
found that pMad staining was strongly reduced in mutant cells that lacked
either sotv, ttv or both genes
(Fig. 7). As Hh regulates Dpp
expression in cells adjacent to the AP boundary, the loss of pMad staining in
posterior clones could be an indirect consequence of lowered Hh activity.
However, pMad levels were also reduced in clones anterior to the Dpp stripe,
where Hh is normally absent (Fig.
7C). Consequently, the reduction in pMad is a direct effect of
sotv and ttv on the Dpp gradient. Furthermore, the lower
pMad levels reflect diminished signal transduction, as shown by reduced
expression of spalt (sal) a direct transcriptional target of
the Dpp pathway in the wing pouch (de
Celis et al., 1996
). Sal levels were strongly diminished in mutant
clones, consistent with the reduction in pMad levels described above
(Fig. 7D-F).
|
To determine the effect of loss of sotv on HS synthesis, we
examined all five sotv alleles as homozygotes (data not shown) and
hemizygotes over Df(2R)Jp8. As a control, we tested a
transheterozygous combination of the null ttv alleles
(ttvK06619/ttv02055). GAGs prepared from third
instar larvae were digested into disaccharide units using either heparin or
chondroitin lyases and separated by HPLC with a reverse phase-ion pair column.
The disaccharides were then labeled by covalent fluorescence derivatisation
and measured with an inline fluorescence detector. In wild type, six
HS-derived disaccharide species are detected: the unsulfated uronic
acid-GlcNAc, two monosulfated species, UA-GlcNS and UA-Glc6S, two disulfated
disaccharides, UA-GlcNS6S, UA2S-GlcNS and a single tri-sulfated unit,
UA2S-GlcNS6S (Fig. 8A).
Fluorescence intensities permit reliable quantification of each of these
disaccharides, with a sensitivity of 1 ng/mg of dried tissue
(Toyoda et al., 2000b
;
Toyoda et al., 1997
).
We found that the HS-derived disaccharide levels were dramatically reduced
in all sotv alleles. For example, homozygotes for the
sotv1.8.1 null allele had total disaccharide levels
several hundred fold lower than in wild type
(Table 1,
Fig. 8). Larvae null for
ttv also showed severely reduced disaccharide levels (75 fold
lower, Table 1,
Fig. 8)
(Toyoda et al., 2000b
), with
the residual HS probably resulting from perdurance of maternal product. Taken
together, these findings establish that sotv is required for HS
production in vivo and argue that a Ttv/Sotv complex constitutes the
functional HS polymerase.
|
Reducing sotv function alters the sulfation of HS
HSPG GAG chains undergo extensive modification during synthesis, including
N-deacetylation, epimerization of some GlcA residues to iduronic acid, and N-
and O-sulfation. Sulfation patterns vary in a tissue-specific manner and can
differentially affect interactions with proteins such as bFGF, antithrombin
III and HGF (Nakato and Kimata,
2002). Therefore, we analyzed the residual HS chains from
ttv and sotv mutants to determine if the mutations impact
GAG modification as well as synthesis. We found that the individual
sotv alleles produced HS chains with strikingly different
compositions (Fig. 8C). For
example, as the length of the mutant protein increased, levels of the
trisulfated disaccharide UA2S-GlcNS6S rose, with the mildest allele retaining
more than 6% of wild-type levels. In general, milder alleles produced more
sulfated disaccharides. These findings show that, in addition to affecting HS
levels, mutations in sotv can affect the extent and perhaps the
pattern of HS sulfation.
Changes in sotv activity affect the levels and structure of CS polymers
Both HS and CS GAG-chain polymerization are initiated on an identical
tetrasaccharide substrate GlcA-Gal-Gal-Xyl attached to the core protein.
Consequently, disruption of the synthesis of one class of GAG chains could
affect the production of the other. Consistent with this idea, chondroitin
synthesis increases in somatic cells bearing mutations in EXT1
(Wei et al., 2000). We
therefore examined CS levels and disaccharide structure in ttv and
sotv mutants (Fig. 9).
As previously reported, ttv-null mutants display only modestly
reduced levels of CS, demonstrating the selectivity of this
EXT1-related gene for HS biosynthesis. However, the sotv
allelic series revealed an unexpected relationship between HS and CS
biosynthesis. Like the ttv nulls, the sotv1.8.1
null reduced chondroitin-derived disaccharides by a modest amount. However, in
the remaining sotv alleles, the proportion of sulfated disaccharides
increased progressively through the allelic series,
sotv2.1.2 to sotv1.4.1
(Fig. 9B). Thus, both the
composition and levels of CS were altered when sotv function was
partially compromised. Moreover, sotv/+ heterozygotes displayed
similar changes in chondroitin levels and structure, albeit to a lesser degree
(data not shown). These findings emphasize that partial-loss of EXT2
function may alter the levels and sulfation of CS as well as HS.
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Discussion |
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We have shown that, like ttv clones, sotv and ttv,
sotv double mutant clones limit the domain of Ci stabilization,
indicating that the range of Hh signaling is impaired
(Bellaiche et al., 1998). This
reduction is consistent with a requirement for HSPGs in Hh transport. However,
our observations that Hh levels are reduced in posterior compartment clones
(Fig. 6) suggest an alternative
(or additional) mechanism by which HSPGs could affect Hh signaling: by
altering ligand stability. In wild type, hh is transcribed and
expressed uniformly throughout the posterior compartment. Therefore, the
weaker staining in posterior clones is unlikely to be due to failure of
concentration-dependent transport mechanisms. Moreover, lower Hh levels are
not caused by reduced expression, as hh transcription (monitored
through expression of a hh-lacZ transgene) is unaffected in
sotv mutant clones (see Fig.
6). By extension, Hh ligand instability in cells lacking HSPGs
could also contribute to the reduced effectiveness of signaling in clones
along the AP boundary. HSPGs could bind to and stabilize the ligand directly,
or alternatively may act indirectly to reduce the activity of extracellular
proteases. Consistent with the latter model, heparin is known to promote
inhibition of thrombin by acting through the protease inhibitor serpin AT III
(Bernfield et al., 1999
).
Reduced Hh ligand stability would lower the distance over which the growth
factor can signal in a manner difficult to distinguish from compromised ligand
transport. Although these models presume that loss of HS impacts growth factor
signaling by disrupting protein interaction with GAG chains, it is also
possible that without GAG synthesis HSPG core proteins are mislocalized or
less stable, contributing to the observed phenotypes.
It has been suggested that disruption of intracellular transport should
lead to a diagnostic accumulation of ligand on the side of a clone closest to
the morphogen source (Teleman et al.,
2001). Although we do not observe ligand accumulation at the
boundaries of ttv or sotv clones, consistent with a failure
to affect transport, we interpret these results cautiously. As Hh stability is
compromised in mutant cells, it is possible that the rate of ligand
degradation simply exceeds the rate of accumulation. Additionally, it has been
argued that ligand accumulation may not be a reliable indicator of a blockage
in transport, and instead could reflect increased expression of ligand-binding
factors (such as receptors or HSPGs)
(Lander et al., 2002
).
A recent cell culture-based screen to identify novel components in the Hh
pathway demonstrated that RNAi-based degradation of Ttv, Sotv and Botv did not
reduce the transcriptional response to exogenously added Hh ligand
(Lum et al., 2003). These
results are consistent with our findings, as addition of exogenous ligand
bypasses any requirement for ligand stabilization or transport. These data
underscore the idea that GAG chain synthesis in receiving cells is not
essential for transduction of the Hh signal. Loss of Hh in posterior
compartment clones lacking ttv or sotv (see
Fig. 6) also argues against a
current model that implicates HSPGs and the transmembrane protein Dispatched
(Disp) in promoting ligand transport (Burke
et al., 1999
; Ingham and
McMahon, 2001
). Disp is required to release Hh from the membrane
of expressing cells. Thus, disp mutant cells show high levels of Hh
accumulation. A Ttv-modified HSPG has been proposed to aid in releasing Hh-Np
from Disp and prevent reinsertion of the ligand into the membrane, thereby
enhancing its diffusion. However, if a Ttv-modified HSPG were essential for
Hh-Np release from Disp, then ttv and sotv mutant clones in
the posterior compartment should accumulate high levels of Hh, similar to
disp clones. Finally, our results conflict with the proposal that Ttv
could synthesize HSPGs that promote Hh signaling specifically
(Bellaiche et al., 1998
;
The et al., 1999
), as the Dpp
and Wg pathways are also affected by loss of Ttv or Sotv. We have only
examined Wg and Dpp signaling in wing discs, and it remains possible that
these ligands have differential requirements for HS during embryogenesis.
Wg and Dpp signaling are sensitive to loss of HS synthesis
A role for HSPGs in Wg signaling in Drosophila is well
established. Addition of HS or CS GAGs to culture medium causes S2 cells to
release secreted Wg protein, while heparinase treatment reduces their ability
to respond to exogenous Wg (Reichsman et
al., 1996). Furthermore, both Wg levels and signaling efficiency
are sensitive to HSPG concentration, as Wg activity is reduced when either HS
synthesis or the core proteins for the glypicans Dally and Dlp are compromised
(Baeg et al., 2001
;
Binari et al., 1997
;
Hacker et al., 1997
;
Haerry et al., 1997
;
Lin and Perrimon, 1999
;
Tsuda et al., 1999
). Severe
disruption of extracellular Wg distribution and the altered expression of the
downstream target Ac, in ttv and sotv mutant clones
demonstrates that HS chains are essential for establishing and/or maintaining
the Wg gradient.
The effects of HSPGs on signaling are further modulated by secondary
modifications to the GAG chains. For example in Drosophila, loss of
either sfl or slalom (sll) compromises both Wg and
Hh signaling (Lin and Perrimon,
1999; Luders et al.,
2003
). Sfl is an N-deacetyl/N-sulfotransferase, and Sll is
required to transport a high-energy sulfate donor molecule to the golgi, where
HSPGs such as dally and dlp are sulfate modified. Loss of
GAG sulfation results in signaling defects, despite the fact that unsulfated
HS-GAGs are still present in sfl mutants
(Toyoda et al., 2000a
).
Consequently, not only are HS-GAG chains required for signaling, but the
extent of sulfation is also crucial. In vertebrates, Qsulf1 promotes Wg
signaling by catalyzing removal of 6-O sulfate groups from HS chains of HSPGs,
including the Dally homolog glypican 1 (Ai
et al., 2003
; Dhoot et al.,
2001
). It has been proposed that in the absence of Qsulf1, Wg
stays tightly bound to HSPGs, which prevents the ligand from interacting with
receptors. However, in Qsulf1-expressing cells, selective 6-O desulfation
reduces the binding affinity sufficiently to permit ligand-receptor
interaction. In this context, the altered HS disaccharide distribution and
abnormal sulfation patterns encountered in hypomorphic sotv alleles
is intriguing (see Fig. 8,
Table 1). If partial loss of
function EXT2 mutations in individuals with HME causes similar disruption of
GAG modifications, they could also have complex and allele-specific effects on
signaling.
Our data also provide direct evidence that HS chains are required for Dpp
signaling in vivo. The core protein of the Glypican Dally has been implicated
in Dpp signaling based on genetic interactions and recent studies
demonstrating its role in regulating the Dpp morphogen gradient in the wing
(Jackson et al., 1997;
Fujise et al., 2003
). However,
the contribution of HS chains to Dpp signaling has remained unclear. We have
shown that Dpp signaling in the wing disc is reduced in ttv or
sotv mutant clones independent of effects on Hh signaling,
establishing that HS GAG chains are required for optimal activity of the Dpp
pathway. Although Dpp activity is clearly compromised in mutant tissue,
signaling is still detectable in mutant clones located where ligand levels are
the highest, such as near the AP compartment boundary (see
Fig. 7). These results imply
that, like Wg, Dpp can signal in the absence of HSPGs, albeit at lower
efficiency.
A Drosophila HS co-polymerase complex
Initial reports that ttv was required for Hh, but not Wg or FGF,
signaling, prompted speculation that Ttv might generate a Hh-specific HSPG,
and that, unlike its mammalian orthologs, Drosophila Sotv might
retain significant functional activity in the absence of its partner
(Bellaiche et al., 1998;
The et al., 1999
;
Toyoda et al., 2000a
;
Toyoda et al., 2000b
).
However, our demonstration that Hh, Wg and Dpp signaling are affected in
single mutants for ttv and for sotv, together with the
biochemical analysis presented here, suggest that the mammalian model of EXT1
and EXT2 as obligate co-polymerases applies equally well to
Drosophila. The severe and comparable reductions in HS disaccharides
observed in ttv and sotv null mutant larvae lend additional
support to the co-polymerase model. Moreover, the fact that the phenotype of
both single and ttv, sotv double mutants is indistinguishable
strongly suggests that any residual partner activity is not biologically
significant.
In contrast to the null alleles, hypomorphic alleles of sotv that
result in C-terminal truncations retain some biosynthetic activity that varies
directly with the predicted length of the protein (see
Fig. 8,
Table 1). Thus,
sotv1.4.1, which encodes the longest mutant product,
generates HS at 3.1% the wild-type level compared with 0.21% in the null
allele sotv1.8.1 (see
Table 1). Retention of similar
levels of function by truncated human EXT2 proteins could explain why all 27
of the disease-causing dominant mutations in EXT2 are clustered
within the N-terminal two-thirds of the protein, rather than spread throughout
its length (Zak et al., 2002).
Perhaps C-terminal truncations retain sufficient function to supplement the
HSPG levels from the wild-type copy and achieve the critical threshold
required for normal bone growth. Alternatively, reduced levels or altered
ratios of proteoglycans bearing specific sulfate modifications may contribute
to the clinical manifestation of the disease. We note, for example, that the
residual HS produced from null and sotv8.2 alleles contain
little or no UA-GlcNAc6S. However, that species constitutes a substantial
fraction of the HS produced by the less truncated alleles
sotv18.2 and sotv1.4.1 (see
Fig. 8 and
Table 1).
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ACKNOWLEDGMENTS |
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