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INTRODUCTION |
Sulfated glycosaminoglycans
(GAGs),1 including
heparin/heparan sulfate and chondroitin sulfate/dermatan sulfate, are
distributed on the surfaces of most cells and in the extracellular
matrices of virtually every tissue. They are implicated in the
regulation and maintenance of cell proliferation, cytodifferentiation,
and tissue morphogenesis, exhibiting their biological activities by interacting with various proteins through specific saccharide sequences. They are synthesized as proteoglycans, on specific Ser
residues in the so-called GAG-protein linkage region,
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, which is
common to the GAGs (for reviews, see Refs. 1 and 2). The linkage region
synthesis is initiated by the addition of Xyl to Ser followed by the
addition of two Gal residues and is completed by the addition of GlcA,
each reaction being catalyzed by a specific glycosyltransferase (1, 2).
The GAGs are built up on this linkage region by the alternating
addition of N-acetylhexosamine and GlcA residues.
Heparin/heparan sulfate is synthesized once GlcNAc is transferred to
the common linkage region, whereas chondroitin sulfate/dermatan sulfate
is formed if GalNAc is first added. However, biosynthetic sorting
mechanisms of different GAG chains remain enigmatic. Although at least
eight different kinds of glycosyltransferase reactions are required to
synthesize these GAGs, only the GlcA transferase that completes the
tetrasaccharide linkage region and the heparan sulfate-polymerase that
polymerizes GlcA and GlcNAc have been cloned (3, 4).
Recent cDNA cloning of the latter enzyme of bovine origin revealed
its 94% sequence identity to human EXT2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors (4). EXT is an autosomal dominant disorder characterized by cartilage-capped skeletal excrescences, which may lead to skeletal abnormalities and short stature (5). Although the exostoses represent
osteochondromas that are benign bone tumors, malignant transformation
into chondrosarcomas or osteosarcoma occurs in approximately 2% of EXT
patients (5, 6). Genetic linkage of this disorder has been ascribed to
three independent loci on chromosomes 8q24.1 (EXT1),
11p11-13 (EXT2), and 19p (EXT3) (7-9). This
family of EXT genes has recently been extended by the identification of
three additional EXT-like genes, EXTL1,
EXTL2/EXTR2, and EXTL3/EXTR1 (10-13). Sporadic
and exostoses-derived chondrosarcomas are attributable to the loss of
heterozygosity for the markers in EXT1 and EXT2 loci (14, 15), indicating that the genes responsible for EXTs and the
EXT-like genes may encode tumor suppressors.
While searching for the key enzyme involved in biosynthetic sorting of
chondroitin sulfate/dermatan sulfate from heparin/heparan sulfate, we
found a novel
-N-acetylgalactosaminyltransferase (
-GalNAcT) in fetal bovine sera and in the mouse mastocytoma cells,
which transferred an
-GalNAc residue to
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser derived from
the GAG-protein linkage region (16, 17). In addition, Miura and Freeze
(18) detected a substantial amount of
-GalNAcT activity in Golgi
fractions prepared from several cells. The enzyme activity was also
found in the culture medium of a human sarcoma cell line and was an
1,4-GalNAc transferase (19). Mysteriously, however, the structure of
the reaction product has not been found in any natural GAG chain. In
this study, we purified the enzyme and demonstrated 100% peptide
sequence identity to the protein encoded by the multiple exostoses-like
gene EXTL2/EXTR2 (10, 11), a unique member of the EXT gene
family. A recombinant enzyme had a dual catalytic activity of
1,4-GalNAc transferase and
1,4-GlcNAc transferase that determines
and initiates the heparan sulfate synthesis on the common GAG-protein
linkage region (20).
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EXPERIMENTAL PROCEDURES |
Materials--
UDP-[3H]GalNAc (10 Ci/mmol) and
UDP-[3H]GlcNAc (30 Ci/mmol) were purchased from NEN Life
Science Products. Unlabeled UDP-GalNAc and UDP-GlcNAc were obtained
from Sigma. Flavobacterium heparinum heparitinase I, jack
bean
-N-acetylhexosaminidase, and Acremonium sp.
-N-acetylgalactosaminidase were purchased from
Seikagaku Corp. (Tokyo, Japan). The chemically synthesized linkage
tetrasaccharide-serine GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (21) was
provided by T. Ogawa (RIKEN, The Insitute of Physical and Chemical
Research, Saitama, Japan). N-Acetylchondrosine
GlcA
1-3GalNAc was a gift from K. Yoshida (Seikagaku Corp.).
GlcA
1-3Gal
1-O-naphthalenemethanol (20) was provided
by J. D. Esko (University of California, San Diego, CA).
Enzyme Purification and Peptide Sequencing--
The
-GalNAcT
was purified from the serum-free culture medium of a human sarcoma
(malignant fibrous histiocytoma) cell line using an extension of the
procedure described previously (19). The details of the isolation
procedure will be reported
elsewhere.2 The purified
-GalNAcT was resolved by SDS-polyacrylamide gel electrophoresis
transferred to a polyvinylidene difluoride membrane (Bio-Rad, Tokyo,
Japan), and the resolved protein bands were stained with Coomassie
Brilliant Blue (Sigma). The membrane strip containing the major protein
band of 38 kDa was excised and was subjected to an
NH2-terminal amino acid sequence analysis (Takara, Kyoto, Japan).
Construction of a Soluble Form of the Enzyme--
A truncated
form of
-GalNAcT, lacking the first NH2-terminal 57 amino acids of EXTL2/EXTR2 (10, 11), was amplified with human fetal
liver cDNA (CLONTECH) as a template by PCR
using a 5' primer (5'-CGGGATCCCAGGGCAAGTCCACCAT-3') containing an
in-frame BamHI site and a 3' primer
(5'-CGGGATCCAAGCTACTCAAATGCCAAGCA-3') containing an in-frame
BamHI site located 54 base pairs downstream of the stop
codon. PCR reactions were carried out with Pfu polymerase (Stratagene, La Jolla, CA) by 30 cycles of 96 °C for 30 s,
55 °C for 30 s, and 72 °C for 75 s. The PCR fragment
was subcloned into the BamHI site of pGIR201protA (22),
resulting in the fusion of
-GalNAcT to the insulin signal sequence
and the protein A sequence present in the vector. A NheI
fragment containing the fusion protein was inserted into the
XbaI site of the expression vector pSVL (Amersham Pharmacia
Biotech, Tokyo, Japan).
Expression of the Soluble Enzyme and the Assay--
The
expression plasmid (6 µg) was transfected into COS-1 cells on 100-mm
plates using FuGENETM 6 (Roche Molecular Biochemicals,
Tokyo, Japan) according to the instructions provided by the
manufacturer. Two days after transfection, 1 ml of the culture medium
was collected and incubated with 10 µl of IgG-Sepharose (Amersham
Pharmacia Biotech) for 1 h at 4 °C. The beads recovered by
centrifugation were washed with and then resuspended in the assay
buffer and tested for
-GalNAcT and GlcNAc transferase-I (GlcNAcT-I)
activities using the tetrasaccharide-serine (1 nmol) representing the
GAG-protein linkage region, N-acetylchondrosine (5 nmol),
and GlcA
1-3Gal
1-O-naphthalenemethanol (250 nmol) as acceptor substrates as described (16, 19, 20).
Identification of the Enzyme Reaction Products--
The
isolation of the products from the
-GalNAcT reaction using
N-acetylchondrosine as an acceptor was carried out by gel filtration on a Superdex 30 column (Amersham Pharmacia Biotech) equilibrated with 0.25 M NH4HCO3,
7% 1-propanol. The radioactive peak containing the product was pooled
and evaporated to dryness. The isolated product (about 10 pmol) was
digested with 15 mIU of
-N-acetylhexosaminidase or 39 mIU
of
-N-acetylgalactosaminidase in a total volume of 20 µl of 50 mM sodium citrate buffer, pH 4.5, respectively,
at 37 °C overnight. The enzyme digest was analyzed using the same
Superdex 30 column as that noted above.
The isolation of the products from the GlcNAcT-I reaction using
GlcA
1-3Gal
1-O-naphthalenemethanol was performed by
HPLC on a Nova-Pak® C18 column (3.9 × 150 mm;
Waters, Tokyo, Japan) in an LC-10AS system (Shimadzu Co., Kyoto,
Japan). The column was developed isocratically for 15 min with
H2O at a flow rate of 1.0 ml/min at room temperature;
thereafter, a linear gradient was applied to increase the methanol
concentration from 0 to 100% over a 5-min period, and the column was
then developed isocratically for 40 min with 100% methanol. The
radioactive peak containing the product was pooled and evaporated to
dryness. The isolated product (about 74 pmol) was incubated with 14 mIU
of
-N-acetylhexosaminidase in a total volume of 20 µl
of 50 mM sodium citrate buffer, pH 4.5, or with 3 mIU of
heparitinase I for testing the digestability in a total volume of 30 µl of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM Ca(OAc)2 at 37 °C overnight. The enzyme
digest was analyzed using the same Nova-Pak® C18 column as
that noted above.
Expression Levels of the Enzyme in Human Tissues--
Human
Multiple Tissue cDNA Panels (CLONTECH) were
used for the analysis. The manufacturer normalizes each cDNA sample
against six housekeeping genes. To verify this, we determined the
levels of amplification of the glyceraldehyde-3-phosphate
dehydrogenase, whose transcript is always present in the tissues at a
constant level. The amplification reaction was carried out in a total
volume of 50 µl using the 5' primer, 5'-ACCACTGTCCATGCCATCAC-3', and the 3' primer, 5'-TCCACAACACGGTTGCTGTA-3', by 25 cycles of 95 °C for
30 s, 55 °C for 30 s, and 72 °C for 90 s. A
10-µl aliquot of the amplified products was visualized by
electrophoresis on a 1.0% agarose gel containing ethidium bromide.
Using the normalized cDNA input, we then performed the
amplification of a transcript, using a serial number of cycles
(25-30-35) to find the conditions for a semiquantitative amplification.
The best results were obtained by carrying out 30 cycles of 96 °C
for 30 s, 55 °C for 30 s, and 72 °C for 75 s using
the 5' and 3' primers described above, which were designed to span the
two introns in the EXTL2 gene (10) to discriminate a PCR product
amplified from cDNA from, if any, one amplified from contaminating
genomic DNA. PCR products were then visualized by electrophoresis on a
1.0% agarose gel containing ethidium bromide. To confirm that the
amplified DNAs were derived from the EXTL2 mRNA, the amplified
fragments were gel-purified, subcloned into the pGEM®-T Easy vector
(Promega, Madison, WI), and sequenced. The nucleotide sequences of the
amplified DNAs were identical to that of the human EXTL2 cDNA (10)
(data not shown).
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RESULTS |
The Enzyme Is Encoded by a Tumor Suppressor-like
Gene--
-GalNAcT was purified from the serum-free culture medium
of a human sarcoma (malignant fibrous histiocytoma) cell line to near
homogeneity mainly by affinity chromatographies on heparin-Sepharose and UDP-hexanolamine-Sepharose. Upon SDS-polyacrylamide gel
electrophoresis under reducing conditions, the purified
-GalNAcT
preparation gave a major protein band of 38-kDa (data not shown), which
was excised and subjected to NH2-terminal amino acid
sequence analysis. The obtained sequence of thirty amino acid residues
(Fig. 1) was 100% identical to that of
residues 54-83 of the protein encoded by EXTL2/EXTR2 except
for the unidentified amino acid corresponding to the
N-glycosylation site (10, 11), as demonstrated by data base
searches. The EXTL2/EXTR2 sequence indicated a single open reading frame of 990-base pair coding for a protein of 330 amino acids,
including one potential N-glycosylation site (10, 11), which
has a type II transmembrane protein topology characteristic of many
other glycosyltransferases cloned to date. Since the
NH2-terminal amino acid sequence of the purified
-GalNAcT was found 54 amino acids away from the putative start site
for translation of EXTL2/EXTR2, the purified
-GalNAcT
seems to be a truncated form that has lost its transmembrane domain and
subsequently been released from the enzyme-producing cell, as has been
observed for several other glycosyltransferases (23).

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Fig. 1.
Comparison of the NH2-terminal
amino acid sequence of the purified -GalNAcT
with the corresponding EXTL2/EXTR2 sequence. X
represents an unidentified amino acid residue that is most likely
glycosylated, and one potential N-glycosylation site is
marked by an asterisk.
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The Expressed Enzyme Is an Active
-GalNAc Transferase--
A
soluble form of the protein encoded by EXTL2/EXTR2 cDNA was
generated by replacing the first 57 amino acids of EXTL2/EXTR2 with the
cleavable insulin signal sequence and the IgG-binding domain of protein
A as described under "Experimental Procedures." When the expression
plasmid containing the EXTL2/protein A fusion was expressed in COS-1
cells, an approximately 66-kDa protein was secreted (data not shown).
The apparent molecular mass of the fused protein was reduced to 60-kDa
after N-glycosidase treatment (data not shown), indicating
that the one potential N-linked glycosylation site of
EXTL2/EXTR2 is being utilized. The fused enzyme expressed in the medium
was absorbed on IgG-Sepharose beads to eliminate endogenous
glycosyltransferases and then the enzyme-bound beads were used as an
enzyme source for further studies. The bound fusion protein was assayed
for
-GalNAcT activity using a variety of acceptor substrates. As
shown in Table I, marked
glycosyltransferase activity was detected with
N-acetylchondrosine (GlcA
1-3GalNAc), GlcA
1-3Gal
1-O-naphthalenemethanol, and the
tetrasaccharide-serine representing the GAG-protein linkage region as
the acceptor substrates. In addition, no detectable
-GalNAcT
activity was recovered by the affinity purification from the control
pSVL transfection sample. To identify the
-GalNAcT reaction
products, N-acetylchondrosine was labeled by an enzyme
reaction using UDP-[3H]GalNAc as a donor substrate and
the enzyme-bound beads as an enzyme source. The labeled products were
completely digested by
-N-acetylgalactosaminidase, but
not by
-N-acetylhexosaminidase, quantitatively yielding a
3H-labeled peak at the elution position of free
[3H]GalNAc, as demonstrated by gel filtration (Fig.
2A). These results indicated
that a GalNAc residue had been transferred to
N-acetylchondrosine through an
-linkage and that the
expressed protein was
1,4-GalNAc transferase.
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Table I
Acceptor specificity of the 1,4-N-acetylhexosaminyltransferase
secreted into the culture medium by transfected COS-1 cells
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Fig. 2.
Characterization of the
-GalNAcT and GlcNAc transferase reaction products
using various glycosidases. A, 3H-labeled
-GalNAcT reaction products recovered from a Superdex 30 column were
subjected to digestion with -N-acetylgalactosaminidase or
-N-acetylhexosaminidase as described under
"Experimental Procedures." The
-N-acetylgalactosaminidase digest ( ),
-N-acetylhexosaminidase digest ( ), or the undigested
sample ( ) was applied to a column of Superdex 30 (1.6 × 60 cm), and the respective effluent fractions (1 ml each) were analyzed
for radioactivity. V0 and Vt
were around fractions 40 and 120, respectively (not shown). An
arrow indicates the elution position of free GalNAc.
B, isolated 3H-labeled GlcNAc transferase
reaction products were subjected to digestion with heparitinase I or
-N-acetylhexosaminidase as described under
"Experimental Procedures." The heparitinase I digest ( ),
-N-acetylhexosaminidase digest ( ), or the undigested
sample ( ) was analyzed by HPLC on a Nova-Pak® C18
column as described, and the respective effluent fractions (2 ml each)
were analyzed for radioactivity. An arrow indicates the
elution position of free GlcNAc.
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1,4-GlcNAc Transferase for Heparan Sulfate Synthesis--
In
view of the recent findings that EXT1 and EXT2, the carboxyl halves of
both of which shared significant homology with EXTL2/EXTR2, were
heparan sulfate-polymerases required for the heparan sulfate biosynthesis (4), it was of particular interest to investigate the
involvement of EXTL2/EXTR2 in the heparan sulfate biosynthesis. Although our enzyme showed an
-GalNAcT activity toward the
tetrasaccharide-serine derived from the common GAG-protein linkage
region, the structure of the reaction product has not been found in
naturally occurring GAG chains, which prompted us to hypothesize that
the enzyme may have an
-GlcNAc tansferase activity toward the
linkage region structure taking into account the tetrasaccharide
structure of the acceptor substrate for the
-GalNAcT activity.
Hence, the purified fusion protein was assayed for GlcNAc transferase
activity using UDP-[3H]GlcNAc as a sugar donor and two
oligosaccharide acceptor substrates whose structures represent the
GAG-protein linkage region. As shown in Table I, a significant GlcNAc
transferase activity was detected with
GlcA
1-3Gal
1-O-naphthalenemethanol as an acceptor, whereas no activity was detected using the linkage
tetrasaccharide-serine. The observed substrate specificity was
consistent with that reported for GlcNAcT-I (17, 20), which is involved
in the heparan sulfate biosynthesis. No detectable GlcNAc transferase
activity was recovered by affinity purification from a control pSVL
transfection sample, excluding the possibility of an artifact or of an
endogenous origin of the activity. To identify the GlcNAc transferase
reaction products, GlcA
1-3Gal
1-O-naphthalenemethanol
was labeled with [3H]GlcNAc using the enzyme bound to
beads. The labeled products were completely digested by heparitinase I
that cleaves an
1,4-glucosaminide linkage in an eliminative fashion,
quantitatively yielding a 3H-labeled peak at the position
of free [3H]GlcNAc, as demonstrated by HPLC (Fig.
2B), whereas they were inert to the action of
-N-acetylhexosaminidase. These results clearly indicated
that a GlcNAc residue had been transferred exclusively to the
nonreducing terminal GlcA of
GlcA
1-3Gal
1-O-naphthalenemethanol through an
1,4
linkage. Taken together, the present findings demonstrated that
EXTL2/EXTR2 is an
1,4-N-acetylhexosaminyltransferase that
transfers GalNAc/GlcNAc to the artificial yet authentic oligosaccharide acceptor substrate for GlcNAcT-I (20). The failure of the
tetrasaccharide-serine derived from the GAG-protein linkage region to
serve as an acceptor (Table I) is discussed below.
Ubiquitous Expression of the Gene in Human Tissues--
To screen
tissue expression of the
1,4-N-acetylhexosaminyltransferase, we used PCR-based
methods with normalized cDNA pools. A single amplified DNA of the
expected size (875 base pairs) was obtained from each cDNA
preparation of the 18 adult and 8 fetal human tissues examined,
although the amounts of the amplified cDNAs varied (Fig.
3), indicating that the gene was
ubiquitously expressed.

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Fig. 3.
Differential expression of the
1,4-N-acetylhexosaminyltransferase
gene in various human tissues. The procedures used are described
under "Experimental Procedures."
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DISCUSSION |
In the present study, we demonstrated that EXTL2/EXTR2
encoded enzyme with a dual catalytic activity of
-GalNAcT and
GlcNAcT-I, i.e. an
1,4-N-acetylhexosaminyltransferase that transferred
GalNAc/GlcNAc to the core oligosaccharide representing the GAG-protein
linkage region. Thus, the enzyme turned out to be identical to the
previously described GlcNAcT-I that determines and initiates the
biosynthesis of heparan sulfate (20) and most likely heparin as well.
The gene, like other EXT gene family members, was found ubiquitously expressed in virtually every human tissue examined (Fig. 3), which is
in accordance with the observations that heparan sulfate proteoglycans are distributed on the surfaces of most cells and the extracellular matrices in virtually every tissue. In view of the present findings of
the involvement of EXTL2/EXTR2 in the heparan sulfate biosynthesis together with those of Lind et al. (4), who recently
reported that EXT1 and EXT2 both encoded a
heparan sulfate-polymerase required for the heparan sulfate
biosynthesis, the expression of heparan sulfate seems to play an
important role in the tumor suppressor function although the precise
mechanism remains unclear.
The initial characterization of crude GlcNAcT-I preparations showed
that the enzyme exhibited strict specificity toward
GlcA
1-3Gal
1-O-naphthalenemethanol (20),
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-naphthalenemethanol,
or GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-benzyl
(24). Neither N-acetylheparosan (-4GlcA
1-4GlcNAc
1-)n nor the
tetrasaccharide-serine, GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser derived from
the linkage region, was utilized as an acceptor substrate (17, 20).
Hence, it was suggested that the transfer of the first GlcNAc residue
to the linkage tetrasaccharide primer is mediated by GlcNAcT-I,
distinct from the enzyme that has been termed heparan
sulfate-polymerase involved in the formation of the repeating
disaccharide units of heparan sulfate (20) and that GlcNAcT-I directly
recognizes a specific sequence in the core protein or an aglycone
structure attached to the linkage tetrasaccharide (24). These
hypotheses have now been proven by the molecular identification and
characterization of both enzymes in a recent study (4) and in the
present study. The molecular similarity of the two enzymes is
consistent with the fact that both enzymes have
1,4-GlcNAc
transferase activities and recognize the terminal
-GlcA moiety of
their acceptor substrates.
The enzyme protein of the
1,4-N-acetylhexosaminyltransferase composed of 330 amino
acids is about half the size of the other EXT family members that have
676~919 amino acids. The variation in size is due to differences on
the amino-terminal side of the protein. The protein shows significant
homology with the carboxyl termini of the other members of the family.
Based on the facts that both EXT1 and EXT2 encode
a bifunctional heparan sulfate-polymerase catalyzing the GlcA and
GlcNAc transferase reactions and that EXTL2/EXTR2 encodes
GlcNAcT-I, it is reasonable to assume that the carboxyl-terminal side
of EXT1 and EXT2 is the domain catalyzing the GlcNAc transferase
reaction. A phylogenetic tree of the EXT gene family based on the
conserved carboxyl-terminal amino acids shows a close relationship
between EXT2 and EXTL3/EXTR1 and between EXT1 and EXTL1 (13),
suggesting that the remaining two members, EXTL1 and EXTL3/EXTR1 might
be heparan sulfate-polymerases. Notably, the EXTL2/EXTR2 gene is the
most divergent, suggesting that this unique gene family member was the
first to split from a common ancestor during evolution and evolved
separately from the others (13).
The possible role of the
-GalNAcT activity of the enzyme in the GAG
biosynthesis remains unclear, since no
-GalNAc-capped structure has
been reported in naturally occurring GAG chains. In our recent study,
the
-GalNAc-capped pentasaccharide serine GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, a
reaction product of the
-GalNAcT, was not utilized as an acceptor
for the glucuronyltransferase involved in the chondroitin sulfate
biosynthesis (25), suggesting that the addition of an
-GalNAc
residue may serve as a stop signal that precludes further chain
elongation. The cDNA now provides an essential tool for
investigating the biological functions of the
-GalNAcT reaction
products, if any, in naturally occurring glycoconjugates as well as
heparan sulfate with regard to the tumor suppressor activity.
The EXTL2 has been assigned to chromosome 1p11-p12, and this
region has been found to be involved in chromosomal rearrangements in a
variety of tumors as described (10). In view of the tumor suppressor
capacity of the EXT genes, EXTL2 might also be a serious candidate gene involved in one of the tumors associated with this region. In this regard, many different types of tumors are associated with undersulfation and distinct changes in the sulfation pattern of
the heparan sulfate structure (Ref. 26 and the references therein), and
recent studies have demonstrated a critical role for heparan sulfate in
growth factor signaling mediated by Wingless proteins during
Drosophila development (27). In addition,
Drosophila homologues of EXT1 and heparan sulfate
2-O-sulfotransferase (encoded by ttv and
pipe, respectively) were recently implicated in the Hedgehog
diffusion and the formation of embryonic dorsal-ventral polarity,
respectively (28, 29). Thus, considering the probability that deletion
of the gene would cause the complete elimination of heparan sulfate and
heparin unless functional redundancy with other genes exists, it is
likely that germ line mutations inactivating the enzymatic activity
result in embryonic lethality and that somatic mutations cause much
more serious defects than those caused by EXT1 and
EXT2, leading to the progression of various tumors or to
lethal disorders. In fact, congenital deficiency in heparan sulfate
even only in enterocytes results in severe clinical problems and
eventually death (30).