From the Department of Biochemistry, Kobe Pharmaceutical
University, Higashinada-ku, Kobe 658-8558, Japan, the
Department of Environmental Sciences, Faculty of
Education and Regional Sciences, Tottori University, Tottori 680-8551, Japan, and the § Department of Medical Biochemistry and
Microbiology, University of Uppsala, The Biomedical Center, S-751 23 Uppsala, Sweden
Received for publication, November 27, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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The proteins encoded by the EXT1,
EXT2, and EXTL2 genes, members of the
hereditary multiple exostoses gene family of tumor suppressors, are
glycosyltransferases required for the heparan sulfate biosynthesis.
Only two homologous genes, rib-1 and rib-2, of
the mammalian EXT genes were identified in the
Caenorhabditis elegans genome. Although heparan sulfate is
found in C. elegans, the involvement of the rib-1 and rib-2
proteins in heparan sulfate biosynthesis remains unclear. In the
present study, the substrate specificity of a soluble recombinant form
of the rib-2 protein was determined and compared with those of the
recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The
present findings revealed that the rib-2 protein was a unique
Heparan sulfate proteoglycans are distributed on the surfaces of
most cells and in the extracellular matrices of virtually every tissue.
They consist of a protein core to which heparan sulfate
glycosaminoglycan (GAG)1
chains are attached. Heparan sulfate GAGs show a tremendous diversity of structures that are known to play important roles in many biological recognition events of vertebrates and invertebrates, such as cell adhesion, growth factor/cytokine action, and regulation of important signaling pathways (for reviews see Refs. 1 and 2). Their biological
activities are believed to be expressed through interactions with
various proteins through specific saccharide sequences. These GAGs are
synthesized on the so-called GAG-protein linkage region, GlcUA Recent cDNA cloning of the glycosyltransferases involved in the GAG
biosynthesis revealed that the heparin/heparan sulfate biosynthesis in
mammals is associated with the EXT gene family, the
hereditary multiple exostoses (HME) gene family of tumor suppressors (for a review see Ref. 5). HME is an autosomal dominant disorder characterized by cartilage-capped skeletal excrescences, which may lead
to skeletal abnormalities and short stature (6). Although the exostoses
represent osteochondromas that are benign bone tumors, malignant
transformation into chondrosarcomas or osteosarcomas occurs in ~2%
of HME patients (6, 7). Genetic linkage of this disorder has been
ascribed to three independent loci on chromosomes 8q24.1
(EXT1), 11p11-13 (EXT2), and 19p
(EXT3) (8-10). This family of EXT genes has
recently been extended by the identification of three additional
EXT-like genes, EXTL1, EXTL2, and
EXTL3 (11-14). Exostoses-derived and sporadic
chondrosarcomas are attributable to the loss of heterozygosity for the
markers in EXT1 and EXT2 loci (15, 16),
indicating that the EXT genes may encode tumor suppressors.
It has been demonstrated that both mammalian EXT1 and EXT2 proteins are
the heparan sulfate copolymerases that polymerize GlcUA and
GlcNAc alternately (17-19), and the human EXTL2 protein is
GlcNAc transferase I (20), which determines and initiates the heparan
sulfate synthesis on the common GAG-protein linkage region (21).
Interestingly, the EXT gene family has also been identified
in invertebrate genomes such as the nematode worm Caenorhabditis elegans (22) or the fruit fly Drosophila melanogaster
(23), suggesting the presence of heparan sulfate in these organisms. In
fact, heparan sulfate is found in invertebrates, including C. elegans and Drosophila. Recent studies have shown that
both these genetically tractable organisms make heparan sulfate and chondroitin or chondroitin 4-sulfate (24-26). Although analysis of
mutants defective in the Drosophila EXT1 homolog,
ttv, has provided some evidence that Ttv might also be a
heparan sulfate polymerase (25), glycosyltransferase activities have
not yet been reported for these invertebrate EXT family proteins. A
data base search revealed only the two homologous genes,
rib-1 and rib-2, of the mammalian EXT
genes in the C. elegans genome (22). Notably, however, no
C. elegans homolog of the mammalian EXTL2 gene
encoding GlcNAc transferase I required for the chain initiation of
heparan sulfate was found. These observations, therefore, suggested that the biosynthetic mechanism of heparan sulfate in C. elegans might be distinct from that reported for mammals. In the
present study, to clarify the involvement of the rib-2 protein in
heparan sulfate biosynthesis, the substrate specificity of a soluble
recombinant form of the rib-2 protein was determined and compared with
those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The findings demonstrated that the rib-2 protein was a novel
and unique Materials--
UDP-[U-14C]GlcUA
(285.2 mCi/mmol), UDP-[3H]GlcNAc (60 Ci/mmol), and
UDP-[3H]GalNAc (10 Ci/mmol) were purchased from
PerkinElmer Life Sciences. Unlabeled UDP-GlcUA, UDP-GlcNAc, and
UDP-GalNAc were obtained from Sigma. Flavobacterium
heparinum heparitinase I and Jack bean Construction of Expression Vectors Encoding Soluble Forms of the
Rib-2, EXT-1, EXT-2, and EXTL2 Proteins--
The cDNA fragment
encoding a truncated form of rib-2, lacking the first
N-terminal 58 amino acids of the rib-2, was amplified by
reverse transcription PCR with adult C. elegans total RNA as a template using a 5' primer (5'-CGGGATCCGATTATGACGCGTCATGCAGTG-3') containing an in-frame BamHI site and a 3' primer
(5'-CGGGATCCGTAGCACCATTCGACAAGTGAA-3') containing a BamHI
site located 68 bp downstream of the stop codon. The cDNA fragment
encoding a truncated form of EXT-1, lacking the first
N-terminal 43 amino acids, was amplified by PCR with human
EXT-1 cDNA in pcDNA 3.1 (29) as a template using a
5' primer (5'-CCGGAATTCGAATGGCTTGCACCACCCCAG-3') containing an in-frame EcoRI site and a 3' primer
(5'-CGCGGATCCCTGATGAGTGGATCTGCACTG-3') containing a
BamHI site located 84 bp downstream of the stop codon. The
cDNA fragment encoding a truncated form of EXT-2, lacking the first
N-terminal 55 amino acids, was amplified by PCR with bovine
EXT-2 cDNA in pcDNA 3.1zeo (17) as a template
using a 5' primer (5'-CGCGGATCCCTGGAGCGTGGAGAAGCGCA-3')
containing an in-frame BamHI site and a 3' primer
(5'-CGCGGATCCTGTCCGGCATTCGTGTTGAGC-3') containing a BamHI
site located 40 bp downstream of the stop codon. PCR reactions
were carried out with KOD DNA polymerase (TOYOBO, Tokyo, Japan)
by 30 cycles of 96 °C for 30 s, 58 °C for 30 s, and
72 °C for 60 s. The PCR fragment of rib-2 or
EXT-2 was subcloned into the BamHI site of
pGIR201protA (30), resulting in the fusion of rib-2 or
EXT-2 to the insulin signal sequence and the protein A
sequence present in the vector. The PCR fragment of EXT-1
was subcloned into the EcoRI/BamHI site of
pGIR201protA. An NheI fragment containing each of the above
fusion protein sequences was inserted into the XbaI site of
the expression vector pEF-BOS (31). The nucleotide sequence of the
amplified cDNA was determined in a 377 DNA sequencer (PerkinElmer
Life Sciences). The construction of a soluble form of EXTL2 fused with
the cleavable insulin signal sequence and the protein A IgG-binding
domain was carried out as described previously (20).
Expression of the Soluble Forms of the Rib-2, EXT-1, EXT-2, and
EXTL2 Proteins and Enzyme Assays--
Each expression plasmid (6 µg)
was transfected into COS-1 cells in 100-mm plates using
FuGENETM 6 (Roche Molecular Biochemicals)
according to the manufacturer's instructions. 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 each assay buffer described below and tested
for GlcNAc transferase activities using
N-acetylheparosan oligosaccharides
GlcUA Identification of the Enzyme Reaction Products--
The
isolation of the products from the GlcNAc transferase reaction using
N-acetylheparosan as an acceptor was carried out by gel
filtration on a Superdex peptide column (Amersham Pharmacia Biotech)
equilibrated with 0.25 M
NH4HCO3/7% 1-propanol. The radioactive peak
containing the enzyme reaction product was pooled and evaporated to
dryness. The isolated product (about 72 pmol) was digested with 3 mIU
of
The isolation of the products from the GlcNAc transferase I reaction
using
GlcUA Rib-2 Expressed in COS-1 Cells Generates GlcNAc Transferase
Activities for Heparan Sulfate Synthesis--
C. elegans
rib-2 encodes a protein with homology to the human EXT family
members, especially to the EXT2 and EXTL3 proteins (see
"Discussion"). In view of the recent findings that EXT2 is a
heparan sulfate-polymerase required for the heparan sulfate biosynthesis (17, 19), it was important to clarify the involvement of
rib-2 in the heparan sulfate biosynthesis in C. elegans. The rib-2 sequence indicated an open reading frame of 2442 bp coding for a
protein of 814 amino acids, with eight potential
N-glycosylation sites (22) and a type II transmembrane
protein topology characteristic of many glycosyltransferases cloned to
date. A soluble form of the protein encoded by rib-2
cDNA was generated by replacing the first 58 amino acids of rib-2
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 rib-2/protein A fusion was expressed
in COS-1 cells, an approximate 130-kDa protein was secreted as shown by
Western blotting using IgG (data not shown). The apparent
Mr of the fused protein was reduced to about
110-kDa after N-glycosidase treatment (data not shown),
indicating that a few potential N-linked glycosylation sites
of rib-2 were 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 GlcUA transferase or GlcNAc
transferase activities involved in the heparan sulfate biosynthesis
using a variety of acceptor substrates. There is no known C. elegans homolog of the human EXTL2 gene encoding GlcNAc
transferase I required for the chain initiation of heparan sulfate (see
"Introduction"), which suggested that rib-2 may also have
GlcNAc transferase I activity. Hence, the purified fusion protein
was also assayed for GlcNAc transferase I activity, in addition to the
GlcUA and GlcNAc transferase activities for the polymerization
reactions.
GlcUA
To identify these GlcNAc transferase reaction products, the
acceptor substrates, N-acetylheparosan oligosaccharides
GlcUA Comparison of Acceptor Specificity of the Rib-2, EXT1, EXT2,
and EXTL2 Proteins--
To distinguish the specificity of rib-2
from that of other so far characterized mammalian glycosyltransferases
involved in the heparan sulfate biosynthesis, the rib-2 was compared
with the soluble recombinant forms of mammalian EXT1, EXT2, and EXTL2 for their ability to utilize a variety of acceptor substrates (Table
I). Comparison of rib-2 with EXT1 and EXT2 showed that they all
utilized N-acetylheparosan oligosaccharides
GlcUA Although heparan sulfate is found in C. elegans, it was
unknown which enzyme proteins were involved in the heparan sulfate biosynthesis (24, 25). In the present study, we demonstrated that the
C. elegans rib-2 protein was a unique The hitherto unreported specificity revealed for rib-2 can be predicted
to be required for heparan sulfate biosynthesis in C. elegans, because there is no C. elegans homolog of the
mammalian EXTL2 gene that encodes GlcNAc transferase I,
which is responsible for the chain initiation of heparan sulfate (21).
The rib-2 protein, composed of 814 amino acids, is a similar size to
the mammalian four EXT family members, EXT1, EXT2, EXTL1, and EXTL3 that have 676-919 amino acids and is about twice the size of the other
EXT member, EXTL2, which has 330 amino acids. The carboxyl terminus of
the rib-2 protein shows significant homology to these members of the
family and exhibits the highest homology to EXT2 and EXTL3 (44 and 58%
amino acid identities, respectively). Although EXT2
encodes a bifunctional heparan sulfate-cotransferase catalyzing the
GlcUA and GlcNAc transferase II reactions (17, 19), the rib-2
protein exhibited only the GlcNAc transferase II activity and no GlcUA
transferase activity. Instead, the rib-2 protein catalyzed the GlcNAc
transferase I reaction involved in the biosynthetic initiation of
heparan sulfate, whereas EXT2 did not. Based on these findings,
together with the observation that the rib-2 protein is more similar to
EXTL3 than EXT2, it is possible that rib-2 might be a
C. elegans ortholog of mammalian EXTL3 and that
EXTL3 might encode a similar A data base search revealed only two homologous genes, rib-1
and rib-2, of the mammalian EXT genes in the
C. elegans genome (22). In view of the finding that the
rib-2 protein exhibits no GlcUA transferase activity required for the
heparan sulfate chain elongation, it is possible that the rib-1 protein
possesses the GlcUA transferase activity. The rib-1 protein, composed
of 378 amino acids, is about half the size of the rib-2 protein and the
mammalian four EXT family members, EXT1, EXT2, EXTL1, and EXTL3.
Interestingly, the rib-1 protein shows significant homology to the
amino termini of these family members, especially to EXT1 (45% amino
acid identity). In this regard, it should be noted that
EXT1, as well as EXT2, encodes bifunctional
heparan sulfate-cotransferase, which catalyzes the GlcUA transferase
reaction, in addition to the GlcNAc transferase II reaction (17, 19),
and that EXTL2, which shows significant homology to the carboxyl
termini of the other EXT family members, shows the GlcNAc transferase I
activity (20). Very recently, Wei et al. (33) reported that
the GlcUA transferase domain most likely resides in the amino-terminal
portion of the EXT1 protein. Thus, it is suggested that the rib-1
protein might be the GlcUA transferase involved in the chain elongation of heparan sulfate. However, when a soluble recombinant form of the
rib-1 protein was produced and assayed for the GlcUA transferase and
also the GlcNAc transferase I and II activities under the condition
used in the present study, no such glycosyltransferase activities were
detected for the rib-1 protein (data not shown). In this regard, it is
possible that the kinetic properties of C. elegans enzymes,
rib-1 and rib-2, might be very different from those of the mammalian
EXT1 and EXT2 proteins. More efforts to detect the GlcUA transferase
activity for the rib-1 and rib-2 proteins are required.
Nevertheless, the findings from the present study suggest that the
biosynthetic mechanism of heparan sulfate in C. elegans is
distinct from that in mammals. In mammals, it was demonstrated that
EXT1 and EXT2 are both GlcUA/GlcNAc-cotransferases that need to
interact with each other to form an active heparan sulfate polymerase
(18, 19, 34). Coexpression of the two proteins, but not mixing of
separately expressed recombinant EXT1 and EXT2, yields
hetero-oligomeric complexes in mammalian cells, with augmented glycosyltransferase activities (18, 19). The available information suggests that neither of the two cotransferases can substitute for the
other. For example, L-cell or Chinese hamster ovary cell mutants deficient in EXT1 were unable to synthesize heparan
sulfate even after transfection with EXT2 (18, 33).
Moreover, mouse embryonic stem cells derived from EXT1
homozygous null embryos markedly reduced the formation of heparan
sulfate, even though the cells contained an EXT2 ortholog
(35). Thus, although it is obvious that EXT1 has GlcUA/GlcNAc
transferase activities and that it functions in vivo in
heparan sulfate formation, the role of EXT2 is unclear given its low
activity and the lack of mutants. In addition, 1-2% contamination by
EXT1 could easily account for the low level of activity seen for EXT2
in mammalian systems (Refs. 17 and 19; also see Table I) given that the
enzymes interact. We have demonstrated that EXTL2 encodes
GlcNAc transferase I that determines and initiates the heparan sulfate
synthesis (20) but have found no EXT homolog that encodes
only GlcNAc transferase I or GlcUA/GlcNAc-cotransferase in C. elegans as shown in the present study. Moreover, coexpression of
the rib-1 and rib-2 proteins did not result in the promotion of the
GlcNAc transferase activities or the expression of the GlcUA
transferase activity required for heparan sulfate polymerization (data
not shown). Although the possibility cannot be ruled out that
nonhomologous genes to the EXT gene family may encode
GlcUA/GlcNAc-cotransferases, these findings suggest that the
biosynthetic mechanism of heparan sulfate in C. elegans is
distinct from that in mammals.
Recent studies have demonstrated a critical role for heparan sulfate in
growth factor signaling mediated by Wingless proteins during
Drosophila development (2). In addition, a
Drosophila homolog of EXT1 (encoded by ttv) was
recently implicated in the Hedgehog diffusion (2). C. elegans is also a genetically tractable organism ideally suited
for the analysis of glycan function during tissue assembly and
morphogenesis. In fact, three proteins required for wild-type vulval
invagination in C. elegans, which are encoded by
sqv-3, -7, and -8 (squashed
vulva), respectively, have been reported to be similar to
components of a glycosylation pathway (36). sqv-3,
-7, and -8 are three of the eight genes in the sqv class, identified on the basis of a common vulval
invagination defect. Recently, Bulik et al. (26) showed that
sqv-3, -7, and -8 mutants all affected
the biosynthesis of GAGs and that sqv-3 and -8 genes encoded enzymes with Gal transferase I and GlcUA transferase I
activity, respectively. Because both enzymes are required for the
synthesis of the GAG-protein linkage tetrasaccharide common to all
GAGs, including chondroitin sulfate and heparan sulfate (37-39), it is
reasonable that sqv-3 and -8 mutants showed significantly reduced levels of both chondroitin and heparan sulfate (26). Thus, if rib-2 mutants become available, they would be useful tools for determining which GAG, heparan sulfate or chondroitin, is required for C. elegans vulval invagination.
1,4-N-acetylglucosaminyltransferase involved in the
biosynthetic initiation and elongation of heparan sulfate. In contrast,
the findings confirmed the previous observations that both the EXT1 and
EXT2 proteins were heparan sulfate copolymerases with both
1,4-N-acetylglucosaminyltransferase and
1,4-glucuronyltransferase activities, which are involved only in the
elongation step of the heparan sulfate chain, and that the EXTL2
protein was an
1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These
findings suggest that the biosynthetic mechanism of heparan sulfate in
C. elegans is distinct from that reported for the mammalian system.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, attached to
specific Ser residues of core proteins, which is common to the GAGs
including heparin/heparan sulfate and chondroitin sulfate/dermatan
sulfate (for reviews see Refs. 3 and 4). 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 GlcUA, each
reaction being catalyzed by the respective specific glycosyltransferase (3, 4). The GAGs are built up on this linkage region by the alternate
addition of N-acetylhexosamine and GlcUA 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,
the biosynthetic sorting mechanisms for different GAG chains remain enigmatic.
1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylhexosaminidase were purchased from Seikagaku
Corp. (Tokyo, Japan). The chemically synthesized linkage
tetrasaccharide-serine GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (27) and
linkage pentasaccharide-serine GlcNAc
1-4GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(27) were provided by T. Ogawa (RIKEN, The Institute of Physical and
Chemical Research, Saitama, Japan).
GlcUA
1-3Gal
1-O-C2H4NHCbz was
chemically synthesized.2
N-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5 were prepared as
described previously (28). Adult C. elegans total RNA was
from J. Hirabayashi (Teikyo University, Kanagawa, Japan).
1-(4GlcNAc
1-4GlcUA
1-)n (50 µg),
GlcUA
1-3Gal
1-O-C2H4NHCbz
(10 or 250 nmol), or
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (10 nmol) for
GlcUA transferase activities using N-acetylheparosan oligosaccharides, GlcNAc
1-(4GlcUA
1-4GlcNAc
1-)n (50 µg), or GlcNAc
1-4GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(10 nmol) and for GalNAc transferase activities using
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (1 nmol) as an
acceptor substrate as described below. The assay mixture for GlcNAc
transferase contained 10 µl of the resuspended beads, an
acceptor substrate, 250 µM UDP-[3H]GlcNAc
(8.21 × 105 dpm), 100 mM MES buffer, pH
6.5, 10 mM MnCl2, and 171 µM the sodium salt of ATP in a total volume of 30 µl. The assay mixture for
GlcUA transferase contained 10 µl of the resuspended beads, an
acceptor substrate, 250 µM UDP-[14C]GlcUA
(5.66 × 105 dpm), 50 mM HEPES buffer, pH
7.2, 10 mM MnCl2, 10 mM
MgCl2, 5 mM CaCl2, 0.04% Triton
X-100, and 171 µM sodium salt of ATP in a total volume of
30 µl. The assay mixture for GalNAc transferase contained 10 µl of
the resuspended beads, an acceptor substrate, 8.57 µM
UDP-[3H]GalNAc (5.28 × 105 dpm), 50 mM MES buffer, pH 6.5, 20 mM MnCl2,
and 171 µM sodium salt of ATP in a total volume of 30 µl. Reaction mixtures were incubated at 37 °C for 1 h, and
then radiolabeled products were separated from
UDP-[3H]GlcNAc, UDP-[14C]GlcUA, or
UDP-[3H]GalNAc by gel filtration using a syringe column
packed with Sephadex G-25 (superfine) or using a Superdex peptide
column (Amersham Pharmacia Biotech) or by HPLC on a
Nova-Pak® C18 column (3.9 × 150 mm; Waters, Tokyo,
Japan) as described previously (17, 20, 21, 32). The recovered labeled
products were quantified by liquid scintillation spectrophotometry.
-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 calcium acetate at 37 °C overnight. The enzyme digest
was analyzed using the same Superdex peptide column as described above.
1-3Gal
1-O-C2H4NHCbz 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 1 mIU
of
-N-acetylhexosaminidase in a total volume of 20 µl
of 50 mM sodium citrate buffer, pH 4.5, or with 1 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 calcium acetate at 37 °C overnight. The enzyme digest was analyzed using the same Nova-Pak® C18 column as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-O-C2H4NHCbz used
as an acceptor substrate for the GlcNAc transferase I reaction shares the disaccharide sequence with the GAG-protein linkage region tetrasaccharide. It served as a good acceptor for the EXTL2 protein (see Table I) and was comparable with
GlcUA
1-3Gal
1-O-naphthalenemethanol, an
artificial, yet authentic oligosaccharide acceptor substrate for GlcNAc
transferase I.2 As shown in Table I, the marked GlcNAc
transferase activity was detected with
N-acetylheparosan oligosaccharides
GlcUA
1-(4GlcNAc
1-4GlcUA
1-)n and
GlcUA
1-3Gal
1-O-C2H4NHCbz
but not with the tetrasaccharide-serine GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser as
acceptor substrates, whereas no GlcUA transferase activity was
observed using N-acetylheparosan oligosaccharides
GlcNAc
1-(4GlcUA
1-4GlcNAc
1-)n or the
pentasaccharide-serine GlcNAc
1-4GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
No detectable GlcNAc transferase activity was recovered by
the affinity purification from the control pEF-BOS transfection sample,
and it therefore seems unlikely that the results are due to an artifact
or an endogenous activity.
Comparison of acceptor specificity of the rib-2, EXT1, EXT2, and EXTL2
fusion proteins secreted into the culture medium by transfected
COS-1 cells
1-(4GlcNAc
1-4GlcUA
1-)n and
GlcUA
1-3Gal
1-O-C2H4NHCbz,
were individually labeled by the respective transferase reaction using
UDP-[3H]GlcNAc as a donor substrate and the enzyme-bound
beads as an enzyme source. Both labeled products were completely
digested by heparitinase I, which cleaves an
1,4-N-acetylglucosaminidic linkage in an eliminative
fashion, quantitatively yielding a 3H-labeled peak at the
elution position of free [3H]GlcNAc, as demonstrated by
gel filtration (Fig. 1A) or
hydrophobic HPLC (Fig. 1B). In contrast, they were inert to
the action of
-N-acetylhexosaminidase. These findings
clearly indicated that a GlcNAc residue had been transferred
exclusively to the nonreducing terminal GlcUA of
N-acetylheparosan oligosaccharides or
GlcUA
1-3Gal
1-O-C2H4NHCbz through an
1,4 linkage. The present findings demonstrated that rib-2
was a novel and unique
1,4-GlcNAc transferase involved in the
biosynthetic initiation and elongation of heparan sulfate.
View larger version (22K):
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Fig. 1.
Characterization of the GlcNAc transferase
reaction products using various GAG-degrading enzymes.
A, 3H-labeled GlcNAc transferase reaction
products obtained using N-acetylheparosan as an acceptor
substrate were subjected to digestion with heparitinase I or
-N-acetylhexosaminidase as described under
"Experimental Procedures." The heparitinase I digest (filled
circles),
-N-acetylhexosaminidase digest (open
circles), or the undigested sample (filled squares) was
applied to a column of Superdex peptide (1.0 × 30 cm), and the
respective effluent fractions (0.4 ml each) were analyzed for
radioactivity as described under "Experimental Procedures." An
arrow indicates the elution position of free GlcNAc.
B, 3H-labeled GlcNAc transferase reaction
products, which were obtained using
GlcUA
1-3Gal
1-O-C2H4NHCbz as
an acceptor substrate, were subjected to digestion with heparitinase I
or
-N-acetylhexosaminidase as described under
"Experimental Procedures." The heparitinase I digest (filled
circles),
-N-acetylhexosaminidase digest (open
circles), or the undigested sample (filled squares) was
analyzed by HPLC on a Nova-Pak® C18 column as described
under "Experimental Procedures," and the respective effluent
fractions (2 ml each) were analyzed for radioactivity. An
arrow indicates the elution position of free GlcNAc.
1-(4GlcNAc
1-4GlcUA
1-)n as their preferred acceptor
substrates when UDP-[3H]GlcNAc was used as a donor
substrate. However, both EXT1 and EXT2 exhibited a strict specificity,
showing no [3H]GlcNAc incorporation into
GlcUA
1-3Gal
1-O-C2H4NHCbz.
Equally striking was that both EXT1 and EXT2 showed the GlcUA
transferase activity required for heparan sulfate polymerization,
whereas rib-2 showed no [14C]GlcUA incorporation when
tested with N-acetylheparosan oligosaccharides GlcNAc
1-(4GlcUA
1-4GlcNAc
1-)n or the pentasaccharide-serine GlcNAc
1-4GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
as acceptor substrates. Thus, both EXT1 and EXT2 have marked activities
of GlcNAc transferase II and GlcUA transferase required for heparan
sulfate polymerization but no GlcNAc transferase I activity for the
heparan sulfate initiation. In addition, comparison of rib-2 to EXTL2
revealed that both utilized GlcUA
1-3Gal
1-O-benzyloxycarbonyl, whereas EXTL2
showed negligible [3H]GlcNAc incorporation into
N-acetylheparosan GlcUA
1-(4GlcNAc
1-4GlcUA
1-)n. Thus, EXTL2 harbors only GlcNAc transferase I activity for the heparan
sulfate initiation. Previously, we demonstrated that EXTL2 encoded the enzyme with a dual catalytic activity of GlcNAc transferase I and
1,4-GalNAc transferase, i.e. an
1,4-N-acetylhexosaminyltransferase that transferred
GlcNAc/GalNAc to GlcUA
1-3Gal
1-O-naphthalenemethanol or the core tetrasaccharide-serine representing the GAG-protein linkage
region (20). Hence, each of the purified recombinant proteins was
assayed for GalNAc transferase activity using
UDP-[3H]GalNAc as a sugar donor and the
tetrasaccharide-serine
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser as an
acceptor. As shown in Table I, a significant GalNAc transferase activity was detected for EXTL2 but not for rib-2. These findings indicate that the rib-2 exhibits properties clearly distinct from the
three mammalian glycosyltransferases characterized to date.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-GlcNAc
transferase involved in the biosynthetic initiation and elongation of
heparan sulfate. As shown in Table I, rib-2, characterized in the
present study, exhibited distinct but overlapping acceptor substrate
specificities when compared with those of the mammalian
glycosyltransferases responsible for the heparan sulfate biosynthesis,
EXT1/EXT2 and EXTL2. Indeed, the two primary products of rib-2,
GlcNAc
1-(4GlcUA
1-4GlcNAc
1-)n and
GlcNAc
1-4GlcUA
1-3Gal
1-, are also products of EXT1/EXT2 (17,
19) and of EXTL2 (20), respectively. In addition, rib-2 appears to
recognize a specific sequence in the core protein or an aglycone
structure attached to the linkage region tetrasaccharide as in the case
of EXTL2 (20), because the tetrasaccharide-serine, GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
derived from the linkage region, was not utilized as an acceptor substrate.
1,4-GlcNAc transferase
involved in the biosynthetic initiation and elongation of heparan
sulfate in mammals.
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ACKNOWLEDGEMENT |
---|
We thank Dr. T. Ogawa for the synthetic enzyme substrates.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Uehara Memorial Foundation (to H. K.), the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and a Grant-in-aid for Scientific Research on Priority Areas 10178102 (to K. S.) from the Ministry of Education, Science, Sports, and Culture of Japan and by Human Frontier Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7569; E-mail: k-sugar@kobepharma-u.ac.jp.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.C000835200
2 H. Shimakawa, Y. Kano, H. Kitagawa, J. Tamura, J. D. Esko, and K. Sugahara, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: GAG(s), glycosaminoglycan(s); bp, base pair(s); Cbz, benzyloxycarbonyl; HME, hereditary multiple exostoses; HPLC, high-performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; PCR, polymerase chain reaction.
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