From the Glycogene Function Team, Research
Center for Glycoscience, National Institute of Advanced Industrial
Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, § Amersham
Biosciences KK, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan, the ¶ Seikagaku Corporation, 1253 Tateno 3-Chome,
Higashi-yamato, Tokyo 207-0021, Japan, the
Institute for
Molecular Science of Medicine, Aichi Medical University, Nagaute,
Aichi 480-1195, Japan, ** JGS Japan Genome Solutions, Inc.,
51 Kamiyacho, Hachioji, Tokyo 192-0031, Japan, the
§§ Cell Regulation Team, Age Dimension Research
Center, AIST, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan,
the
New Energy and Industrial Technology
Development Organization (NEDO), Sunshine 60 Bldg., 3-1-1 Higashi
Ikebukuro, Toshima-ku Tokyo, 170-6028, Japan
Received for publication, August 30, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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By a tblastn search with
Proteoglycans (PGs),1
molecules consisting of a core protein and at least one
glycosaminoglycan (GAG) chain, exist as one of the major components of
extracellular matrix and on the cell surface. A variety of proteoglycan
functions are exerted depending on the GAG chains. These chains are
usually highly sulfated, and can be classified into several groups
including chondroitin sulfate (CS)/dermatan sulfate, heparan
sulfate (HS)/heparin, and keratan sulfate based on the GAG composition.
HS has been shown to be involved in signal transduction and development
together with certain growth factors, cytokines, and extracellular
matrices. In Drosophila melanogaster, HS deficiencies caused
by mutations in the genes encoding enzymes involved in the synthesis of
HS result in abnormal developmental phenotypes (1). HS has been demonstrated to bind to a variety of cell growth factors such as the
fibroblast growth factor (FGF) family molecules and to modulate their
activities in various ways (2). Hepatocyte growth factor (HGF) and some
interleukins (interleukins 3 and 7) bind to HS for efficient signal
transduction (3, 4). Recent studies have demonstrated that CS also
plays various important roles in cell adhesion, migration, and
recognition, especially of neuronal cells (5-7). The sulfation
profiles of CS vary with aging in the cartilage (8, 9). Disulfated
disaccharide units, CS-D (GlcA(2S) The initial stage in the biosynthesis of both CS and HS involves a
linkage tetrasaccharide structure
(GlcA For the synthesis of HS, five glycosyltransferase genes,
EXT1, EXT2, EXTL1, EXTL2,
and EXTL3, were cloned and their products characterized.
They retain conserved motifs of short amino acid stretches in their
COOH terminus and belong to one family. The EXT1 and
EXT2 genes, which have been identified as tumor suppressor genes and were implicated in hereditary multiple exostoses, were found
to encode HS polymerases having the activity of both
Very recently, three glycosyltransferases, chondroitin synthase (CSS)
(14), CS glucuronyltransferase (CSGlcAT) (15) and CSGalNAcT (16, 17),
which are involved in the synthesis of CS, have been reported. CSS was
found to be a CS polymerase having the activity of both
In this study, we report the cloning and characterization of a novel
N-acetylgalactosaminyltransferase, CSGalNAcT-2, that is the
fourth member participating in the synthesis of CS. In considering the
substrate specificities of these two CSGalNAcTs in
vitro, we suggest their differential roles in vivo.
Isolation of Human CSGalNAcT-2 cDNA--
We performed a
tblastn search of the GenBankTM data base using
Construction and Purification of CSGalNAcT Proteins Fused with
FLAG Peptide--
The putative catalytic domain of CSGalNAcT-2 (amino
acids 37 to 542) was expressed as a secreted protein fused with a FLAG peptide in insect cells according to the instruction manual of GATEWAYTM Cloning Technology (Invitrogen, Groningen,
Netherlands). An ~1.6-kb DNA fragment was amplified by PCR using the
Marathon ReadyTM cDNA derived from human bone marrow
(Clontech) as a template, and two primers,
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAAGGTGACGAGGAGCAGCTGGCAC-3' and
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATGTTTTTTTGCTACTTGTCTTCTGT-3'. The amplified fragment was inserted into the vector pDONRTM
201 (Invitrogen), then transferred into the expression vector pFBIF to
construct pFBIF-CSGalNAcT-2 as described previously (18). A CSGalNAcT-1
expression vector, pFBIF-CSGalNAcT-1, was also constructed as reported
elsewhere (17). The catalytic domains of CSGalNAcT-1 and
CSGalNAcT-2 were expressed in Sf21 insect cells. A 50-ml volume of culture medium was mixed and incubated with anti-FLAG M1 antibody resin (SIGMA). The resin was washed twice with 50 mM
Tris-buffered saline (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 1 mM CaCl2 and
suspended in 100 µl of each assay buffer described below.
Construction of Assay of CSGalNAcT Activity--
To determine the enzymatic
activity, UDP-galactose (UDP-Gal) and
UDP-N-acetylgalactosamine (UDP-GalNAc) (Sigma) were utilized as donor substrates. UDP-[3H]GalNAc (7.0 Ci/mmol) was
purchased from PerkinElmer Life Sciences. For acceptor substrates,
Gal-
For the GalT assay, 14 mM Hepes buffer (pH 7.4) containing
250 µM UDP-Gal, 12.5 mM MnCl2,
and 500 µM of each acceptor substrate was used. For the
GalNAcT assay, 50 mM MES buffer (pH 6.5) containing 0.1%
Triton X-100, 1 mM UDP-GalNAc, 10 mM
MnCl2, and 500 µM of each acceptor substrate
was used. A 10-µl volume of enzyme solution for 20 µl of each
reaction mixture was added and incubated at 37 °C for 2 h for
the GalT and 16 h for the GalNAcT assay. After incubation, the
mixture was filtrated with an Ultrafree-MC column (Millipore, Bedford,
MA) and a 10-µl aliquot was subjected to reversed-phase high
performance liquid chromatography on an ODS-80Ts QA column (4.6 × 250 mm; TOSOH, Tokyo, Japan). 0.1% Trifluoroacetic acid/H2O was used as a running solution and the products
were eluted with a 0-15 (for GlcA- Assay of CSGalNAcT Activity with a Tetrasaccharide-bikunin
Peptide--
A Xyl-bikunin peptide (VLPQEEEGS(-Xyl)GGGQLVT) was
purchased from the Peptide Institute Inc. (Osaka, Japan). The Cy5
(Amersham Biosciences)-labeled Xyl-peptide was incubated with 5 µl of
three glycosyltransferases, Determination of Products by CSGalNAcT-2 with Mass Spectrometry
(MS)--
An additional peak detected by reversed-phase chromatography
was isolated and analyzed by an electrospray ionization (ESI) or
matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)
MS (ESI-MS, esquire3000plus, and MALDI-TOF-MS, Reflex IV; Bruker
Daltonics, Billerica, MA). Then, 25 pmol of product was dissolved in 5 µl of H2O, and 45 µl of 0.1% formic acid and 50 µl
of methanol were added. The product solution was infused at a rate of 3 µl/min with a capillary voltage of 3 kV. The hypothetical product
peaks obtained by ESI-MS were analyzed by ESI tandem mass spectrometry
(ESI-MS/MS). These analyses were done in both positive-ion and
negative-ion ESI modes. For MALDI-TOF MS analysis, 10 pmol of product
was dried, dissolved in 1 µl of H2O, and applied.
In Vivo CS/HS Synthesis on a Syndecan-4/FGF-1 Chimera
Protein--
For the expression in CHO-K1 cells, the full-length
cDNAs of CSGalNAcT-1 and CSGalNAcT-2 were amplified by PCR with
primers CSGalNAc-T1, 5'-CCCAAGCTTATGATGATGGTTCGCCGGGGGCT-3' and
5'-GCTCTAGATCATGTTTTTTTGCTACTTGTCTTCTGT-3', and CSGalNAc-T2,
5'-CCCAAGCTTATAAGAATGCCTAGAAGAGGACTGA-3' and 5'-GCTCTAGATCAACCAACAGCTTCACTGTTTGTC-3'. The amplified fragments were inserted into pcDNA3.1/hygromycin(+) (pcDNA3.1/Hygro,
Invitrogen) after HindIII and XbaI digestion. The
resulting plasmid, pcDNA3.1/HygroCSGalNAcT-1,-CSGalNAcT-2, and the
pcDNA3.1/Hygro expression vector were transfected into CHO-K1 cells
harboring pMEXneo-PG-FGF-1 that were expressing the syndecan-4/FGF-1
chimera protein (PG-FGF-1) using LipofectAMINE 2000 (Invitrogen)
according to the manufacturer's instructions. Medium conditioned by
the CHO transfectant containing the secreted PG-FGF-1 protein with GAGs
was collected. Fractionation of PG-FGF-1 using a DEAE-Sepharose column
(Amersham Biosciences) and endoglycosidases: heparitinase (HSase),
heparanase (HPase), and chondroitinase ABC (CSase) (Seikagaku Corp.)
digestion and Western blotting with monoclonal antibody against FGF-1
(mAb1) (20) were performed as described previously (21). The PG-FGF-1
bands digested by glycosidases were quantified by densitometric
scanning of the digitized image using NIH Image (version 1.60) software
(National Institutes of Health, Bethesda, MD).
Quantitative Analysis of CSGalNAcT-1 and CSGalNAcT-2 Transcripts
in Human Tissues by Real-time PCR--
For quantification of the two
CSGalNAcT transcripts, we employed the real-time PCR method,
as described in detail previously (18, 22, 23). Marathon
ReadyTM cDNAs derived from various human tissues and
cells were purchased from Clontech. Standard curves
for the endogenous control, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNAs, were generated by serial dilution of a pCR2.1
(Invitrogen) DNA containing the GAPDH cDNA. The primer sets and the probes for CSGalNAcT-1 and CSGalNAcT-2 were as follows. The forward primer for CSGalNAcT-1 was
5'-GACTTCATCAATATAGGTGGGTTTGAT-3', the reverse primer,
5'-GTCCGTACCACTATGAGGTTGCT-3', and the probe, 5'-ACCTTTATCGCAAGTATCT-3'
with a minor groove binder (24). The forward primer for CSGalNAc-T2 was
5'-CTGACCATTGGTGGATTTGACAT-3', the reverse primer,
5'-AACCGGAGTCCGAATCACAA-3', and the probe, 5'-CATCTTTATCGAAAATACTTACATGG-3' with a minor groove binder. PCR products were continuously measured with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amount of each CSGalNAcT transcript was normalized to the
amount of GAPDH transcript in the same cDNA.
Determination of Nucleotide and Amino Acid Sequence of
CSGalNAcT-2--
We determined a novel full-length cDNA sequence
by the 5'-rapid amplification of cDNA ends method and registered it
in the GenBankTM data base with accession number AB079252.
The nucleotide sequence and the putative amino acid sequence are shown
in Fig. 1. This cDNA sequence
consisted of a 254-bp 5'-untranslated region, 1629-bp coding region,
and 1791-bp 3'-untranslated region that contained a poly-A tail (Fig.
1). Although the original expressed sequence tag was obtained in a
GenBankTM data base search with
A genome sequence identical to that of the CSGalNAcT-2 cDNA was
found in a genome clone (GenBankTM AC011890), which is
localized on chromosome 10. The genomic structure of the
CSGalNAcT-2 gene was determined to be composed of at least 7 exons by comparison between the cDNA and genome sequences (Fig. 1).
The exon/intron junctions of CSGalNAcT-2 were identical to those of
CSGalNAcT-1 (data not shown).
Determination of CSGalNAcT-2 Activity in Elongation and Initiation
of Chondroitin Poly- and Oligosaccharides--
The truncated soluble
forms of CSGalNAcT-1 and CSGalNAcT-2 were expressed in insect cells and
employed for all experiments. On Western blotting using anti-FLAG
antibody, each enzyme was detected as a single band corresponding to
the predicted size (Fig. 3B).
An additional band appeared by Coomassie staining at ~50 kDa shared
by all lanes (Fig. 3A), however, the protein recovered from
mock transfectants showed no activity for any donors and acceptor
substrates (data not shown). At first, we screened the transfer
activity of CSGalNAcT-2 using nine donor substrates and multiple
monosaccharide-pNP acceptors. In screening of donor and acceptor
substrates, CSGalNAcT-2 transferred a GalNAc residue to GlcA-pNP,
however, no activity was observed with other combinations of donor and
acceptor substrates (data not shown). It was identified in a previous
report that CSGalNAcT-1 is
Two kinds of GalNAc-GlcA linkages are known in CS, one in its polymer
structure (3GalNAc Comparison of Acceptor Substrate Specificity between CSGalNAcT-1
and CSGalNAcT-2--
In view of these results, CSGalNAcT-2 was
suggested to be GalNAcT involved in the synthesis of CS as well as
CSGalNAcT-1. To distinguish the functions of the two CSGalNAcTs, we
compared GalNAcT activity toward GlcA found at the nonreducing terminus of various kinds of chondroitin-related acceptor substrates. The amount
of enzyme was estimated by Western blotting, and approximately equal
amounts of enzyme were used for the GalNAcT reaction. The results are
summarized in Table I. Regarding the
elongation activity, CSGalNAcT-2 utilized chondroitin polysaccharide as
an acceptor more than any other substrate examined, and showed higher
levels of enzymatic activity than CSGalNAcT-1 toward all substrates
except for the linkage tetrasaccharide (Table I). Furthermore,
CSGalNAcT-2 showed remarkably strong activity, compared with
CSGalNAcT-1, toward sulfated substrates such as CS poly- and
oligosaccharides (Table I). CS-A and CS-B, both of which are sulfated
at position C-4 of GalNAc, were better substrates for CSGalNAcT-2 than
CS-C, which is sulfated at position C-6 of GalNAc and CS-D, which is sulfated at C-6 of GalNAc and C-2 of GlcA (Fig.
4). CSGalNAcT-2 exhibited much
stronger activity toward the longer oligosaccharides, prepared from
chondroitin and CS, than toward the shorter ones. These results
strongly indicated that CSGalNAcT-2 is much more active in the
elongation of chondroitin and CS than is CSGalNAcT-1.
Regarding the initiation activity, CSGalNAcT-1 preferred the linkage
tetrasaccharide as substrate and showed a much higher level of activity
for the linkage tetrasaccharide than did CSGalNAcT-2. This indicated
that CSGalNAcT-1 is the enzyme mainly responsible for the initiation of
chondroitin and CS synthesis, not for elongation. To examine whether
two GalNAcTs show a synergistic effect for initiation and elongation
activities, two enzymes were mixed in the enzyme reaction toward the
linkage tetrasaccharide or chondroitin. We observed additional effects
of GalNAcT activity, but not synergistic effect, to any acceptor
substrates (data not shown).
Comparison of Initiation Activity with Linkage Tetrasaccharide
Peptide between Two CSGalNAcTs--
We performed an in
vitro enzymatic synthesis of a chondroitin pentasaccharide-bikunin
peptide (VLPQEEEGS*GGGQLVT; the peptide sequence encodes the
NH2-terminal sequence of bikunin and the serine residue
with an asterisk is attached with a saccharide chain) for determination
of the initiation activity by CSGalNAcTs. Peak S in Fig.
5A, which was identified to
possess the linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-bikunin peptide)
in a previous study, was used as the acceptor substrate. To obtain the
relative activity of each enzyme, equal amounts of enzyme were
employed, and the incubation was terminated before a large amount of
acceptor substrate remained. As seen in Fig. 5, B and
C, the reaction product (Peak P) appeared at 30.1 min with
CSGalNAcT-1 or -2, respectively. Peak P was identified as
GalNAc-GlcA-Gal-Gal-Xyl-bikunin peptide in a previous study (17). The
peak area of the CSGalNAcT-1 product (Fig. 5C) was 3-fold
larger than that of the CSGalNAcT-2 product (Fig. 5B). The
value of this relative activity was consistent with that toward the
methoxyphenyl-linkage tetrasaccharide in Table I, i.e. the
activity of CSGalNAcT-1 was 2.3-fold that of CSGalNAcT-2. These results
indicated that the presence of bikunin peptide does not influence the
relative activity of the two CSGalNAcTs in vitro.
Change of GAG Composition of PG-FGF-1 by CSGalNAcT-1 or CSGalNAcT-2
Transfection--
Previously, the gene construct encoding a chimera
protein (PG-FGF-1) consisting of syndecan-4 and FGF-1 was transfected
into CHO-K1 cells to express the chimera protein (21). Syndecan-4 is a
well analyzed proteoglycan possessing chains of HS and/or CS. The GAG
composition of PG-FGF-1 secreted from the stable transfectant cells
with PG-FGF-1 had been analyzed. It was found that GAG on PG-FGF-1
consisted of large populations of HS and small populations of CS. The
initiation of the synthesis of HS or CS is determined by
PG-FGF-1 secreted into the medium conditioned by the transfectants was
detected by immunoblotting with anti-FGF-1 monoclonal antibody (mAb1)
(20). PG-FGF-1 from CHO-K1 mock transfectants showed a major band with
a large molecular mass of more than 200 kDa (Fig.
6, lane 1). The composition of
GAGs on PG-FGF-1 was analyzed by glycosidase digestion. Digestion with
chondroitinase ABC (CSase) yielded a protein band at 32 kDa (lane
2), which corresponds to the molecular mass of PG-FGF-1 with
linkage saccharides, although most proteins remained at a large
molecular mass. The ratio of CS to CS plus HS on PG-FGF-1 was obtained
by measurement of the intensity of the 32-kDa band, for example, the
ratio of CS/CS + HS on PG-FGF-1 produced by the mock transfectants was
9.5%, which was calculated as the intensity of the 32-kDa band in
lane 2 divided by that in lane 4. This indicated
that PG-FGF-1 containing only CS without HS occupied only 9.5% of
total PG-FGF-1, and HS occupied most of the PG-FGF-1 produced by
the mock-transfected CHO-K1 cells. Digestion of PG-FGF-1 (lane
3) with a mixture of heparitinase (HSase) and heparanase (HPase)
yielded a strong 32-kDa band, however, faint smear bands, which
probably carry CS, were detected at a molecular mass of approximately
40-70 kDa. CSase treatment of PG-FGF-1 recovered from the CSGalNAcT-2
transfectants did not change significantly the intensity of the 32-kDa
band, compared with the control (9.5 in lane 2 versus 4.3% in lane 6). In contrast, CSase
treatment of PG-FGF-1 recovered from the CSGalNAcT-1 transfectants
apparently increased the intensity of the 32-kDa band (9.5 in
lane 2 versus 39.1% in lane 10). The
above results indicated that CSGalNAcT-1 effectively initiates the
synthesis of CS and increases the number of CS chains by transferring
GalNAc to the linkage tetrasaccharide on syndecan-4, but CSGalNAcT-2 did not exert initiation activity in the cells.
Tissue Distribution of CSGalNAcT-2 Transcripts--
We determined
the tissue distribution and expression levels of CSGalNAcT-2
transcripts, in comparison with CSGalNAcT-1 transcripts, by the
real-time PCR method. The expression levels of both genes in various
human tissues were shown as the relative amount versus GAPDH
transcripts (Fig. 7). Both genes were
expressed ubiquitously in all tissues examined. However, the expression
profiles differed from each other, i.e. CSGalNAcT-2 was
highly expressed in the small intestine, leukocyte, and spleen, whereas
CSGalNAcT-1 was highly expressed in the placenta and thyroid gland.
The investigation of CS has progressed to the relationship between
its structural properties including saccharide composition and
sulfation, and its biological function. However, the mechanisms of its
biosynthesis, such as the number of enzymes participating and their
roles, still remained to be elucidated. To date, three glycosyltransferases, CSS (14), CSGlcAT (15), and CSGalNAcT-1 (16, 17),
which participate in the biosynthesis of CS, have been reported. In
this study, a novel enzyme CSGalNAcT-2 was found to be a fourth member
involved in the synthesis, and identified to be the second GalNAcT
transferring a GalNAc to GlcA.
The amino acid sequence of CSGalNAcT-2 is highly homologous to that of
CSGalNAcT-1. The Regarding the initiation activity, CSGalNAcT-1 exhibited much stronger
GalNAcT activity toward the linkage tetrasaccharide than CSGalNAcT-2.
In a previous study, we reported that CSGalNAcT-1 has effective
initiation activity, i.e. it transferred GalNAc to the
linkage tetrasaccharide conjugated with a bikunin peptide (GlcA-Gal-Gal-Xyl-bikunin peptide) in vitro (17). The
initiation activity of CSGalNAcT-1 relative to CSGalNAcT-2 was 2.3- and
3.0-fold higher toward methoxyphenyl-linkage tetrasaccharide and
tetrasaccharide-bikunin peptide, respectively. The presence of a
peptide backbone did not affect the relative initiation activity. The
in vivo assay system using the syndecan-4/FGF-1 chimera
protein (PG-FGF-1) demonstrated that CSGalNAcT-1 exhibits initiation
activity for syndecan-4 by competing with the synthesis of HS (Fig. 6).
Syndecan-4 is a complex-type PG that has four putative GAG attachment
sites, and both CS and HS can bind to these sites (36). In our
construction of PG-FGF-1, a part of syndecan-4 containing three GAG
attachment sites was ligated to the NH2 terminus of FGF-1
(21). The intensity of the 32-kDa band after CSase or HSase/HPase
treatment of PG-FGF-1 reflects the CS and HS composition of PG-FGF-1 as
demonstrated in our previous study (21). In this assay system, we
identified an increase in CS on PG-FGF-1 only in
CSGalNAcT-1-transfected CHO-K1 cells, not in CSGalNAcT-2 transfectants.
The increase of CS is probably directed by the initiation activity of
CSGalNAcT-1. This in vivo observation is consistent with
previous results in vitro of the effective initiation
activity of CSGalNAcT-1 as we reported (17). Taken together with the
previous findings in vitro, the in vivo results
of the present study strongly support that CSGalNAcT-1 is the enzyme
most responsible for the initiation of CS synthesis in the cells.
On the other hand, CSGalNAcT-2 transfected into CHO-K1 cells showed no
increase in CS, although it had some initiation activity in
vitro toward the linkage tetrasaccharide. This contradiction between the in vitro and in vivo activities of
CSGalNAcT-2 suggests that the initiation of CS synthesis might be
controlled by some unknown mechanisms. One possibility is the effect of
sulfation on the linkage tetrasaccharide. In vertebrates, CS is
specifically sulfated at position C-6 of the inner Gal (Gal The tissue distribution of transcripts for three glycosyltransferase
genes, i.e. CSS, CSGlcAT, and
CSGalNAcT-1, have been reported to be ubiquitously expressed
in all tissues examined (14-17). CSGalNAcT-2 also showed a ubiquitous
expression like the others. However, characteristic of that CSGalNAcT-2
was a high level of expression in the small intestine, leukocytes, and
spleen, whereas the others are not so highly expressed in these
tissues. Hiraoka et al. (31) reported that the transcripts
of C4ST-1 and C4ST-2, which produce CS-A, are also highly expressed in
the small intestine, spleen, and leukocytes. In contrast, C6ST-2, which
produces CS-C, is expressed significantly in the spleen, but not so in
small intestine (34). The preference of CSGalNAcT-2 for CS-A and CS-B
to CS-C and CS-D, as demonstrated in Table I, led us to speculate of a
cooperative CS synthesis by CSGalNAcT-2 and C4STs in the cells. The
similar profile of the tissue distribution of CSGalNAcT-2 to that of
C4STs further suggested this cooperative synthesis. In the case of HS,
it has been proposed that EXTL2 and EXTL3 are involved in the
initiation of the synthesis (12, 13), however, the exact role of each
enzyme in vivo remains to be elucidated.
Finally, the catalytic activities of each enzyme involved in the
synthesis of HS and CS are schematically summarized in Fig. 8. Four glycosyltransferases
responsible for the synthesis of the linkage tetrasaccharide have been
cloned and analyzed (41-46). Using these enzymes, it is now possible
to synthesize enzymatically the linkage tetrasaccharide bound to the
peptide (17). The initiation of the CS chain is also feasible using
CSGalNAcT-1 (17). Three enzymes, i.e. CSS, CSGlcAT, and
CSGalNAcT-2, have been identified as involved in the polymerization of
the CS chain. In addition to the four enzymes, there still may be
unknown enzymes, which remain to be cloned and analyzed, for the
synthesis of CS.
1,4-galactosyltransferases as query sequences, we found an expressed
sequence tag that showed similarity in
1,4-glycosyltransferase
motifs. The full-length complementary DNA was obtained by a method of
5'-rapid amplification of complementary DNA ends. The predicted open
reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin
sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger
N-acetylgalactosaminyltransferase activity toward
glucuronic acid of chondroitin poly- and oligosaccharides, and
chondroitin sulfate poly- and oligosaccharides with a
1-4 linkage,
i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of
the glycosaminoglycan linkage structure
(GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e.
initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells
yielded a change of glycosaminoglycan composition, i.e. the
replacement of heparan sulfate on a syndecan-4/fibroblast growth
factor-1 chimera protein by chondroitin sulfate, however, transfection
of the CSGalNAcT-2 gene did not. The above results
indicated that CSGalNAcT-1 is involved in the initiation of chondroitin
sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the
elongation, not initiation. Quantitative real-time PCR analysis
revealed that CSGalNAcT-2 transcripts were highly expressed in the
small intestine, leukocytes, and spleen, however, both CSGalNAcTs were
ubiquitously expressed in various tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3GalNAc(6S)) and CS-E
(GlcA
1-3GalNAc(4S,6S)), promote the outgrowth of neurites in
rat brain (10). These reports suggest that HS and CS have different
functions. Thus, it is of interest to clarify the mechanism for the
biosynthesis of HS and CS, the molecules responsible for these diverse
biological phenotypes.
1-4Gal
1-3Gal
1-4Xyl
1-), which binds covalently to
serine residues of core proteins. To initiate the synthesis of HS, a
GlcNAc residue is transferred to GlcA of the linkage tetrasaccharide
with an
1-4 linkage. On the other hand, GalNAc is transferred to
the same acceptor with a
1-4 linkage for the initiation of CS
synthesis. Thus, because the acceptor substrate is identical, it is
possible that the initiation enzyme for HS or CS synthesis compete with
the acceptor substrate, i.e. the linkage tetrasaccharide
bound to core proteins, in the cells. If this is the case, the
initiation enzymes may play a key role in determining the species of
the GAG chain, HS or CS, on the core proteins. After the initiation
reaction, the addition of disaccharide units of
GlcNAc
1-4GlcA
1-4 are repeated for elongation of the HS
chain, whereas GalNAc
1-4GlcA
1-3 units are repeatedly added for polymerization of the CS chain.
1,4-glucuronyltransferase (
4GlcAT) and
1,4-N-acetylglucosaminyltransferase (
4GlcNAcT) (11).
Both enzymes are responsible for the elongation of HS chains. Three
enzymes, EXTL1-3, exhibit only
4GlcNAcT activity, but their
substrate specificities were different. EXTL1 showed
4GlcNAcT
activity toward GlcA in elongation. EXTL2 showed activity in
initiation. EXTL3 showed activity in both initiation and elongation for
the synthesis of HS (12, 13).
1,3-glucuronyltransferase (
3GlcAT) and
4GalNAcT.
CSGlcAT or CSGalNAcT exhibits the activity of only one
glycosyltransferase,
3GlcAT and
4GalNAcT, respectively. Furthermore, CSGalNAcT was found to exhibit apparent
4GalNAcT activity toward the linkage tetrasaccharide for the initiation of CS
synthesis. Given the many enzymes involved in the synthesis of HS, it
is easy to speculate that multiple glycosyltransferases would also
participate in the synthesis of CS.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-glycosyltransferase motifs as queries and identified an expressed sequence tag with GenBankTM accession number
NM_018590, which contained a partial open reading frame (ORF), but
showed high homology to the carboxyl-terminal region of CSGalNAcT-1. An
additional search of the Human Genome Project data base revealed that
the genome sequence with GenBankTM accession number
NT_008776 was identical to the expressed sequence tag. To obtain the
complete ORF, the 5'-rapid amplification of complementary DNA
(cDNA) ends method was employed using a Marathon ReadyTM cDNA Amplification Kit
(Clontech, Palo Alto, CA). Two reverse primers were
designed for the 1st PCR: GP245, 5'-GTCAGGAAATCTGAACGATGCTGA-3', and
for the nested PCR, GP244, 5'-GCAGCTGTTAAGGAATTCGGCTGA-3'. The sequence
of the DNA fragment obtained by the 5'-rapid amplification of
complementary DNA method was determined using a DYEnamic ET Terminator
Cycle Sequencing Kit (Amersham plc, Amersham Place, UK). Finally, a
cDNA sequence encoding the ORF was obtained by PCR using the
Marathon ReadyTM cDNA of human bone marrow tissue
(Clontech) as a template.
4GalT-7,
3GalT-6, and GlcAT-I Fused with
FLAG Peptides--
The putative catalytic domain of
4GalT-7,
3GalT-6, or GlcAT-I was expressed as secreted protein fused with
FLAG peptide in insect cells or COS-1 cells as described in detail
previously (17). Each enzyme was purified as described (17) and
suspended in 100 µl of the glycosylation buffer described below.
-para-nitrophenyl (pNP), Gal-
-pNP, GalNAc-
-benzyl, N-acetylglucosamine (GlcNAc)-
-pNP,
GlcNAc-
-pNP, glucose-
-pNP, glucose-
-pNP, GlcA-
-pNP,
fucose-
-pNP, mannose-
-pNP, xylose (Xyl)-
-pNP, and Xyl-
-pNP
were obtained from Calbiochem (La Jolla, CA) and Sigma. Chondroitin and
CS-A, -B, -C, and -D were purchased from Seikagaku Corporation (Tokyo,
Japan). Chondroitin hexasaccharide and methoxyphenyl-linkage
tetrasaccharides were kindly donated by Seikagaku Corporation.
Oligosaccharides of CS and chondroitin were prepared as previously
described (19).
-pNP) or 7-10% (for linkage
tetrasaccharide-para-methoxypheny) acetonitrile gradient at
a flow rate of 1.0 ml/min at 50 °C. For glycosylated peptides,
H2O containing 0.1% trifluoroacetic acid and 21%
acetonitrile was utilized as the running solution. An ultraviolet
spectrophotometer (absorbance at 210 nm), SPD-10AVP (Shimadzu, Kyoto, Japan) was used for detection of the peaks. In the
analysis of glycosylated peptide, labeling was done with Cy5 (Amersham
Biosciences) and fluorescence was detected with a fluorescence
detector, RF-10AXL (Shimadzu). For the analysis of
elongation activity, the CSGalNAcTs reaction mixture containing 100 µg of chondroitin or CS and 40,000-55,500 dpm of
UDP-[14C]GalNAc was used. After a 1-h incubation at
37 °C, the reaction mixture was filtrated and fractionated with a
G2500PW column (TOSOH, Tokyo, Japan) or Superdex 30 pg column (Amersham
Biosciences). The radioactivity of each fraction was monitored by
liquid scintillation spectrophotometry.
4GalT-7,
3GalT-6, and GlcAT-I, and 1 mM donor substrates, UDP-Gal and UDP-GlcA, at 37 °°C
for 16 h as described in detail previously (17). A 50 mM MES buffer (pH 6.5) containing 0.1% Triton X-100, 1 mM UDP-Gal, 1 mM UDP-GlcA, 10 mM
MnCl2, and 500 µM Xyl-bikunin peptide was
used for the reaction. The glycosyltransferases for the synthesis of
the tetrasaccharide-bikunin peptide were inactivated by heating at
95 °C for 5 min. Then, the reaction mixture was filtrated with an
Ultrafree-MC column (Millipore), and a 10-µl aliquot was incubated
with 1 mM donor substrate, UDP-GalNAc, and each CSGalNAcT
at 37 °C for 8 h for the assay of initiation activity of each
CSGalNAcT. The reaction products of CSGalNAcTs were filtrated with an
Ultrafree-MC column and a 10-µl aliquot was subjected to
reversed-phase high performance liquid chromatography on an ODS-80Ts QA
column as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-glycosyltransferase
motifs as query sequences, the full-length ORF sequence was identified
as highly homologous to CSGalNAcT as previously reported by us (17). We designated this novel cDNA as CSGalNAcT-2, and renamed the previous CSGalNAcT, CSGalNAcT-1. An alignment of CSGalNAcT-1 and CSGalNAcT-2 is
shown in Fig. 2. A hydropathy profile of
the putative amino acid sequence based on Kyte and Doolittle
hydrophobicity plots indicates that the ORF of CSGalNAcT-2 encodes a
typical type II membrane protein, which is consistent with the topology
of other glycosyltransferases, with a cytoplasmic tail of 14 amino
acids, a transmembrane domain of 20 amino acids, and a large catalytic portion of 508 amino acids. CSGalNAcT-2 contains a DXD motif, which is
conserved in many glycosyltransferases and functions as a key sequence
for divalent cation binding, and a GWGGED motif, which is highly
conserved among some of the
4GalT family.
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Fig. 1.
Nucleotide and amino acid sequences of
CSGalNAcT-2. The predicted start and stop codons are
underlined. The splicing junctions are indicated with
arrowheads. A poly(A) signal is double
underlined.
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Fig. 2.
Multiple alignment of amino acid sequences of
CSGalNAcT-2 and CSGalNAcT-1 by GENETYX. Introduced gaps are shown
with hyphens. The putative transmembrane domains are
underlined. The DXD motif and the 1,4-glycosyltransferase
motif are boxed. Identical amino acids are shown with
asterisks. Possible N-glycosylation sites are
indicated by arrowheads.
4GalNAcT, which transfers GalNAc to GlcA
for initiation and elongation in the synthesis of CS (17). So far, the
linkage of GalNAc with GlcA has been identified only in CS. We
considered that CSGalNAcT-2 was also a GalNAcT involved in the
synthesis of CS, and performed further analysis using CS-related
substrates as acceptors.
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Fig. 3.
Determination of CS elongation and initiation
activities of CSGalNAcT-2. CSGalNAcT-1 and -2 were purified with
anti-FLAG M1 antibody resin and were applied to SDS-PAGE. The detection
of proteins was performed with Coomassie staining (A) and
Western blotting using anti-FLAG antibody (B). C,
chondroitin was used as an acceptor substrate for CSGalNAcT-2. The
purified reaction mixtures were applied to a G2500PW column with 0.2 M NaCl as a running buffer at a flow rate of 0.6 ml/min.
The radioactivity of each fraction (340 µl each) was quantified by
liquid scintillation spectrophotometry. The peak of the reaction
product is indicated with an arrow. D, linkage
tetrasaccharide-O-methoxyphenyl was used as an acceptor, and
the product was analyzed by reversed-phase chromatography. Each peak of
substrate (peak S) and reaction product (peak P)
was isolated and subjected to MALDI-TOF-MS analysis (E and
F).
1-4GlcA
1-)n and the other between the
polymer CS and the linkage tetrasaccharide
(GlcA
1-3Gal
1-3Gal
1-4Xyl). At first, chondroitin was
utilized as an acceptor substrate to examine the elongation activity of
CSGalNAcT-2. As shown in Fig. 3C, CSGalNAcT-2 apparently
transferred GalNAc to chondroitin to produce an additional peak
(indicated by an arrow in Fig. 3C) as a reaction
product. This peak was isolated and identified to have GalNAc on its
nonreducing terminus with chondroitinase ACII treatment (data not
shown), the method used having been described in a previous study (17).
Second, a linkage tetrasaccharide (GlcA
1-3Gal
1-3Gal
1-4Xyl
-O-methoxyphenyl)
synthesized chemically was utilized as an acceptor substrate to
identify the initiation activity of CSGalNAcT-2. As shown in Fig.
3D, the peak P appeared at a 30.3-min retention time in
addition to the acceptor substrate peak (peak S) at 31.1 min. Peaks S
and P were isolated by reversed-phase chromatography and their
molecular weights were determined by MALDI-TOF MS. Peak S gave a
molecular mass of 779.14 m/z, the same as that of
the linkage tetrasaccharide with Na+ (Fig. 3E).
Peak P gave two peaks of 982.28 and 998.25 m/z as shown in Fig. 3F. The molecular
mass of 982.28 or 998.25 m/z was the same
molecular weight as a GalNAc-added linkage
tetrasaccharide-O-methoxyphenyl with Na+ or with
K+, respectively. Moreover, the peak P was identified to
have GalNAc at its nonreducing terminus with chondroitinase ACII
treatment (data not shown). These results suggested that CSGalNAcT-2
has two GalNAcT activities, i.e. elongation of chondroitin
and initiation of the CS synthesis by transferring GalNAc to the
linkage tetrasaccharide.
Comparison of acceptor specificity between two CSGalNAcTs
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Fig. 4.
Determination of chondroitin and CS polymer
elongation activity of CSGalNAcT-2. Chondroitin (open
circle), CS-A (closed circle), CS-B (open
square), CS-C (closed square), and CS-D (open
triangle) were used as acceptor substrates. The fractions
containing the CSGalNAcT product are indicated by an
arrow.
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Fig. 5.
Comparison of initiation activity between two
CSGalNAcTs. A, the GlcA-Gal-Gal-Xyl-bikunin peptide
(peak S) was synthesized enzymatically with three
glycosyltransferases, 4GalT-7,
3GalT-6, and GlcAT-I, and detected
by a reversed-phase chromatography. The product (peak S) was
used as an acceptor substrate for CSGalNAcT-2 (B) or
CSGalNAcT-1 (C). The products (peak P) were
identified as described previously (17).
4GlcNAcT or
4GalNAcT, respectively. If the two enzymes compete with the common
linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-Ser) as an acceptor in the
cells, they would be key enzymes differentially regulating the number
of HS or CS chains. To test this hypothesis, the CSGalNAcT-1
or CSGalNAcT-2 genes were introduced into CHO-K1 cells that
were producing PG-FGF-1. The transcripts for CSGalNAcT-1 and
CSGalNAcT-2 in the stable transfectant cells were quantified by
real-time PCR. Their expression levels were almost 20 times higher than
the physiological level in human placenta, in which the transcripts for
the genes involved in the synthesis of CS are expressed at the highest
level among many tissues (data not shown).
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Fig. 6.
Change of GAG composition in Syndecan-4/FGF-1
(PG-FGF-1) secreted by CHO-K1 cells transfected with the
CSGalNAcT-2 or CSGalNAcT-1
genes. PG-FGF-1 recovered from the culture medium of CHO-K1
cells transfected with the mock vector (lanes
1-4), the CSGalNAcT-2 gene (lanes
5-8), and the CSGalNAcT-1 gene (lanes
9-12) were resolved by SDS-PAGE and immunoblotted with
mAb1. Aliquots of the sample were subjected to endoglycosidase
digestion before being subjected to SDS-PAGE. PG-FGF-1 was left
untreated (lanes 1, 5, and 9), or
digested with CSase (lane 2, 6, and
10), with a mixture of HPase and HSase (lane 3,
7, and 11), or with a mixture of all enzymes
(lane 4, 8, and 12). Positions of
molecular mass standards are indicated in kilodaltons. The density of
each band was measured with a film exposed for 2 min.
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Fig. 7.
Quantitative analysis of CSGalNAcT-2 and
CSGalNAcT-1 transcripts in human tissues by real-time PCR.
Standard curves for CSGalNAcTs and GAPDH were generated by serial
dilution of each plasmid DNA. The expression level of the CSGalNAcT-2
(open bars) and CSGalNAcT-1 (closed bars)
transcripts was normalized to that of the GAPDH transcript, which was
measured in the same cDNAs. Data were obtained from triplicate
experiments and are given as the mean ± S.D. PBMC,
peripheral blood mononuclear cell.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4GT motif (GWGGED) and DXD sequence, which is a
divalent cation binding motif, are conserved with a good alignment (Fig
2). Six
4GalTs,
4GalT-1 to -6, possess a WGWGGEDDD
sequence as the motif (25-28), whereas both of the CSGalNAcTs possess
KGWGGEDVH in place of WGWGGEDDD. Very recently,
Ramakrishnan et al. (29) reported that Tyr289 of
bovine
4GalT-1 is essential for its donor binding based on its
crystallized structure. The elimination of a hydrogen bond by mutating
the Tyr289 residue to Leu, Ile, or Asn enhances the GalNAcT
activity in place of the GalT activity. In the case of
4GalNAcT of
Caenorhabditis elegans, the amino acid at the same position
is Ile (47). In the primary sequence of CSGalNAcT-1 and -2, it was
difficult to find the amino acid residue corresponding to the position
of Tyr289 on
4GalT-1 because of a gap in the amino acid
alignment between
4GalT-1 and CSGalNAcTs. However, CSGalNAcT-2 has
an almost identical sequence to that of CSGalNAcT-1 around this region
that probably determines the donor substrate specificity. Therefore,
the
4GalNAcT activity of CSGalNAcT-2 was easily predicted before the
experiments. In fact, CSGalNAcT-2, as well as CSGalNAcT-1, exhibited
GalNAcT activity in both the initiation and elongation of the CS
synthesis in vitro (Table I). However, CSGalNAcT-2 showed
remarkably different activity from CSGalNAcT-1, i.e. much
stronger activity toward chondroitin and CS substrates, particularly
CS-A and CS-B, than CSGalNAcT-1 (Table I and Fig. 4). Sulfation of
chondroitin is important to exhibit various biological functions.
Sulfation at position C-4 of GalNAc is directed by
chondroitin-4-O-sulfotransferase-1 (C4ST-1) (30), C4ST-2
(31), and C4ST-3 (32), that at C-6 of GalNAc by
chondroitin-6-O-sulfotransferase-1 (C6ST-1) (33) and C6ST-2
(34), and that at C-2 of GlcA by uronyl 2-sulfotransferase (35) in the
GalNAc-GlcA backbone. It is still unclear whether the sulfation occurs
after the synthesis of the long chondroitin chain or the sulfation and
GalNAc-GlcA polymerization occur simultaneously. The preference of
CSGalNAcT-2 for sulfated substrates indicates that the elongation of
sulfated chondroitin is directed by CSGalNAcT-2, not by CSGalNAcT-1.
CSGalNAcT-2 may be responsible for the elongation of polymers that are
simultaneously coupled with sulfation.
1-4) and
at C-4 and C-6 of the outer Gal (Gal
1-3) in the linkage
tetrasaccharide (37, 38). These sulfations might influence the
CSGalNAcTs in terms of recognition. Another possibility is the effect
of the peptide sequence of the core protein on the enzyme recognition. A currently proposed GAG attachment motif is the Ser-Gly-Ser-Gly sequence and surrounding acidic amino acids (39, 40). CSGalNAcT-2 might
recognize CS binding peptide sequences of core proteins other than
syndecan-4.
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Fig. 8.
A schematic diagram of catalytic activities
of glycosyltransferases involved in the synthesis of HS and CS.
The scale of the letters of enzyme names indicates the strength of
activity. Xylosyltransferase (XT) (41, 42),
1,4-galactosyltransferase VII (
4GalT7) (43, 44),
1,3-galactosyltransferase VI (
3GalT6) (45), and
1,3-glucuronyltransferase I (GlcAT-I) (46) work sequentially for the
synthesis of linkage tetrasaccharide. In the case of HS/HP synthesis,
EXT1 and EXT2, which are HS polymerases having both
1,4-glucuronyltransferase and
1,4-N-acetylglucosaminyltransferase activities (11), and
EXTL1, EXTL2, and EXTL3, which are HS
1,4-N-acetylglucosaminyltransferases (12, 13),
participate. In the case of CS/DS synthesis, CSS, which is CS synthase
having both
1,4-glucuronyltransferase and
1,4-N-acetylgalactosaminyltransferase activities (14),
and CS
1,3-glucuronyltransferase (CSGlcA-T) (15) and CS
1,4-N-acetylgalactosaminyltransferase 1 and 2 (CSGalNAcT-1 and CSGalNAcT-2) (Ref. 17 and this paper)
participate.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Seikagaku Corporation for providing an acceptor substrate of the linkage tetrasaccharide, GlcA-Gal-GalXyl-O-methoxyphenyl.
![]() |
FOOTNOTES |
---|
* This work was performed as part of the R&D Project of Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization (NEDO).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB079252.
¶¶ To whom correspondence should be addressed: Glycogene Function Team, Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Central-2 C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Tel.: 81-298-61-3200; Fax: 81-298-61-3201; E-mail: h.narimatsu@aist.go.jp.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208886200
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ABBREVIATIONS |
---|
The abbreviations used are:
PG, proteoglycan;
GAG, glycosaminoglycan;
CS, chondroitin sulfate;
HS, heparan
sulfate;
FGF, fibroblast growth factor;
GlcA, glucuronic acid;
Gal, galactose;
Xyl, xylose;
GlcNAc, N-acetylglucosamine;
GalNAc, N-acetylgalactosamine;
GT, glycosyltransferase;
(4,
3,
or CS)GlcAT, (
1,4-,
1,3- or CS)glucuronyltransferase;
4GlcNAcT,
1,4-N-acetylglucosaminyltransferase;
(
4 or CS)GalNAcT, (
1,4- or
CS)N-acetylgalactosaminyltransferase;
(
4)GalT, (
1,4-)galactosyltransferase;
CSS, chondroitin synthase;
ORF, open
reading frame;
UDP, uridine diphosphate;
pNP, para-nitrophenyl;
MES, 2-morpholinoethanesulfonic acid;
ESI, electrospray ionization;
MS, mass spectrometry;
MALDI-TOF, matrix-assisted laser desorption ionization-time of flight;
HSase, heparitinase;
HPase, heparanase;
CSase, chondroitinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CHO, Chinese hamster ovary;
mAb, monoclonal antibody;
C4ST, chondroitin-4-O-sulfotransferase.
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