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 ¶ RIKEN (The Institute of Physical and
Chemical Research), Wako-shi, Saitama 351-0198, Japan
Received for publication, September 16, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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We identified a novel human chondroitin
N-acetylgalactosaminyltransferase, designated
chondroitin GalNAcT-2 after a BLAST analysis of the
GenBankTM data base using the sequence of a
previously described human chondroitin
N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading
frame encoding a protein of 542 amino acids with a type II
transmembrane protein topology. The amino acid sequence displayed 60%
identity to that of human chondroitin GalNAcT-1. Like chondroitin
GalNAcT-1, the expression of a soluble form of the protein in COS-1
cells produced an active enzyme, which not only transferred
Chondroitin sulfate is one of the sulfated glycosaminoglycan
(GAG)1 chains that are
covalently attached to various core proteins as proteoglycans. Most if
not all vertebrate cells produce chondroitin sulfate proteoglycans to a
variable extent as major components of the connective tissue matrix,
and they are also found at the surface of many cell types. GAGs play
fundamental roles in growth factor signaling, cellular differentiation,
morphogenesis, and pathophysiology (for reviews see Refs. 1-3). Recent
studies of chondroitin/dermatan sulfate chains have shown that they
play important roles in neural network formation in the developing mammalian brain (for reviews see Refs. 4 and 5) and are major
inhibitory molecules affecting axon growth after spinal cord injury in
the central nervous system of adult mammals (6). Although GAGs are
ubiquitously expressed, they exist in discrete structural forms
generated by varying patterns of sulfation and epimerization, which may
account for the functional specificity of tissue-derived proteoglycans.
Both chondroitin/dermatan sulfate and heparin/heparan sulfate are
produced on the so-called GAG protein linkage region
(GlcUA Recent cDNA cloning of GAG glycosyltransferases has led to
unexpected developments (for reviews see Refs. 9-11), providing several important clues to help determine the molecular mechanisms of
the biosynthetic sorting of chondroitin/dermatan sulfate and heparin/heparan sulfate chains, as well as the mechanisms of chain elongation and polymerization of these GAGs. The glycosyltransferases responsible for heparin/heparan sulfate biosynthesis are encoded by the
EXT gene family, the hereditary multiple exostoses gene family of tumor suppressors (9, 10). The EXT gene family has
five members. EXT1 and EXT2 encode heparan
sulfate copolymerases, which catalyze the alternate addition of GlcNAc
and GlcUA residues (12-14), whereas EXTL1, EXTL2, and
EXTL3, called EXT-like genes, are highly
homologous to EXT1 and EXT2 (9, 10). The human EXTL1 protein is a GlcNAc transferase II involved in elongation of the
heparan sulfate chain (15); the human EXTL2 protein is a GlcNAc
transferase I (16) that determines and initiates the synthesis of
heparan sulfate on the common GAG-protein linkage region (17); and the
human EXTL3 protein exhibits both GlcNAc transferase I and II
activities (15).
Three homologous glycosyltransferases responsible for
chondroitin/dermatan sulfate biosynthesis have been cloned (18-21).
The first chondroitin glycosyltransferase cloned was chondroitin
synthase consisting of a single large polypeptide with dual
glycosyltransferase activities of GlcUA transferase II (GlcAT-II)
and GalNAc transferase II (GalNAcT-II) that is responsible for
synthesizing the repeating disaccharide units of chondroitin sulfate
(18). Chondroitin GalNAcT-1, the second chondroitin glycosyltransferase
cloned, exhibits GalNAcT-II activity for chain elongation and GalNAc
transferase I (GalNAcT-I) activity that determines and initiates the
synthesis of chondroitin sulfate on the common GAG-protein linkage
region (19, 20, 22). Chondroitin GlcUA transferase, the third
chondroitin glycosyltransferase cloned, has only GlcAT-II activity,
which has been proposed to be involved in chain elongation (21).
Therefore, more than three enzymes are likely responsible for
chondroitin/dermatan sulfate biosynthesis, and these likely form a gene
family as in the case of heparin/heparan sulfate biosynthesis. To
search for additional members of the glycosyltransferase gene family
involved in chondroitin/dermatan sulfate biosynthesis, the chondroitin GalNAcT-1 protein sequence was used to screen a data base. Here, we
describe the cloning of a human cDNA encoding a novel chondroitin GalNAc transferase, designated chondroitin GalNAcT-2, that is similar
to but distinct from chondroitin GalNAcT-1.
Materials--
UDP-[U-14C]GlcUA (285.2 mCi/mmol) and UDP-[3H]GalNAc (10 Ci/mmol) were purchased
from PerkinElmer Life Sciences. Unlabeled UDP-GlcUA and
UDP-GalNAc were obtained from Sigma. Chondroitin (a chemically desulfated derivative of whale cartilage chondroitin sulfate A), Acremonium sp. In Silico Cloning of the Novel Chondroitin GalNAcT
cDNA--
A tBLASTn analysis of the GenBankTM data
base using the sequence of human chondroitin GalNAcT-1 (19), revealed
highly homologous clones. Analysis of one clone (GenBankTM
accession number BC030268) revealed a single open reading frame with
significant sequence similarity to human chondroitin GalNAcT-1. In
addition, a data base search of the Human Genome Project, which
recently became available, identified a genome sequence
(GenBankTM accession number NT033985.2) identical to the
cDNA sequence. Comparison of the cDNA and genome sequences
revealed the genomic organization of the novel chondroitin GalNAcT gene.
Construction of a Soluble Form of the Novel Chondroitin
GalNAcT--
A cDNA fragment of a truncated form of the novel
chondroitin GalNAcT lacking the first 36 N-terminal amino acids was
amplified by reverse transcription PCR with total RNA derived from G361 human melanoma cells (ATCC CRL-1424) as a template using a 5'-primer (5'-CGCGGATCCTTGTTAGGCAAATACACATTAATAAG-3') containing an in-frame BamHI site and a 3'-primer
(5'-CGCGGATCCGTTTTGTGGTTCATACAGTAACGC-3') containing a BamHI
site located 37 bp downstream from the stop codon. PCR was carried out
with Pfu polymerase (Stratagene, La Jolla, CA) for 30 cycles
of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for
120 s in 5% (v/v) dimethyl sulfoxide. The PCR fragments were
subcloned into the BamHI site of pGIR201protA (28),
resulting in the fusion of the putative GalNAcT with the insulin signal sequence and the protein A sequence present in the vector. An NheI fragment containing this fusion protein sequence was
inserted into the XbaI site of the expression vector pEF-BOS
(29). The nucleotide sequence of the amplified cDNA was determined
in a 377 DNA sequencer (PE Applied Biosystems).
Expression of a Soluble Form of the Novel Chondroitin GalNAcT and
Enzyme Assays--
The expression plasmid (6.7 µg) was transfected
into COS-1 cells on 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 Biosciences) 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 GlcUA transferase activities as described below. The acceptors used
for GalNAcT included polymer chondroitin (167 µg),
chondro-hexasaccharide (GlcUA Identification of the Enzyme Reaction Products--
The products
of the GalNAcT reaction using polymer chondroitin as the acceptor were
isolated by gel filtration on a Superdex peptide column equilibrated
with 0.25 M NH4HCO3, 7%
1-propanol. The radioactive fractions containing the enzyme reaction
product were pooled and evaporated to dryness. The isolated GalNAcT
reaction product (about 150 µg) was digested with 100 mIU of
chondroitinase AC-II to assess digestibility in a total volume of 30 µl of 50 mM sodium acetate buffer, pH 6.0, at 37 °C
overnight. The enzyme digest was analyzed using the Superdex peptide
column described above.
The products from the GalNAcT reaction using
GlcUA Northern Blot Analysis--
A commercial human 12-lane multiple
tissue Northern blot (Clontech) membrane was used
for the analysis. The membrane was probed with a gel-purified,
radiolabeled (>1 × 109 cpm/µg), 1.0-kb chondroitin
GalNAcT-2-specific fragment corresponding to nucleotides 109-1123 of
the chondroitin GalNAcT-2 cDNA (GenBankTM accession
number AB090811).
In Silico Cloning of a Human cDNA Homologous to Chondroitin
GalNAcT-1--
We recently identified and characterized human
chondroitin GalNAcT-1 (19). Screening of the nonredundant data base at
the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) using the deduced amino acid sequence of human
chondroitin GalNAcT-1 identified a clone (GenBankTM
accession number BC030268) containing a 335-bp 5'-untranslated region,
a single open reading frame of 1626 bp encoding a protein of 542 amino
acids with two potential N-glycosylation sites (Fig. 1), and a 3'-untranslated region of
approximately 1.8 kb with one presumptive polyadenylation signal.
Northern blot analysis indicated that the mRNA was about 3.9 kb
long in various human tissues (see below), suggesting that the cDNA
was approximately full-length. The deduced amino acid sequence
corresponded to a 62,571-Da polypeptide. The predicted translation
initiation site conformed to the Kozak consensus sequence for
initiation (34), and an in-frame stop codon was present
upstream from the assigned ATG initiating codon. A Kyte-Doolittle
hydropathy analysis (35) revealed one prominent hydrophobic segment of
19 amino acid residues in the N-terminal region, predicting that the
protein has the type II transmembrane topology characteristic of many
of the Golgi-localized glycosyltransferases cloned to date (Fig. 1).
Data base searches indicated that the amino acid sequence displayed
60% identity to human chondroitin GalNAcT-1 (GenBankTM
accession number AB071403) (Fig. 1); the highest sequence identity was
found in the C-terminal domain, which has catalytic activity. Notably,
each protein shared a conserved DVD motif (Fig. 1), which appears to
correspond to the conserved DXD motif found in most
glycosyltransferases (36). Therefore, the features of the identified
protein sequence suggest that the identified gene product possesses
Genome Organization and Chromosome Localization--
Comparison of
the identified cDNA sequence with the genome sequence deposited in
the Human Genome Project data base revealed the genomic structure and
chromosome localization of the gene. The gene spans more than 47 kb,
and the coding region of the gene is divided into seven discrete exons
as shown in Fig. 2. Its genomic organization is very similar to that of the chondroitin GalNAcT-1 gene,
which consists of seven discrete exons in the coding region. The
intron/exon junctions follow the G(T/A)G rule (37) and are flanked by
conserved sequences (data not shown). This gene is located on human
chromosome 10q11.22.
Expression of a Soluble Form of the Novel Glycosyltransferase and
Its Characterization as Chondroitin GalNAcT-2--
To facilitate the
functional analysis of the putative GalNAcT, a soluble form of the
protein was generated by replacing the first 36 amino acids of the
putative glycosyltransferase with a cleavable insulin signal sequence
and the protein A IgG-binding domain, as described under
"Experimental Procedures." Then, the soluble putative
glycosyltransferase was expressed in COS-1 cells as a recombinant
enzyme fused with the protein A IgG-binding domain. When the expression
plasmid containing the putative glycosyltransferase/protein A fusion
construct was expressed in COS-1 cells, an ~95-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 87 kDa after N-glycosidase treatment (data not shown),
suggesting that either one or both of the two potential
N-linked glycosylation sites of the putative
glycosyltransferase was utilized. The fused enzyme expressed in the
medium was adsorbed onto IgG-Sepharose beads to eliminate endogenous
glycosyltransferases, and then the enzyme-bound beads were used as an
enzyme source. The bound fusion protein was assayed for
glycosyltransferase activity using a variety of acceptors and either
UDP-GalNAc or UDP-GlcUA as a donor substrate. GlcUA
To identify the GalNAcT reaction products, representative acceptor
substrates, polymer chondroitin and
GlcUA Pattern of Chondroitin GalNAcT-2 Expression--
Northern blot
mRNA analysis demonstrated a single ~3.9-kb band in all human
tissues examined (Fig. 4). The gene
exhibited a ubiquitous but differing expression in all the human
tissues examined. Notably, the expression pattern differed from those of chondroitin GalNAcT-1 and chondroitin synthase (Fig. 4). The strongest signals were seen in the skeletal muscle, lung, and peripheral blood leukocytes. These findings are in accord with the
observations that chondroitin/dermatan sulfate proteoglycans are
distributed on the surfaces of most cells and in the extracellular matrices of virtually every tissue.
In this study we identified a second human chondroitin GalNAcT,
chondroitin GalNAcT-2, which is homologous to but distinct from the
previously cloned chondroitin GalNAcT-1. Chondroitin GalNAcT-2 is the
fourth chondroitin-synthesizing glycosyltransferase cloned to date. As
summarized in Table II, the four
glycosyltransferases exhibit distinct but overlapping acceptor
substrate specificities, except for the two chondroitin GalNAcTs that
harbor both GalNAcT-I and -II activities. Therefore, at least four
distinct enzymes forming this novel gene family are responsible for
initiation and elongation of chondroitin/dermatan sulfate chains. The
situation is similar to that of the heparan sulfate
glycosyltransferases in which five homologous EXT family members with
distinct but overlapping acceptor specificity have been identified
(14-16). It is still possible that other glycosyltransferases are also involved in chondroitin/dermatan sulfate biosynthesis.
1,4-N-acetylgalactosamine (GalNAc) from
UDP-[3H]GalNAc to a polymer chondroitin representing
growing chondroitin chains (
-GalNAc transferase II activity) but
also to
GlcUA
1-3Gal
1-O-C2H4NHCbz, a
synthetic substrate for
-GalNAc transferase I that transfers the
first GalNAc to the core tetrasaccharide in the protein-linkage region
of chondroitin sulfate. In contrast, the tetrasaccharide serine
(GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser) derived
from the linkage region, which is an inert acceptor substrate for
chondroitin GalNAcT-1, served as an acceptor substrate. The coding
region of this enzyme was divided into seven discrete exons, which is
similar to the genomic organization of the chondroitin GalNAcT-1 gene,
and was localized to chromosome 10q11.22. Northern blot analysis
revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the
biosynthetic initiation and elongation of chondroitin sulfate, which is
important for understanding the biosynthetic mechanisms leading to the
selective chain assembly of chondroitin/dermatan sulfate on the linkage
region tetrasaccharide common to various proteoglycans containing
chondroitin/dermatan sulfate and heparin/heparan sulfate chains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-3Gal
1-4Xyl
1-O attached to
specific Ser residues of core proteins) common to GAGs (for reviews,
see Refs. 7 and 8). Synthesis of this region is initiated by the
addition of Xyl to Ser, followed by the addition of two Gal residues,
and completed by the addition of GlcUA; each reaction is catalyzed by a
specific glycosyltransferase (7, 8). The GAGs are built up on this
linkage region tetrasaccharide by the alternate addition of
N-acetylhexosamine and GlcUA residues. Chondroitin/dermatan
sulfate is synthesized once GalNAc is transferred to the common linkage
region, whereas heparin/heparan sulfate is formed if GlcNAc is added
first. Therefore, the first hexosamine transfer is critical in
determining whether chondroitin/dermatan sulfate or heparin/heparan
sulfate chains are selectively assembled on the common linkage region.
Although such mechanisms have long been proposed based on data from
conventional structural and enzymological studies (8), the molecular
mechanisms underlying the selective chain assembly of different GAG
chains have not been clarified.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylgalactosaminidase (EC
3.2.1.49), and Arthrobacter aurescens chondroitinase AC-II
(EC 4.2.2.5) were purchased from Seikagaku Kogyo (Tokyo, Japan).
Purified
-thrombomodulin (23) was provided by the research institute
of Dai-ichi Pure Chemicals (Tokyo, Japan) and contained the linkage
tetrasaccharide GlcUA
1-3Gal
1-3Gal
1-4Xyl (24). The
chemically synthesized linkage tetrasaccharide serines
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, GlcUA
1-3Gal(4-O-sulfate)
1-3Gal
1-4Xyl
1-O-Ser,
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-(Gly)Ser-(Gly-Glu), and
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser-(Gly-Trp-Pro-Asp-Gly)
were chemically synthesized (25, 26).
GlcUA
1-3Gal
1-O-C2H4NHCbz was
also synthesized.2
Chondro-hexasaccharide (GlcUA
1-3GalNAc)3 was prepared
from chondroitin as previously described (27). The
SuperdexTM peptide HR10/30 column was obtained from
Amersham Biosciences.
1-3GalNAc)3 (10 nmol),
-thrombomodulin (1 nmol),
GlcUA
1-3Gal
1-O-C2H4NHCbz (1 or 100 nmol),
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (1 nmol), GlcUA
1-3Gal(4-O-sulfate)
1-3Gal
1- 4Xyl
1-O-Ser
(1 nmol),
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-(Gly)Ser-(Gly-Glu) (1 nmol), and
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser-(Gly-Trp-Pro-Asp-Gly) (1 nmol), whereas that for GlcUA transferase was polymer chondroitin (167 µg). The assay mixture for GalNAcT contained 10 µl of the resuspended beads, an acceptor substrate, 8.57 µM
UDP-[3H]GalNAc (3.60 × 105 dpm), 50 mM MES buffer, pH 6.5, 10 mM MnCl2,
and 171 µM sodium salt of ATP in a total volume of 30 µl (30, 31). The assay mixture for GlcAT-II contained 10 µl of the
resuspended beads, 167 µg of polymer chondroitin, 14.3 µM UDP-[14C]GlcUA (1.46 × 105 dpm), 50 mM sodium acetate buffer, pH 5.6, and 10 mM MnCl2 in a total volume of 30 µl
(32). The reaction mixtures were incubated at 37 °C for 4 h,
and then the radiolabeled products were separated from
UDP-[3H]GalNAc or UDP-[14C]GlcUA by gel
filtration using a syringe column packed with Sephadex G-25
(superfine), or a Superdex peptide column, or by HPLC on a
Nova-Pak® C18 column (3.9 × 150 mm, Waters, Tokyo,
Japan) as described previously (16, 30-33). The recovered labeled
products were quantified by liquid scintillation spectrophotometry.
1-3Gal
1-O-C2H4NHCbz were
isolated by HPLC on a Nova-Pak® C18 column (described
above) in an LC-10A system (Shimadzu, Kyoto, Japan). The column was
developed isocratically for 15 min with H2O at a flow rate
of 1.0 ml/min at room temperature; then, a linear gradient was applied
to increase the methanol concentration from 0 to 100% over a 5-min
period; the column was then developed isocratically for 40 min with
100% methanol. The radioactive fractions containing the product were
pooled and evaporated to dryness. The isolated product (about 80 pmol)
was incubated with 100 mIU of chondroitinase AC-II to assess the
digestibility in a total volume of 30 µl of 50 mM sodium
acetate buffer, pH 6.0, at 37 °C overnight or 40 mIU of
-N-acetylgalactosaminidase in a total volume of 20 µl
of 50 mM sodium citrate buffer, pH 4.5, at 37 °C
overnight (16). The enzyme digest was analyzed using the Nova-Pak® C18 column described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-GalNAc transferase (GalNAcT-I and/or -II) activities like
chondroitin GalNAcT-1. Intriguingly, a homologue of the identified
human gene was found in the Drosophila genome but not in the
Caenorhabditis elegans genome. The human sequence shared
38% identity with that of Drosophila (Fig. 1).
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Fig. 1.
Comparison of the predicted amino acid
sequence of the novel human glycosyltransferase (chondroitin
GalNAcT-2), human chondroitin GalNAcT-1 (GenBankTM
accession number AB071403), and the putative chondroitin
GalNAcT in Drosophila (GenBankTM accession
number CG12913). The predicted amino acid sequences
were aligned using the program GENETYX-MAC (version 10).
Closed and shaded boxes indicate that the
predicted amino acid in the alignment is identical in all three or any
two sequences, respectively. Gaps introduced for maximal alignment are
indicated by dashes. The putative membrane spanning domains
are boxed. The conserved DXD motif is indicated
by underlines. Two potential N-glycosylation
sites for the human putative glycosyltransferase (chondroitin
GalNAcT-2) are marked with asterisks.
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Fig. 2.
Comparison of the genomic organization of the
novel human glycosyltransferase (chondroitin GalNAcT-2) and human
chondroitin GalNAcT-1 genes. Exon regions are denoted by
boxes. Closed boxes represent the coding
sequence, and open boxes denote the 5'- and 3'-untranslated
sequences. The translation initiation (ATG) and termination
(TGA) codons are also shown. Black horizontal
bars denote the introns.
1-3Gal
1-O-C2H4NHCbz,
which was used as an acceptor substrate for the GalNAcT-I reaction,
shares the disaccharide sequence with the GAG protein linkage region
tetrasaccharide. As shown in Table I,
marked GalNAc transferase activity was detected with polymer chondroitin, chondro-hexasaccharide (GlcUA
1-3GalNAc)3,
-thrombomodulin containing a linkage region tetrasaccharide,
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1 on the native core protein
(24),
GlcUA
1-3Gal
1-O-C2H4NHCbz, the
tetrasaccharide serine,
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, and the two
tetrasaccharide peptides,
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-(Gly)Ser-(Gly-Glu) and
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser-(Gly-Trp-Pro-Asp-Gly), but not with the sulfated tetrasaccharide serine
GlcUA
1-3Gal(4-O-sulfate)
1-3Gal
1-4Xyl
1-O-Ser, as acceptor substrates. In contrast, no GlcUA transferase activity was
observed using polymer chondroitin. No detectable glycosyltransferase activity was recovered by affinity purification from a control pEF-BOS transfection sample. These findings clearly indicate
that the expressed protein is a GalNAcT responsible for chondroitin biosynthesis.
Comparison of the acceptor specificity of chondroitin GalNAcT-1 and -2 secreted into culture medium by transfected COS-1 cells
1-3Gal
1-O-C2H4NHCbz, were individually labeled via the respective transferase reaction using
UDP-[3H]GalNAc as a donor substrate and the enzyme-bound
beads as an enzyme source. Both labeled products were completely
digested by chondroitinase AC-II, which cleaves
1,4-N-acetylgalactosaminidic linkages in an eliminative
fashion, quantitatively yielding a 3H-labeled peak at the
position of free [3H]GalNAc as demonstrated by gel
filtration (Fig. 3A) or
hydrophobic HPLC (Fig. 3B). In contrast, both products were
inert with respect to
-N-acetylgalactosaminidase, which
is in marked contrast to the product of the
1,4-GalNAc transferase
reaction catalyzed by EXTL2 (16). These findings indicate that a GalNAc
residue was indeed transferred to the nonreducing terminal GlcUA of the polymer chondroitin or
GlcUA
1-3Gal
1-O-C2H4NHCbz
through a
1-4 linkage. Combined, these results indicate that the
identified protein is a novel
1,4-GalNAc transferase I/II involved
in the initiation and elongation of chondroitin/dermatan sulfate.
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Fig. 3.
Identification of the reaction products of a
novel human glycosyltransferase. A,
3H-labeled GalNAcT-II reaction products obtained using
polymer chondroitin as the acceptor substrate were digested with
chondroitinase AC-II as described under "Experimental Procedures."
The chondroitinase AC-II digest (solid circles) or the
undigested sample (solid squares) was applied to a Superdex
peptide column (1.0 × 30 cm), and the radioactivity in the
effluent fractions (0.4 ml each) was analyzed as described under
"Experimental Procedures." The arrowhead indicates the
elution position of free GalNAc. B, 3H-labeled
GalNAcT-I reaction products obtained using
GlcUA 1-3Gal
1-O-C2H4NHCbz as
the acceptor substrate digested with chondroitinase AC-II or
-N-acetylgalactosaminidase as described under
"Experimental Procedures." The chondroitinase AC-II digest
(solid circles),
-N-acetylgalactosaminidase
digest (solid squares), and undigested sample (solid
triangles) were analyzed by HPLC on a Nova-Pak® C18
column as described under "Experimental Procedures," and the
radioactivity in the effluent fractions (2 ml each) was analyzed. The
arrowhead indicates the elution position of free
GalNAc.
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Fig. 4.
Northern blot analysis of chondroitin
GalNAcT-2, chondroitin GalNAcT-1, and chondroitin synthase in human
tissues. Northern blots with RNA from various human tissues were
hybridized with probes for chondroitin GalNAcT-2 (upper
panel), chondroitin GalNAcT-1 (middle panel), or
chondroitin synthase (lower panel), as described under
"Experimental Procedures." Lane 1, brain; lane
2, heart; lane 3, skeletal muscle; lane 4,
colon; lane 5, thymus; lane 6, spleen; lane
7, kidney; lane 8, liver; lane 9, small
intestine; lane 10, placenta; lane 11, lung;
lane 12, peripheral blood leukocytes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of the human glycosyltransferases involved in chondroitin
sulfate biosynthesis
Our findings demonstrated that chondroitin GalNAcT-2, like chondroitin GalNAcT-1, possesses both GalNAcT-I and -II activities responsible for chain initiation and elongation in chondroitin/dermatan sulfate. These results are reminiscent of the heparan sulfate GlcNAc transferase encoded by human EXTL3, Drosophila DEXT3, and C. elegans rib-2, which have both GlcNAc transferase-I and -II activities responsible for chain initiation and elongation in heparin/heparan sulfate (15, 38, 39). Therefore, there are at least two types of enzymes involved in the synthesis of both chondroitin/dermatan sulfate and heparin/heparan sulfate, one for both chain initiation and elongation (chondroitin GalNAcT-1 and -2 and heparan sulfate GlcNAc transferase EXTL3, respectively) and the other for chain polymerization via the alternate transfer of GlcUA and GalNAc or GlcNAc (chondroitin synthase and heparan sulfate copolymerases EXT1 and EXT2, respectively). Intriguingly, these combined findings suggest that the assembly of the polysaccharide backbones of chondroitin sulfate and heparan sulfate shares a similar biosynthetic mechanism irrespective of the involvement of the two distinct glycosyltransferase gene families.
As shown in Table I, the two recombinant chondroitin GalNAcTs exhibited
both GalNAcT-I and -II activities, and polymer chondroitin was the best
substrate for both enzymes. However, chondroitin GalNAcT-2 showed
broader acceptor specificity than chondroitin GalNAcT-1, as the former
utilized all of the acceptor substrates tested that contained terminal
1,3-linked GlcUA residues except for the sulfated tetrasaccharide
serine,
GlcUA
1-3Gal(4-O-sulfate)
1-3Gal
1-4Xyl
1-O-Ser (Table I). Notably, no such chondroitin GalNAcT has been reported. Chondroitin GalNAcT-2 is a unique
1,4-GalNAc transferase that utilizes the tetrasaccharide serine,
GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser, from the
linkage region as an acceptor substrate. Previously, when this compound
was tested as an acceptor together with a sugar donor, UDP-GalNAc, to
search for such GalNAcTs using fetal bovine sera or mouse mastocytoma
cell extracts as the enzyme source, only
-GalNAc transferase
activity was detected, resulting in the exclusive production of an
-GalNAc-capped pentasaccharide, GalNAc
1-4GlcUA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(27, 40, 41). Therefore, high expression of
-GalNAc transferase
might have interfered with the identification of a
1,4-GalNAc
transferase, such as chondroitin GalNAcT-2.
Chondroitin GalNAcT-2, like chondroitin GalNAcT-1, transfers the first
1,4-GalNAc residue to initiate chondroitin/dermatan sulfate chains
on the common linkage region by segregating chondroitin/dermatan sulfate synthesis from heparin/heparan sulfate synthesis. One explanation for the existence of two distinct GalNAcT-I molecular species that share enzyme activity critical for the differential assembly of chondroitin and heparan chains is that they initiate chondroitin/dermatan sulfate chains on different core proteins by
discriminating the amino acid sequences. The notion of the importance
of amino acid sequences in heparin/heparan sulfate biosynthesis was
well documented by Esko and Zhang (42) based on the specificity of
GlcNAcT-I in Chinese hamster ovary cells. Artificial
-D-xylosides with lipophilic aglycons containing two fused aromatic rings, such as estradiol and naphthalene, efficiently initiate heparin/heparan sulfate biosynthesis, whereas
chondroitin/dermatan sulfate chains are initiated on less lipophilic
xylosides (43, 44). Consistent with this concept were our observations
that chondroitin GalNAcT-1 and -2 differed in how they transferred a
GalNAc residue to an
-thrombomodulin proteoglycan bearing the truncated linkage region tetrasaccharide
(GlcUA
1-3Gal
1-3Gal
1-4Xyl
1) (24), chemically synthesized
disaccharides
GlcUA
1-3Gal
1-O-C2H4NHCbz, and
the tetrasaccharide-peptides
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser-(Gly-Trp-Pro-Asp-Gly) derived from the chondroitin sulfate/heparan sulfate attachment site of
betaglycan (26, 45) and
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-(Gly)Ser-(Gly-Glu) derived from the chondroitin sulfate attachment site of
-thrombomodulin (23, 25). The aglycone part of
GlcUA
1-3Gal
1-O-C2H4NHCbz probably mimics the required peptide sequence as observed for GlcNAc
transferase I reactions catalyzed by EXTL2 and EXTL3, both of which
also utilize
GlcUA
1-3Gal
1-O-C2H4NHCbz as
an acceptor for heparan synthesis (15, 16). Interestingly, the
chondroitin GalNAcT-2 activity was inhibited when the sulfated
tetrasaccharide serine was used as an acceptor (Table I). The
4-O-sulfated Gal residue has been identified in the linkage
region of chondroitin/dermatan sulfate but not in that of
heparin/heparan sulfate (46, 47). Hence, it is hypothesized that it is
recognized by a chondroitin GalNAcT specific for the linkage region and
might be a molecular signal that promotes chondroitin chain synthesis
segregating it from heparan chain (9). However, the negative results
obtained in this study do not appear to support this
possibility, although the function of the 4-O-sulfated Gal
residue remains unclear.
In addition, the respective roles of the GalNAcT-II activity of chondroitin GalNAcT-1 and -2 in the chain polymerization of chondroitin/dermatan sulfate remain to be clarified, especially where it requires GlcAT-II as a partner. It is not known whether chondroitin synthase or chondroitin GlcUA transferase cooperates with chondroitin GalNAcT-1 and/or -2 to synthesize the repeating disaccharide region or in chain polymerization, or whether an as yet unidentified GlcAT-II with no homology to the known genes exists in addition to chondroitin synthase and chondroitin GlcUA transferase. A multimeric complex might also exist. In this context, no chondroitin chain polymerizing enzyme activity has been demonstrated in vitro for chondroitin synthase (18). If either chondroitin GalNAcT-1 or -2 is associated with chondroitin synthase, chondroitin/dermatan sulfate chains might possibly be initiated and polymerized efficiently on the tetrasaccharide core. However, such hetero-oligomeric complex formation has not been reported and is the subject of a future study.
The chondroitin GalNAcT-1 and -2 genes exhibit unique tissue-specific patterns of expression. Particularly striking is the abundant expression of the chondroitin GalNAcT-2 gene in the skeletal muscle, lung, and peripheral blood leukocytes, with modest expression in the brain, thymus, and placenta (Fig. 4). In contrast, chondroitin GalNAcT-1 is abundantly expressed in the placenta, followed by the heart and small intestine. Very little is expressed in the colon and thymus (19), similar to the pattern of chondroitin synthase expression (18). Therefore, chondroitin GalNAcT-2 may play a unique role in the biosynthesis of chondroitin sulfate in certain tissues in view of the different specificities (Table I) and distinct tissue expression of chondroitin GalNAcT-1 and -2 (Fig. 4). The production and analysis of chondroitin GalNAcT-1 or -2 knock-out mice would provide further insights into the possible distinct functions of these genes.
The genomic organization of the four cloned chondroitin glycosyltransferases involved in chondroitin backbone formation has been elucidated. The protein-coding sequences of the human chondroitin GalNAcT-1 and -2 genes are distributed among seven exons that span ~120 (19) and 30 kb (this study), respectively. Comparison of the genomic organizations of these two genes shows a quite similar genetic exon-intron organization within the coding sequences (Fig. 2). In contrast, the genomic organizations of the human chondroitin synthase and chondroitin GlcUA transferase genes are simpler; their protein-coding sequences are divided into 3 and 4 exons that span ~76 and 6 kb, respectively (18, 21). Interestingly, chromosomal assignments of the four human chondroitin glycosyltransferases (chondroitin synthase, chondroitin GalNAcT-1 and -2, and chondroitin GlcUA transferase) indicate that these genes are localized on different chromosomes, 15q26.3, 8p21.3, 10q11.22, and 7q35 (Table II), respectively, despite the significant homology in the nucleotide and amino acid sequences of the four genes. These findings suggest that the four chondroitin glycosyltransferases diverged from an ancestor gene early in evolution.
As described previously (18, 19), a homologue of human chondroitin synthase is present in C. elegans and Drosophila, which is consistent with the finding of chondroitin and chondroitin 4-sulfate in C. elegans and Drosophila, respectively (48-50). Notably, however, a homologue of human chondroitin GalNAcT-1 or -2 is absent from C. elegans but is present in "higher" species, such as Drosophila (Fig. 1). It is possible that the biosynthetic mechanism for chondroitin sulfate in C. elegans is similar to but different from that in mammals, although the conventional linkage region tetrasaccharide sequence has recently been identified not only in Drosophila but also in C. elegans (51).
The mechanism for the selective chain assembly of chondroitin/dermatan
sulfate and heparin/heparan sulfate on the common linkage region
tetrasaccharide has been an enigma since the discovery of the common
linkage region tetrasaccharide in the mid-1960s (7, 9). cDNA probes
for enzymes harboring GalNAcT-I or GlcNAc transferase I activity (15,
16, 19) are now available for investigating the biological functions of
chondroitin/dermatan sulfate as well as the mechanism of selective
chain assembly of chondroitin/dermatan sulfate and heparin/heparan sulfate.
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ACKNOWLEDGEMENT |
---|
We thank Y. Kato for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by the Science Research Promotion Fund of the Japan Private School Promotion Foundation and Grants-in-aid for Scientific Research C-14572086 (to H. K.) and for Scientific Research on Priority Areas 14082207 (to K. S.) from the Ministry of Education, Science, Culture, and Sports of Japan.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) AB090811.
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: 81-78-441-7571; E-mail:
k-sugar@kobepharma-u.ac.jp.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209446200
2 J. Tamura, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
GAG, glycosaminoglycan;
bp, base pair(s);
Cbz, benzyloxycarbonyl;
EXT, hereditary multiple exostoses gene;
GalNAc, N-acetyl-D-galactosamine;
GalNAcT, N-acetylgalactosaminyltransferase;
GlcNAc, N-acetyl-D-glucosamine;
GlcAT, 1,3-glucuronyltransferase;
GlcUA, D-glucuronic acid;
HPLC, high-performance liquid chromatography;
MES, 2-(N-morpholino)ethanesulfonic acid.
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