From the Department of Biochemical Cell Research,
Tokyo Metropolitan Institute of Medical Science (RINSHOKEN), Tokyo
113-8613, Japan, the ¶ Department of Plastic and Reconstructive
Surgery, Graduate School of Medicine, University of Tokyo, Tokyo
113-0033, Japan, and the § Division of Biochemistry and Cell
Biology, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira,
Tokyo 187-8502, Japan
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ABSTRACT |
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We isolated a cDNA encoding a novel
glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis
of the HNK-1 carbohydrate epitope from rat embryo cDNA by the
degenerate polymerase chain reaction method. The new cDNA sequence
revealed an open reading frame coding for a protein of 324 amino acids
with type II transmembrane protein topology. The amino acid sequence of
GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in
the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of
GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope
on the cell surface. The enzyme expressed in COS-7 cells transferred a
glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to
paragloboside (lacto-N-neotetraosylceramide), a precursor
of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity
analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D
transfers a GlcA not only to
Gal HNK-1, also known as CD57, is recognized as an important epitope
involved in neurogenesis. Expression of HNK-1 epitope is spatiotemporally regulated, and the epitope is found on migrating neural crest cells (1), odd-numbered rhombomeres (2), and myelinating
Schwann cells in motor neurons (3). The HNK-1 epitope is a glycan
expressed on glycoproteins, glycolipids, and proteoglycans (reviewed in
Refs. 4 and 5). In particular, the epitope has been discovered on a
series of cell adhesion molecules, such as myelin-associated
glycoproteins (6), L1 (7), neural cell adhesion molecule (7), P0 (8),
and transiently expressed axonal glycoprotein-1 (9). It is interesting
that only a subpopulation of these molecules expresses HNK-1 epitope.
The epitope has also been identified as a ligand for P0 (10), laminin
(11), and L- and P-selectins (12). Moreover, it was demonstrated that HNK-1 antibody or isolated HNK-1 glycan interferes with cell-cell or
cell-substrate interaction (13-15). These observations indicate that
HNK-1 epitope plays a significant role in cell-cell and cell-matrix interaction.
Structures of HNK-1 epitope determined to date almost invariably carry
HSO3-3GlcA We examined the enzymatic features of glycosyltransferases, including
Materials--
UDP-[14C]GlcA (10.1 GBq/mol) was
purchased from NEN Life Science Products. Unlabeled UDP-GlcA,
CDP-choline, and GlcA were purchased from Sigma.
Lacto-N-neotetraose (LNnT), lacto-Ntetraose
(LNT), lacto-N-fucopentaose III (LNFP III), and
lacto-N-neodifucohexaose I (LNnDFH I) were purchased from
Seikagaku. Lacto-N-neotetraosylceramide (LNnT-Cer)
and glucuronylneolactotetraosylceramide (GlcA-LNnT-Cer) were
purchased from Dia-Iatron and Wako Pure Chemicals, respectively. Asialo-orosomucoid was prepared by mild acid hydrolysis (in 0.05 M H2SO4 at 80 °C for 1 h)
of PCR-based Cloning of a New Glucuronyltransferase--
Based on
the amino acid sequence alignment of rat GlcAT-P (21) and human GlcAT-I
(24), degenerate oligonucleotides to elements conserved in motifs II
and IV were synthesized. The sequences of the 5'- and 3'-primers
were 5'-TSGTSTAYTTYGCYGAYGAYGA-3' and 5'-TTYTCNGTNCKNGTRTGCCANA-3'
(K = A + C, N = A + C + G + T, R = A + G, S = C + G, and Y = C + T), respectively. PCR was performed using first
strand cDNA synthesized with total RNA of rat E13 brain. Thirty
cycles (94 °C for 45 s, 50 °C for 45 s, and 72 °C for 90 s) were run using AmpliTaq Gold polymerase (PE Applied Biosystems). The PCR product of around 380 base pairs in length was
subcloned into the pGEM-T Easy vector (Promega), and 10 clones were
sequenced. Two of the 10 clones contained a novel putative glucuronyltransferase (GlcAT-D) fragment that was used as a probe for
screening a rat E15 brain cDNA library (25). One positive clone,
58-2-2, was isolated and sequenced.
To obtain the entire coding sequence of GlcAT-D, rapid amplification of
cDNA 5'-end was employed with 5'-AmpliFINDER RACE Kit
(CLONTECH) using rat E15 brain total RNA as a
template. The amplified cDNA of about 1.6 kilobase pairs in length
was subcloned into pGEM-T Easy and sequenced. Several clones were
sequenced to compensate for misreading by AmpliTaq Gold polymerase.
Northern Blot Analysis--
Total RNA was extracted from embryo,
newborn, and adult rat brain using ISOGEN reagent (Nippon gene). Equal
amounts of total RNA (20 µg in each lane) were run on formaldehyde
1.2% agarose gel and transferred to a nitrocellulose membrane.
Multiple tissue Northern blot of rat poly(A)+ RNA was
purchased from CLONTECH. The blots were hybridized
overnight with 32P-labeled EcoRI fragment of
58-2-2 at 42 °C in hybridization buffer. Then the filters were
washed in 0.5× SSC plus 0.1% SDS for 1 h at 65 °C.
In Situ Hybridization--
For the construction of riboprobes
for in situ hybridization analysis, we obtained the cDNA
fragment of GlcAT-D (nucleotide positions 285-1005) by digestion of
58-2-2 plasmid with EcoRI and subcloned into pBluescript II
SK(
In situ hybridization with probes of GlcAT-D and GlcAT-P was
performed as described previously (26) with slight modifications. Briefly, the head region of E13.5 Sprague-Dawley rat embryos was fixed
in 4% paraformaldehyde in phosphate-buffered saline overnight at
4 °C. Cryosections, 14 µm in thickness, were thaw-mounted onto VECTABOND® (Vector Laboratories)-coated glass slides. After
rehydration in phosphate-buffered saline with 0.1% Tween 20, the
sections were postfixed in 4% paraformaldehyde for 20 min, treated
with proteinase K (1 µg/ml) at 37 °C for 4 min, postfixed again in 4% paraformaldehyde for 20 min, and hybridized with
digoxigenin-labeled probes (1 µg/ml) overnight at 65 °C. Then the
sections were washed in 50% formamide, 5× SSC, and 1% SDS for 30 min
at 65 °C, subsequently twice in 50% formamide and 2× SSC for 45 min at 65 °C, and finally in Tris-buffered saline containing 0.1%
Tween 20. The slides were then immersed in blocking solution and
incubated overnight at 4 °C with anti-digoxigenin Fab-alkaline
phosphatase conjugate diluted to 1:2000 by blocking solution. The
hybrids were visualized by the alkaline phosphatase reaction with nitro
blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate.
Immunohistochemistry--
Immunostaining patterns of HNK-1
antibody were examined basically as described previously (27, 28).
Frozen sections were prepared as described above, rehydrated in
Tris-buffered saline containing 0.1% Tween 20, and boiled in 0.01 M sodium citrate for antigen enhancement. After
preincubation with the blocking solution for 30 min, the sections were
incubated with Leu7 monoclonal antibody (Becton Dickinson) diluted to
1:25 by the blocking solution overnight at 4 °C. Biotinylated
anti-mouse IgM (Zymed Laboratories Inc.) was used as a
secondary antibody, and the sections were quenched with hydrogen
peroxide. Immunoreactivity was detected using the ABC kit (Vector
Laboratories) and the metal enhanced diaminobenzidine kit (Pierce).
Construction of Expression Vector Containing a Full-length
GlcAT-D cDNA--
A DNA fragment, containing the open reading
frame of GlcAT-D, from nucleotide positions Construction of Soluble Form of GlcAT-D--
A truncated form
of GlcAT-D, lacking the first 35 amino acids of GlcAT-D, was
amplified by PCR with pBS-GlcAT-D as a template using a 5'-primer
(5'-GGAATTC CCGACCCTACTTCTCTCCGCATA-3') containing an EcoRI
site and the same 3'-primer as used in the amplification of the
fragment containing the open reading frame of GlcAT-D. The PCR product
was digested with EcoRI and cloned into the EcoRI site of pPROTA vector (29). A recombinant plasmid with the correct orientation, pPROTA-GlcAT-D, was used for expression.
Flow Cytometry Analysis--
COS-7 cells (5 × 106 cells) were transfected with pCDM8-GlcAT-D or pCDM8 by
electroporation using a Bio-Rad Gene Pulser at 300 V, 960 microfarad.
After 2 days, the cells were harvested, stained with fluorescein
isothiocyanate-conjugated anti-CD57 (Pharmingen), and analyzed on
FACSCalibur (Becton Dickinson).
Assays of Glucuronyltransferase Activity to Glycoproteins and
Glycolipids--
COS-7 cells (1 × 106 cells) were
transfected with one of the expression plasmids, pCDM8-GlcAT-D or pCDM8
(10 µg each) using LipofectAMINE (Life Technologies, Inc.) according
to the manufacturer's instructions. After 3 days, the cells were
harvested, washed with phosphate-buffered saline, homogenized in 100 µl of 0.3% Triton X-100, and used as an enzyme source.
Glucuronyltransferase activity to glycoproteins was measured in 0.1 M sodium cacodylate, pH 6.5, 100 µg of
asialo-orosomucoid, 100 µM UDP-[14C]GlcA, 5 mM CDP-choline, 10 mM GlcA, 10 mM
MnCl2, and 10 µl of the enzyme source (total volume, 30 µl). After incubation at 37 °C for 2 h, the reaction mixtures
were spotted onto Whatman number 1 paper (2 × 2 cm). The papers
were dipped in 10% (w/v) trichloroacetic acid and then rinsed twice
with fresh 10% (w/v) trichloroacetic acid, once with
ethanol/ethylether (2:1, v/v), and finally with ethylether. After being
air-dried, the radioactivity on the papers was counted using a liquid
scintillation counter.
When a glycolipid was used as the acceptor, glucuronyltransferase
activity was measured in 0.1 M MES, pH 6.0, 6 µg of
LNnT-Cer, 100 µM UDP-[14C]GlcA, 5 mM CDP-choline, 10 mM GlcA, 10 mM
MnCl2, and 10 µl of the enzyme source (total volume, 30 µl). After incubation at 37 °C for 2 h, the reaction mixtures
were applied to a 1cc Sep-Pak Vac Cartridge (Millipore) preactivated
sequentially with methanol, chloroform/methanol (2:1) and then methanol
again and subsequently equilibrated with water. The column was washed
with 50 ml of water and eluted with 0.9 ml of methanol and 1.0 ml of
chloroform/methanol (2:1). The eluant was evaporated by SpeedVac and
resuspended with 30 µl of chloroform/methanol (2:1). The product was
applied to a silica gel 60 high performance thin-layer chromatography
plate (Merck) and developed with chloroform/methanol/0.5%
CaCl2 (55:45:10). The glycolipids were visualized with
orcinol reagent, and the mobility was compared with that of
GlcA-LNnT-Cer purchased from Wako Pure Chemicals. The position and
radioactivity of the product were estimated with a Fujix BAS 2000 Bioimage Analyzer (Fuji Photo Film).
Preparation of Pyridylaminated
Glycans--
Gal Assays of Glucuronyltransferase Activity to Pyridylaminated
Glycans--
For the expression of the soluble form of GlcAT-D, 10 µg of pPROTA-GlcAT-D or pPROTA plasmid was transfected to COS-7 as
described above. After 3 days, 20 µl of the 50% suspension of
IgG-Sepharose (Pharmacia) was added to 1 ml of culture medium and
rotated overnight at 4 °C. The beads were washed twice with reaction
buffer and used as an enzyme source.
Glucuronyltransferase activity to pyridylaminated glycans was measured
in 0.1 M sodium cacodylate, pH 6.5, 0.1 mM
pyridylaminated glycan, 1 mM UDP-GlcA, 5 mM
CDP-choline, 10 mM GlcA, 20 mM
MnCl2, and 10 µl of the enzyme source (total volume, 30 µl). After incubation at 37 °C for 4-16 h, the reactions were
stopped by boiling for 5 min. After centrifugation 10,000 × g for 5 min, 10 µl of the supernatant was subjected to
HPLC analysis using PALPAK Type N column (Takara), equilibrated, and
eluted with acetonitrile/200 mM acetic acid-triethylamine,
pH 7.3 (55:45) at a flow rate of 1.0 ml/min at 40 °C. The elution
profile was monitored with a fluorescence spectrophotometer (Shimadzu).
The amounts of the products were determined from their fluorescence
intensities using PA sugar chain 041 (Takara).
Smith Degradation of the Reaction Products--
The products
from the glucuronyltransferase reaction using
Gal Cloning of a New Glucuronyltransferase--
Our preliminary
experiment of the enzymatic characterization of glycosyltransferases
from rat embryo suggested that a glucuronyltransferase other than
GlcAT-P, which is involved in the biosynthesis of HNK-1 epitope, may be
expressed in the rat embryonic nervous system. To clone new members of
the glucuronyltransferase gene family, we designed two degenerate
primers to the sequences conserved in motifs II and IV of rat GlcAT-P
and human GlcAT-I. The PCR using these primers with rat E13 brain
cDNA as a template resulted in the amplification of a product
around 380 base pairs in length. After subcloning the PCR product, we
sequenced and characterized 10 individual clones: one clone was
GlcAT-P; seven clones were putative rat GlcAT-I, a sequence of which
had 93% identity with human
GlcAT-I2; and the other two
clones had sequences that were similar to but distinct from those of
GlcAT-P and GlcAT-I, suggesting that these clones contained a novel
glucuronyltransferase (GlcAT-D) fragment. Using the PCR fragment as a
probe, we screened a rat E15 brain cDNA library and obtained one
clone, 58-2-2. The sequence of 58-2-2 contained a putative stop codon
but no in-frame ATG. To obtain the entire coding sequence of GlcAT-D,
rapid amplification of cDNA 5'-end was employed. The nucleotide
sequence of the overlapping cDNA fragments revealed that GlcAT-D
has a single open reading frame consisting of 324 amino acids, with a
molecular mass of 37,177 Da, and two potential
N-glycosylation sites (Fig.
1A). The predicted translation
initiation site conformed to Kozak's consensus sequence (32), and the
upstream region contained an in-frame stop codon. Hydropathy analysis
(Fig. 1B) indicated the presence of a potential
transmembrane domain at the N-terminal region (from Ala-4 to Val-25),
suggesting that the protein has type II transmembrane topology, which
has been found in almost all glycosyltransferases cloned to date. The
domain (from Asp-26 to Pro-81) next to the transmembrane region was
characterized by its high proline content (20%; 11 of 56 amino acids),
as seen in several other glycosyltransferases including rat GlcAT-P and human GlcAT-I. The putative catalytic domain (from Leu-80 to Val-324) showed a very high sequence identity with those of both rat GlcAT-P and
human GlcAT-I (Fig. 2). A data base
search indicated that a hypothetical protein of Drosophila
melanogaster (accession number AL033125) has high homology with
these three GlcATs in addition to those of C. elegans and
S. mansoni, as pointed out by Terayama et al.
(21).
Northern Blot Analysis--
Of the adult rat tissues examined,
GlcAT-D was expressed only in the brain (Fig.
3A). Three transcripts of 1.1 kb (major), 2.3 kb (minor), and 4.0 kb (faint) were detected in the
adult whole brain (Fig. 3A), cerebral cortex, and cerebellum
(Fig. 3B). In the postnatal (P1 and P7) cerebral cortex, the
1.1-kb transcript was more intense than the 2.3-kb one, whereas these
two transcripts in the corresponding cerebellum showed almost equal
intensity. In the E18 brain, the 1.1- and 2.3-kb transcripts showed
similar intensity. A faint single 2.3-kb transcript was detected in the E13 brain.
Localization of HNK-1 Epitope and Gene Expression of GlcATs in
Embryonic Brain--
HNK-1 immunohistochemistry was performed on E13.5
rat brain sections. Intense staining was observed in the outer layer
(preplate) of the cerebral cortex, the lateral and medial ganglionic
eminences, and the broad subventricular area of the basal ganglia.
Specific staining was also detected in the retina and slightly in the
lens (Fig. 4A).
Adjacent brain sections were used for in situ hybridization
to detect the in vivo expression patterns of GlcAT-D and
GlcAT-P. Expression of GlcAT-D was observed in the preplate of the
cerebral cortex and ventral regions of the basal ganglia (Fig.
4B). A focal staining spot was also noted in the pallidal
subventricular zone (arrow in Fig. 4B). GlcAT-P
basically showed similar expression in the telencephalon
(arrowheads in Fig. 4C), although no signal was
observed in the pallidum (compare with arrow in Fig.
4B). In the eye primordium, GlcAT-D mRNA was detected in
the retina, whereas GlcAT-P was very weakly expressed in the lens. The
regions that expressed GlcAT-D and/or GlcAT-P were always
HNK-1-positive, although transcripts of both GlcATs were below the
detection level in the putative piriform cortex (white
arrowheads in Fig. 4C).
Expression of GlcAT-D in COS-7 and Characterization as
Glucuronyltransferase Involved in Biosynthesis of HNK-1
Epitope--
To prove the enzymatic activity of the cDNA product,
expression plasmid containing GlcAT-D cDNA was transfected into
COS-7 cells. The cell homogenate was assayed for glucuronyltransferase activity using a glycoprotein and a glycolipid, both of which contained
Gal Expression of a Soluble Form of GlcAT-D--
To clarify the
function of the cDNA product, a soluble form of GlcAT-D was
generated by fusing the putative stem and catalytic domain of the
protein to a secreted form of the protein A IgG-binding domain. The
fused protein was expressed in COS-7 cells and absorbed on
IgG-Sepharose beads from culture medium, and then the enzyme-bound beads were used as an enzyme source.
The glucuronyltransferase activity of the bound fusion protein was
determined using a variety of pyridylaminated glycans as acceptor
substrates. An aliquot of the reaction mixture was subjected to HPLC.
The elution pattern is shown in Fig. 7.
Compared with the elution pattern of the reaction mixture without
UDP-GlcA (Fig. 7, B and E), the enzymatic
products using Gal Characterization as a Galactoside
Products using Gal
To prove whether a GlcA was transferred to the C-3 position of Gal of
Gal We cloned a new member of the glucuronyltransferase gene family
that transfers a GlcA to the glycan chain terminated by a Gal residue.
We named the cloned enzyme GlcAT-D, meaning a glucuronyltransferase with dual specificity for both glycolipid and glycoprotein acceptors as
mentioned below. The GlcAT-D showed high homology with the two
glucuronyltransferases cloned to date, rat GlcAT-P (21) and human
GlcAT-I (24) (overall amino acid identity of 50.0 and 45.7%,
respectively). These three enzymes may constitute a family of
galactoside The The GlcAT-D catalyzed the transfer of a GlcA to both glycoprotein and
glycolipid containing Gal The glucuronyltransferase activity involved in the biosynthesis of
HNK-1 epitope has been studied using brain extracts from chick embryo
(33), rat embryo (34), and postnatal rat (35, 36) as well as purified
enzyme from postnatal rat forebrain (20). The present study showed that
GlcAT-D acts on both glycoprotein and glycolipid acceptors in
vitro, whereas GlcAT-P has been reported to be specific to
glycoprotein acceptors (20, 35). It remains to be determined what these
glucuronyltransferases use as acceptor substrates in the nervous
system. Our study examined the substrate specificity as to the
carbohydrate sequence using pyridylaminated glycans as acceptor
substrates. The GlcAT-D transferred a GlcA to not only type 2 (Gal The Northern blot analysis showed that the ratio of the two major
transcripts (1.1 and 2.3 kb) changes according to the developmental stage. Combined with the structure of the cloned cDNA, we
speculated that the major initiation site of the transcription might
differ between the early embryo and adult brain, although the structure of transcripts is yet to be analyzed.
In situ hybridization and immunohistochemistry on the
developing brain revealed that regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive. This fact, along with the above-mentioned in vitro data, indicates that both GlcATs
are involved in the synthesis of the HNK-1 epitope in vivo.
The GlcAT-D expression pattern was slightly different from that of
GlcAT-P. GlcAT-D transcripts were positive in the pallidum and retina
where GlcAT-P expression was not specifically observed, suggesting
different in vivo functions between the two GlcATs. GlcAT-D
had catalytic activity on both glycoprotein and glycolipid acceptors in
our in vitro assay, whereas GlcAT-P was reported to be
active on only glycoprotein acceptors (20). This may reflect different
localization patterns of the GlcATs, where the GlcAT-D is expressed in
a wider region than GlcAT-P. It should be noticed that there were some HNK-1-positive areas where both GlcATs were negative. This observation suggests several possible mechanisms: 1) transcripts below the detection level by in situ hybridization, 2) existence of
unknown GlcAT genes, or 3) proliferation or migration of HNK-1-positive neurons into transcript-negative areas. Further studies will reveal the
mechanism of spatiotemporally restricted expression of HNK-1 epitope.
1-4GlcNAc
1-3Gal
1-4Glc-pyridylamine but also to
Gal
1-3GlcNAc
1-3Gal
1-4Glc-pyridylamine. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a
1,3-linkage to a terminal galactose. The
GlcAT-D transcripts were detected in embryonic, postnatal, and adult
rat brain. In situ hybridization analysis revealed that the
expression pattern of GlcAT-D transcript in embryo is similar to
that of GlcAT-P, but distinct expression of GlcAT-D was observed in
the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs
are involved in the synthesis of the HNK-1 epitope in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-4GlcNAc at nonreducing termini (16-19). The precursor of the epitope, Gal
1-4GlcNAc sequence, is
commonly found on glycoproteins and glycolipids, but expression of
HNK-1 epitope is spatially and temporally restricted. The key enzymes
in the biosynthesis of HNK-1 are a
1,3-glucuronyltransferase, which
transfers a GlcA to a terminal galactose, and a sulfotransferase, which
adds a sulfate group to the GlcA. Recently, GlcAT-P, a
glucuronyltransferase involved in the biosynthesis of HNK-1 on
glycoprotein, was purified, and its cDNA was cloned (20, 21).
Subsequently, a sulfotransferase that directs a final step of the
biosynthesis of HNK-1 was cloned by an expression cloning strategy that
involved cotransfection of GlcAT-P cDNA (22, 23). On the other
hand, GlcAT-I, another glucuronyltransferase involved in the
biosynthesis of the linkage region of proteoglycans (EC 2.4.1.135), was
cloned by PCR1 strategy based
on motifs conserved in GlcAT-P with putative proteins in
Caenorhabditis elegans and Schistosoma mansoni
(24).
1,3-glucuronyltransferase, expressed in rat. We found
1,3-glucuronyltransferase activity to glycolipids as well as glycoproteins in rat embryonic brain, suggesting that this enzyme is a
novel glucuronyltransferase. To clone cDNA of the novel enzyme, we
used the RNA of rat embryonic day 13 (E13) brain as a template of
reverse transcription-PCR and designed degenerate primers to the highly
conserved regions found in the alignment of amino acid sequence of
GlcAT-P with GlcAT-I. In this study, we describe the cDNA cloning
of a new member of the glucuronyltransferase family, GlcAT-D. The
expression of cDNA revealed that GlcAT-D is a glucuronyltransferase involved in the biosynthesis of HNK-1 epitope on both glycolipid and
glycoprotein. We also demonstrate that GlcAT-D transfers a GlcA to a
Gal residue of Gal
1-3/4GlcNAc by
1,3-linkage.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-acid glycoprotein purchased from Sigma.
-Glucuronidases from bovine liver and Helix pomatia were purchased from Seikagaku and Sigma, respectively.
1,3/4-Fucosidase from Streptomyces sp. 142 and endo-
-galactosidase from
Escherichia freundii were purchased from Seikagaku. Ceramide
glycanase from leech and
-galactosidases from Diplococcus
pneumoniae and bovine testes were purchased from Roche Molecular Biochemicals.
) (Stratagene). The GlcAT-P cDNA was obtained by reverse
transcription-PCR in which the cDNA template was synthesized from
the total RNA taken from E13 rat embryo brain. Oligonucleotides used to
amplify the GlcAT-P cDNA (21) are 5'-AAACCTGCTGCCACAATGGGTAA-3'
(nucleotides
15 to 8) and 5'-ATGGGAGAGGATGGAAGCCAAGAT-3' (nucleotides
1297 to 1274). The identity of the PCR product was confirmed by
sequencing the subcloned fragment. The cDNA fragment of GlcAT-P was
cloned into pBluescript II SK(
) (Stratagene). The antisense RNA
probes were generated with T3 or T7 RNA polymerase. Corresponding sense
probes were used to check the specificity of hybridization signals.
34 to 1004 was
amplified by PCR with cDNA reverse-transcribed from total RNA of
rat E13 brain using a 5'-primer (5'-CCCAAGCTTGAGGGTGGTGTCCGAGACGCT-3')
containing a HindIII site and a 3'-primer
(5'-GGAATTCCCTTCTCTCCTCAGCGGCTGCTC-3) containing an EcoRI
site. After restriction enzyme digestion, the PCR fragment was
subcloned into HindIII and EcoRI sites of pBluescript II SK(
) (Stratagene), yielding pBSGlcAT-D.
pBS-GlcAT-D was digested with HindIII and NotI
and then cloned into HindIII and NotI sites of
pCDM8, yielding pCDM8-GlcAT-D.
1-4GlcNAc
1-3Gal
1-4Glc-PA,
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA, and
Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4Glc-PA were synthesized by
pyridylamination of LNnT, LNT, and LNFP III, respectively, with a
Palstation and pyridylamination reagent kit (Takara) according to the
manufacturer's instructions.
GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA was synthesized from
GlcA-LNnT-Cer using 1 milliunit of ceramide glycanase from leech
according to the method of Shimamura et al. (30).
GlcNAc
1-3Gal
1-4Glc-PA was prepared by
-galactosidase digestion (from D. pneumoniae, 5 milliunits in 100 µl of
50 mM sodium acetate buffer, pH 6.0, containing 0.5 mg/ml
of bovine serum albumin at 37 °C for 2 h) of 50 nmol of
Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA, following purification by gel
filtration on a Superdex Peptide HR 10/30 column (Amersham Pharmacia
Biotech). Fuc
1-2Gal
1- 4GlcNAc
1-3Gal
1-4Glc-PA was
prepared by pyridylamination of LNnDFH I, following
1,3/4-fucosidase digestion (from Streptomyces sp. 142, 20 microunits in 100 µl of 50 mM potassium phosphate buffer, pH 6.0, 37 °C,
overnight) and purification by Superdex Peptide HR 10/30 column.
Gal
1-4GlcNAc
1-3Gal-PA and Gal
1-3GlcNAc
1-3Gal-PA were
synthesized by endo-
-galactosidase digestion (25 milliunits in 100 µl of 0.1 M sodium acetate buffer, pH 5.5, 37 °C,
48 h) of 100 µg of LNnT and LNT, respectively, following
isolation of trisaccharides by gel filtration on a Superdex Peptide
HR 10/30 column and pyridylamination.
Gal
1-3GalNAc
1-4Gal
1-4Glc-PA was purchased from Takara.
-Glucuronidase Digestion of the Reaction Products--
The
products from the glucuronyltransferase reaction using
Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA or
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA were isolated by HPLC using
PALPAK Type N column in the conditions described above. The isolated
products were digested with 1 milliunit of
-glucuronidase from
bovine liver overnight at 37 °C or with 100 units of
-glucuronidase from H. pomatia for 2 days at 37 °C in
a total volume of 50 µl of 0.1 M sodium acetate buffer, pH 5.0. The digests were analyzed by HPLC using the same PALPAK type N
column as that noted above.
1-4GlcNAc
1-3Gal-PA or Gal
1-3GlcNAc
1-3Gal-PA were
isolated by HPLC using PALPAK Type N column eluted with
acetonitrile/200 mM acetic acid-triethylamine, pH 7.3 (66:34). The isolated products (0.5 nmol each) and intact
Gal
1-4GlcNAc
1-3Gal-PA and Gal
1-3GlcNAc
1-3Gal-PA (2.5 nmol each) were subjected to Smith degradation according to the method
of Hase et al. (31) with slight modification. A
pyridylaminated glycan was dissolved in 20 µl of 80 mM
NaIO4 in 50 mM sodium acetate buffer, pH 4.0, and left to stand at 4 °C for 48 h in the dark. The reaction
was terminated by adding 5 µl of 20% ethylene glycol and kept at
room temperature for 1 h. Aldehyde groups produced were reduced
with 1 mg of sodium borohydride in 100 µl of 0.1 M borate
buffer, pH 8.0, overnight at room temperature. After neutralizing with
acetate, the product was desalted on a Superdex Peptide HR 10/30 column
and concentrated. Mild acid hydrolysis of the products was carried out
in 100 µl of 0.05 M H2SO4 at
80 °C for 1 h. After adding 10 µl of 1 M NaOH,
the product was desalted on a Superdex Peptide HR 10/30 column and
concentrated. Half an aliquot of the Smith degradation products thus
obtained was digested with 25 milliunits of
-galactosidase (bovine
testes) overnight at 37 °C in a total volume of 50 µl of 0.1 M sodium citrate phosphate buffer, pH 4.3, containing 10%
glycerol and 1% bovine serum albumin. These products were analyzed by
HPLC using PALPAK Type N column eluted with acetonitrile/200
mM acetic acid-triethylamine, pH 7.3 (75:25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, nucleotide and deduced amino acid
sequences of GlcAT-D. The putative transmembrane domain is
boxed. Potential N-glycosylation sites are
indicated by arrowheads. B, hydropathy analysis
of GlcAT-D according to the method of Kyte and Doolittle (37).
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Fig. 2.
Sequence alignment of GlcAT-D with rat
GlcAT-P (accession number D88035), human GlcAT-I (accession number
AB009598), and putative proteins in C. elegans
(accession number Z47358), D. melanogaster
(accession number AL033125), and S. mansoni
(accession number U30260). Boxes and shaded
backgrounds indicate identical and similar residues, respectively.
Four highly conserved regions named motifs I-IV are indicated by
arrows.
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Fig. 3.
Northern blot analysis of GlcAT-D. The
blots of adult rat multiple tissue (A) and the blots of
total RNA (20 µg/lane) prepared from embryo, postnatal, and adult rat
brain (B) were hybridized with 32P-labeled
GlcAT-D cDNA followed by -actin.
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Fig. 4.
Immunohistochemistry of HNK-1 and in
situ hybridization of GlcAT-D and GlcAT-P in E13.5 rat
brain. HNK-1 immunostaining (A) was observed in the
preplate of the cortex (Ctx), the broad area of the basal
ganglia, lens (L), and retina (R). HNK-1-positive
regions totally included the areas where gene expression of GlcAT-D
(B) and GlcAT-P (C) was observed. Transcripts of
both genes were localized in the cerebral cortex and ventral surface of
the basal ganglia (arrowheads), whereas only GlcAT-D
transcripts was observed in the pallidal subventricular zone
(PSZ) and the retina (arrows). Slight GlcAT-P
expression was detected in the lens (white arrow). Note that
transcripts of the both GlcATs were absent from the piriform cortex
(PC; white arrowheads). LGE, lateral
ganglionic eminence; MGE, medial ganglionic eminence.
1-4GlcNAc sequences at nonreducing termini, as acceptor substrates. As shown in Table I,
significant activity was detected using both glycoprotein and
glycolipid acceptors. Cells transfected with pCDM8 vector showed much
less activity. As shown in Fig. 5, the
reaction product using a glycolipid, LNnT-Cer, as an acceptor was
detected on high performance thin-layer chromatography at a position
corresponding to GlcA-LNnT-Cer. Moreover, a significant portion of
COS-7 cells transfected with GlcAT-D cDNA were stained with
anti-CD57, showing that these cells expressed HNK-1 epitope (Fig.
6). Cells transfected with pCDM8 vector
did not express the epitope. These results are consistent with those of
Terayama et al. (21), who showed that COS-1 cells
transfected with GlcAT-P cDNA expressed HNK-1 epitope. The results
of expression in COS-7 indicate that GlcAT-D is a glucuronyltransferase
involved in the biosynthesis of HNK-1 epitope.
Glucuronyltransferase activity of GlcAT-D expressed in COS-7 cells
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Fig. 5.
Glucuronyltransferase activity to LNnT-Cer in
COS-7 cells transfected with GlcAT-D cDNA. Lane 1,
standard glycosphingolipids visualized with orcinol reagent.
Glucuronyltransferase activity in COS-7 cells transiently transfected
with pCDM8-GlcAT-D (lane 2) or pCDM8 vector (lane
3) was measured using UDP-[14C]GlcA and LNnT-Cer as
donor and acceptor substrate. The reaction products were visualized
using a Fujix BAS 2000.
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Fig. 6.
Flow cytometric analysis of HNK-1 expression
in transiently transfected COS-7 cells. pCDM8-GlcAT-D-transfected
(A) or pCDM8-transfected (B) cells were incubated
with fluorescein isothiocyanate-conjugated anti-CD57.
1-4GlcNAc
1-3Gal
1-4Glc-PA (Fig.
7A) and Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA (Fig.
7D) as acceptors were eluted at 13.7 and 12.9 min,
respectively. The former was eluted at the same time as a synthetic
standard, GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA. In
contrast, no product was detected using the medium from mock transfected COS-7 cells (Fig. 7, C and F). As
shown in Table II, Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA and
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA were good acceptors for GlcA,
whereas no activity was detected using
Gal
1-3GalNAc
1-4Gal
1-4Glc-PA or
1,2- or
1,3-fucosylated or the agalacto-form of
Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA as acceptors. These results
confirm that the cloned cDNA encodes a glucuronyltransferase, which
transfers a GlcA to a carbohydrate chain containing an unsubstituted Gal
1-4GlcNAc or Gal
1-3GlcNAc sequence.
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Fig. 7.
HPLC analysis of glucuronyltransferase
reaction products. The enzyme adsorbed to IgG-Sepharose from COS-7
transfected with pPROTA-GlcAT-D (A, B,
D, and E) was incubated with
Gal 1-4GlcNAc
1-3Gal
1-4Glc-PA (A and B)
or Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA (D and
E) in the presence of UDP-GlcA (A and
D) or in the absence of UDP-GlcA (B and
E). The beads adsorbing the medium from pPROTA-transfected
COS-7 were incubated with Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA
(C) or Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA
(F) in the presence of UDP-GlcA. The arrow
indicates the elution position of the authentic
GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA.
Acceptor specificity of the glucuronyltransferase absorbed on
IgG-Sepharose beads
1,3-Glucuronyltransferase--
Reactions using pyridylaminated
glycans as acceptors were performed on a large scale to identify the
products, and those isolated by HPLC were subjected to further analyses.
1-4GlcNAc
1-3Gal
1-4Glc-PA and
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA as acceptors were subjected to
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry, and both showed molecular ions [M + Na]+ at
m/z 984 and [M-H+2Na]+ at
m/z 1006 (data not shown). These products were
incubated with
-glucuronidase from bovine liver or H. pomatia, and then subjected to HPLC analysis. The product from
Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA was completely digested with
-glucuronidase from bovine liver (Fig.
8A), but the product from
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA was not digested by the same
enzyme (data not shown). By contrast,
-glucuronidase from H. pomatia digested the product from
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA (Fig. 8C), as well
as the product from Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA (data not
shown). These results indicate that a GlcA is transferred to the
Gal
1-4GlcNAc or Gal
1-3GlcNAc sequence through a
-linkage.
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Fig. 8.
-Glucuronidase digestion of
glucuronyltransferase reaction products. The glucuronyltransferase
reaction products using Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA as an
acceptor were incubated with (A) or without
(B)
-glucuronidase from bovine liver. The products using
Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA as an acceptor were incubated
with (C) or without (D)
-glucuronidase from
H. pomatia. The arrows indicate the elution
positions of the authentic pyridylaminated glycans: arrows
1, Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA; arrows 2,
GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc-PA; arrows
3, Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA.
1-4GlcNAc and Gal
1-3GlcNAc sequence by Smith degradation, the glucuronyltransferase reactions using Gal
1-4GlcNAc
1-3Gal-PA and Gal
1-3GlcNAc
1-3Gal-PA as acceptors were performed on a
large scale. The products were detected in the reaction mixture using both trisaccharide-PAs as acceptors (data not shown), and isolated by
HPLC for further analyses. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of both products showed a molecular ion [M-H+2Na]+ at m/z 843. Smith
degradation (SD) of the intact acceptors and isolated products was
carried out as described under "Experimental Procedures," and the
predicted products are shown in Fig. 9.
The terminal galactoses of Gal
1-4GlcNAc
1-3Gal-PA and
Gal
1-3GlcNAc
1-3Gal-PA are cleaved by periodate, and thus the SD
products are both GlcNAc
1-3-L-threosyl-PA (Fig.
9A). When the GlcA was transferred to the C-3 position of the nonreducing terminal galactose by GlcAT-D, as
O-3-substituted Gal was not cleaved, the SD products of the
enzyme products using Gal
1-4GlcNAc
1-3Gal-PA and
Gal
1-3GlcNAc
1-3Gal-PA as acceptors were
Gal
1-4GlcNAc
1-3-L-threosyl-PA (Fig. 9B)
and Gal
1-3GlcNAc
1-3-L-threosyl-PA (Fig.
9C), respectively. When the GlcA was linked at any position other than the C-3 position of galactose, the galactose was cleaved, and thus the SD products were both
GlcNAc
1-3-L-threosyl-PA (Fig. 9A). Fig.
10 shows the HPLC analysis of the SD
products. The SD products of the two acceptors were eluted at the same
retention time (6.1 min, Fig. 10, A and D),
whereas two species of the SD products of the enzyme reaction products
were eluted at different times from those of the acceptors (Fig. 10,
B and E) and eluted at the same time (6.1 min)
after
-galactosidase digestion (Fig. 10, C and
F). These results indicate that the SD products of the glucuronyltransferase reaction products have a
-linked Gal at their
nonreducing termini and therefore that the C-3 position of Gal of the
enzyme reaction products is substituted with a GlcA. In conclusion, the
cloned GlcAT-D is characterized as a galactoside
1,3-glucuronyltransferase.
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Fig. 9.
The predicted structures of Smith degradation
products of the glucuronyltransferase reaction products. The Smith
degradation products of Gal 1-4GlcNAc
1-3Gal-PA and
Gal
1-3GlcNAc
1-3Gal-PA are both
GlcNAc
1-3-L-threosyl-PA (A). The products of
GlcA
1-3Gal
1-4GlcNAc
1-3Gal-PA and
GlcA
1-3Gal
1-3GlcNAc
1-3Gal-PA are
Gal
1-4GlcNAc
1-3-L-threosyl-PA (B) and
Gal
1-3GlcNAc
1-3-L-threosyl-PA (C),
respectively.
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Fig. 10.
HPLC analysis of Smith degradation
products. Smith degradation was carried out as described under
"Experimental Procedures." The Smith degradation products of
Gal 1-4GlcNAc
1-3Gal-PA (A) and
Gal
1-3GlcNAc
1-3Gal-PA (D) and the
glucuronyltransferase reaction products of
Gal
1-4GlcNAc
1-3Gal-PA (B) and
Gal
1-3GlcNAc
1-3Gal-PA (E) were subjected to HPLC
analysis using PALPAK type N column. After
-galactosidase digestion,
the Smith degradation products of glucuronyltransferase reaction
products of Gal
1-4GlcNAc
1-3Gal-PA (C) and
Gal
1-3GlcNAc
1-3Gal-PA (F) were analyzed with the
same HPLC column.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-glucuronyltransferases, which has high homology with
hypothetical proteins of D. melanogaster, C. elegans, and
S. mansoni (Fig. 2). In addition, a data base search
suggested that more than five additional isoforms have been identified
in C. elegans and that plant Arabidopsis thaliana
and rice might have glucuronyltransferases homologous to this family
(data not shown). The galactoside
1,3-glucuronyltransferase appears
to be commonly conserved from plant, nematode, and insect to mammal.
-glucuronidase digestion and Smith degradation of the
glucuronyltransferase reaction products demonstrated that the GlcAT-D catalyzed the formation of GlcA
1-3Gal sequence, which is
consistent with the determined structure of glycans containing the
HNK-1 epitope, sulfated GlcA
1-3Gal (16-19). For the first time in
this cloned glucuronyltransferase family, the enzyme was confirmed to
be a galactoside
1,3-glucuronyltransferase.
1-4GlcNAc sequence at nonreducing termini.
The transfer is assumed to form the precursor of HNK-1 epitope, which
is used by HNK-1-sulfotransferase as an acceptor substrate. We observed
that the COS-7 cells transfected with the cloned cDNA expressed
HNK-1 epitope and that the sites in situ detected with the
transcript were also stained with HNK-1 antibody. These results support
the idea that this enzyme is a glucuronyltransferase involved in the
biosynthesis of HNK-1 epitope.
1-4GlcNAc) but also type 1 (Gal
1-3GlcNAc) glycan chains,
suggesting that the HNK-1 epitope expressed on type 1 glycan chain may
occur, although the existence of such glycans has not previously been
reported. This result is in conflict with those of previous studies,
which showed that glucuronyltransferase from rat brain extract has no
or little activity transferring a GlcA to a glycolipid containing type
1 glycan (34, 36). Because type 3 (Gal
1-3GalNAc) glycan did not
serve as an acceptor of GlcAT-D, it remains to be examined whether the
GlcAT-D can transfer a GlcA to Gal
1-3Gal, which is a good acceptor
for GlcAT-I (24). In addition, the GlcAT-D did not transfer a GlcA to
Fuc
1-2Gal
1-4GlcNAc or Gal
1-4(Fuc
1-3)GlcNAc, the product
formed by
1,2- or
1,3-fucosyltransferase acting on type 2 glycan
chain, suggesting that the glucuronyltransferase may also compete with
these fucosyltransferases for acceptor substrates in
vivo.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Akio Kaneko and Akemi Suzuki for mass spectrometry analysis and Dr. Masahiko Endo for useful suggestions.
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FOOTNOTES |
---|
* This work was supported in part by the Yamada Science Foundation and by Grants-in-Aid for Scientific Research 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/EMBL Data Bank with accession number(s) AF106624.
To whom correspondence should be addressed: Dept. of
Biochemical Cell Research, Tokyo Metropolitan Institute of Medical
Science, RINSHOKEN, Bunkyo-Ku, Tokyo 113-8613, Japan. Tel.:
81-3-3823-2101; Fax: 81-3-3828-6663; E-mail:
sanai{at}rinshoken.or.jp.
2 Y. Shimoda, Y. Tajima, T. Nagase, K. Harii, N. Osumi, and Y. Sanai, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; GlcA, D-glucuronic acid; HPLC, high performance liquid chromatography; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LNFP III, lacto-N-fucopentaose III; LNnDFH I, lacto-N-neodifucohexaose I; LNnT-Cer, lacto-N-neotetraosylceramide; GlcA-LNnT-Cer, glucuronylneolactotetraosylceramide; En, embryonic day n; SD, Smith degradation; MES, 4-morpholineethanesulfonic acid; PA, pyridylamine; kb, kilobase(s).
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REFERENCES |
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