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INTRODUCTION |
Neural cell adhesion molecules undergo critical post-translational
modifications that modulate their affinity and in some cases
alter their specificity for their cognate ligands (1, 2). In
particular, many neural cell adhesion molecules are heavily
glycosylated, and the extent and composition of attached carbohydrates
modify their adhesive properties. Among those, polysialic acid and
HNK-1 are particularly notable. Polysialic acid is mostly attached to
NCAM,1 and polysialylated
NCAM is abundantly present in embryonic brain (1, 2). In the adult
brain, polysialylated NCAM is restricted to certain tissues, such as
the hippocampus and olfactory bulb, where neuronal regeneration
persists. Polysialic acid is a linear homopolymer of 
-2,8-linked
sialic acid residues formed on -2,3- or -2,6-linked sialic acid
in N-acetyllactosamine of N-glycans (3-6).
Polysialic acid is attached to two N-glycosylation sites in
the fifth immunoglobulin-like domain in NCAM and thought to attenuate
the adhesive property of NCAM (7-9).
The HNK-1 carbohydrate epitope was originally defined
by a monoclonal antibody raised against human natural killer (HNK)
cells (10). In nervous tissues, the HNK-1 carbohydrate was first
recognized as an autoantigen involved in peripheral demyelinative
neuropathy (11, 12). The structural analysis of glycolipids reacting with these autoantibodies demonstrated that the HNK-1 epitope is
sulfo
3GlcA
13Gal14GlcNAc1R (11, 12). HNK-1 glycan is widely distributed in glycoproteins, glycolipids, and proteoglycans (2, 11-13). The expression of HNK-1 glycan is spatially and developmentally regulated and found on migrating neuronal crest cells,
cerebellum, and myelinating Schwann cells in motor neurons but not on
those in the sensory neurons (14-16). Although it has been reported
that HNK-1 glycan is attached to the N-glycosylation site in
the third immunoglobulin-like domain of NCAM, the recent studies showed
that HNK-1 glycan is attached to the second, third, fifth, and sixth
N-glycosylation sites (17).
HNK-1 apparently plays critical roles in neural cell
migration and axonal extension. HNK-1 glycolipids coated on plates
facilitated neurite outgrowth, whereas no facilitation of neurite
outgrowth was observed on sulfatide, 3'-sulfogalactosyl ceramide. The
effect of HNK-1 glycan was abolished once the sulfate group was removed (18, 19). The HNK-1 glycan was identified in NCAM and
N-glycans isolated from P0 glycoprotein (20, 21). It was
also found as rather unusual structures of O-linked
oligosaccharides, sulfo3GlcA13Gal14GlcNAc12Man, which then are attached to serine or threonine (22). This glycan can be attached to dystroglycan, and its defect may cause muscular dystrophy (23).
NCAM has numerous isoforms due to alternative splicing of
precursor mRNAs. Among them, NCAM(MSD) contains an additional
domain, the so-called muscle-specific domain (MSD), consisting of 37 amino acids between two fibronectin type III-like domains (24). This NCAM(MSD) is mostly present in skeletal myotubes and often anchored to
the plasma membrane through glycophosphatidylinositol (24). The MSD is
highly enriched with serine, threonine and proline and
O-linked oligosaccharides were shown to attach to this
domain (25). It has been shown that NCAM(MSD) from C2C12 myoblast cell line contains mucin-type O-linked oligosaccharides
±NeuNAc23Gal13(±NeuNAc26)GalNAcThr/Ser (25).
However, no studies have addressed whether O-glycans in NCAM
contain an HNK-1-capping structure.
The HNK-1 glycan is synthesized in a stepwise manner by the
addition of a 1,3-linked glucuronic acid to precursor
N- acetyllactosamine by 1,3-glucosaminyltransferase
(GlcAT-P) followed by the addition of a sulfate group by HNK-1
sulfotransferase (HNK-1ST) to GlcA13Gal14GlcNAcR, forming sulfo3GlcA13Gal14GlcNAcR (26). Recently, the
cDNAs encoding GlcAT-P and HNK-1ST have been cloned (27-29). In
mucin-type O-glycans, N-acetyllactosamine can be
formed when core 2 branch, GlcNAc16(Gal13) GalNAc1R, is synthesized by core 2 1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT,
Fig. 1, Ref. 30). These results prompted us to examine how
the HNK-1 glycan is synthesized in mucin-type O-linked oligosaccharides attached to NCAM.
In this report, we first present evidence that HNK-1 glycan can
be formed in O-glycans attached to the MSD in NCAM. We found that HNK-1 glycan is attached to core 2 branched O-glycans
but not to core 1 O-glycans present in NCAM. We determined
the HNK-1 glycan structure in mucin-type O-linked
oligosaccharides as
sulfo3GlcA13Gal14GlcNAc16(Gal13)GalNAc. Finally, we found that HNK-1 glycans can be formed on
N-glycans more efficiently than O-glycans on
NCAM, although GlcAT-P and HNK-1ST utilize O-glycan and
N-glycan oligosaccharides almost equally as an acceptor.
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EXPERIMENTAL PROCEDURES |
Plasmids Encoding NCAM, NCAM(MSD), GlcAT-P, and
HNK-1ST--
pIG-NCAM·IgG and pIG-NCAM(MSD)·IgG encoding
NCAM·IgG and NCAM(MSD)·IgG chimeric proteins, respectively, were
constructed from pIG-NCAM(VASE, MSD)·IgG, originally provided by Dr.
David Simmons at Oxford University (31), as described previously (32).
All products derived from these different vectors were found to react with anti-NCAM antibody (Eric-1) by Western blotting, confirming the
sequences and sizes of the products (32).
The cDNA encoding rat 1,3-glucuronyltransferase, GlcAT-P (27),
was cloned as described previously using reverse transcription-PCR and
subcloned into pcDNA3, resulting in pcDNA3(neo)-GlcAT-P (29). The cDNA encoding a catalytic domain of GlcAT-P was obtained by PCR
using pcDNA3(neo)-GlcAT-P as a template. 5'- and 3'-primers for
the PCR are 5'-CGGATCCCAGCCTCGCACCTCTGCTTGCT-3' (the
BamHI site is underlined, and the rest is nucleotides
120-141 of GlcAT-P) and
5'-ACTCGAGTCAGATCTCCACCGAGGGGTC-3' (the
XhoI site is underlined, and the rest of the sequence is
nucleotides 1044-1024 of GlcAT-P). After BamHI and
XhoI digestion, the cDNA fragment was ligated into the
same sites of pcDNAI-A, which harbors a signal peptide and
IgG-binding domain of protein A, resulting in pcDNAI-A·GlcAT-P. The cDNA encoding a human sulfotransferase that transfers a sulfate group from PAPS to glucuronylated N-acetyllactosamine
precursors was cloned into pcDNA3 as described previously,
resulting in pcDNA3-HNK-1ST (29). Similarly, pcDNAI-A·HNK-1ST
was prepared as described before (33). pcDNAI encoding core 2 1,6-N-acetylglucosaminyltransferase, Core2GlcNAcT-I (30),
was cloned into the pcDNA3.1(Zeo), resulting pcDNA3.1(Zeo)-Core2GlcNAcT-I. A cDNA encoding
1,2-N-acetylglucosaminyltransferase I (GnT-I) cloned in
pcDNA3.1 (34, 35) was a kind gift of Dr. Pamela Stanley.
Preparation of Lec1 Cells Expressing HNK-1 Precursors and HNK-1
Glycans--
To avoid the formation of HNK-1 in N-glycans,
the majority of the experiments were carried out using a mutant Chinese
hamster ovary cell line, Lec1. Since Lec1 cells are deficient in GnT-I, all of the N-glycans synthesized in this cell line remain as
high mannose oligosaccharides (36). Lec1 cells were found to lack core
2 1,6-N-acetylglucosaminyltransferase as in other Chinese hamster ovary cells (30). Lec1 cells were stably transfected with
pcDNA3.1(Zeo)-Core2GlcNAcT-I as described before (29). Lec1-core 2 cells were first selected in the presence of zeocin (Invitrogen) and
then by immunofluorescent staining using tomato lectin, which reacts
with N-acetyllactosamine and
poly-N-acetyllactosamines (37). As shown previously, the
formation of core 2 branches results in the formation of small amounts
of poly-N-acetyllactosamine (38). To confirm the integration
of human Core2GlcNAcT-I cDNA into Lec1 cell chromosome, reverse
transcription-PCR was carried out on poly(A)+ RNA derived
from Lec1-core 2 cells. The primers for this reverse transcription-PCR
were designed in exon 1 and exon 2, avoiding the amplification of
hamster genomic DNA harboring Core2GlcNAcT.
Lec1-core 2 cells were transfected with pcDNA3(neo)-GlcAT-P and
selected in the presence of G418 and zeocin, and Lec1-core2·GlcA were
chosen after immunostaining using M6749 antibody (39), which reacts
with both nonsulfated and sulfated forms of HNK-1 carbohydrates (27,
29). Those cells were further transfected with pcDNA3.1(hyg)-HNK-1ST
and selected in the presence of G418, zeocin, and hygromycin. Those
cells expressing a significant amount of HNK-1 glycan, assessed by
immunostaining with anti-HNK-1 antibody (Becton Dickinson), were chosen
and designated as Lec1-core 2·HNK-1. As the second antibody,
fluorescein isothiocyanate-conjugated goat (Fab')2 fragment
specific to mouse IgG (for anti-NCAM antibody) or IgM (for HNK-1
antibody and M6749 antibody) was used. In parallel, Lec1 cells stably
expressing GlcAT-P or HNK-1ST alone or GlcAT-P and HNK-1ST together
were established.
Transient Expression of NCAM and NCAM(MSD)--
Lec1 cells
expressing the HNK-1 glycan were transiently transfected with
pIG-NCAM·IgG or pIG-NCAM(MSD)·IgG with or without pcDNAI-Core2GlcNAcT-I, using LipofectAMINE PlusTM as described previously (29). The medium was changed to serum-free medium 24 h
after the transfection and cultured for an additional 48 h.
NCAM·IgG in the cultured medium was adsorbed to protein A-agarose as
described before (8). NCAM·IgG molecules eluted from the medium were
subjected to SDS-polyacrylamide (5%) gel electrophoresis and
transferred onto a polyvinylidene difluoride membrane. The blot was
then incubated with anti-NCAM antibody, anti-HNK-1 antibody, or M6749
antibody, with a secondary antibody as described before (29).
Preparation of Lec2 Cells Expressing HNK-1 Precursor and HNK-1
Glycans--
Lec2 cells expressing GlcAT-P and HNK-1ST, Lec2-HNK-1,
were established as described previously (29). Lec2-HNK-1 cells were selected using anti-HNK-1 monoclonal antibody. Lec2 cells expressing GlcAT-P were similarly established and selected by immunofluorescent staining using M6749 monoclonal antibody. Lec2 cells do not synthesize sialylated oligosaccharides due to the defect in CMP-sialic acid transporter in the Golgi (40).
Metabolic Labeling of Carbohydrate Moiety of
NCAM-IgG--
Lec1-core 2·HNK-1 cells were transiently transfected
with pIG-NCAM(MSD)·IgG or pIG-NCAM·IgG. Twenty-four hours after the
transfection, the medium was replaced with sulfate-free or glucose-free
RPMI 1640 medium containing dialyzed 10% fetal calf serum in the
presence of Na2[35S]O4 (100 µCi/ml), [6-3H]GlcNH2 (10 µCi/ml),
and [6-3H]Gal (20 µCi/ml) or
[6-3H]GlcNH2 (10 µCi/ml) or
Na2[35S]O4 (100 µCi/ml) alone
(41). After a 48-h incubation, the spent medium was harvested, and
NCAM·IgG was isolated as described above. After confirming the purity
of the isolated samples by SDS-polyacrylamide gel electrophoresis and
fluorography, the samples were subjected to oligosaccharide characterization.
Isolation of O-Linked Oligosaccharides Containing HNK-1
Glycans--
Metabolically labeled NCAM(MSD)·IgG was digested with
Pronase, and labeled glycoproteins were isolated by Sephadex G-25 gel filtration as described previously (41). O-Glycan-containing glycopeptides were separated from high mannose glycopeptides
and other glycans using ConA-Sepharose affinity chromatography. The purified glycopeptides were subjected to alkaline reductive treatment to release O-linked oligosaccharides as described previously
(41). Oligosaccharides were applied to a Poly-Prep column (Bio-Rad) containing 2 ml of anion-exchange QAE-Sephadex A-25. The resin was
equilibrated with 2 mM pyridine-acetate buffer, pH 5.5, and eluted in a stepwise manner with 6 ml each of 70 mM, 250 mM, and 1 M NaCl in 2 mM
pyridine-acetate buffer, pH 5.5. Fractions (1 ml) were collected, and
aliquots were taken for determination of radioactivity.
The oligosaccharides eluted with 1 M NaCl were digested
with Arthrobacter sialidase (Sigma) to remove sialic acid
and subjected to QAE-Sephadex A-25 column chromatography. Those eluted
with 1 M NaCl after sialidase treatment were subjected to
structural characterization.
Structural Characterization of O-Glycans Containing HNK-1
Glycan--
The O-linked glycans were subjected to
solvolysis to remove a sulfate group as described previously (41, 42).
The resultant oligosaccharides, purified by QAE-Sephadex
chromatography, were digested with 125 milliunits of Escherichia
coli -glucuronidase (Sigma) in 50 µl of the reaction mixture
under the same conditions as described previously (11). The obtained
oligosaccharides were analyzed on an AX-5 amino-bonded column using a
Gilson 306 HPLC apparatus at a flow rate of 0.8 ml/min. The column was
equilibrated with 90% solvent A (80% acetonitrile, 20% 15 mM KH2PO4) and 10% of solvent B
(40% acetonitrile, 60% 15 mM
KH2PO4). After 5 min, the elution was carried
out by linear gradient from 10% of solvent B to 100% of solvent B in
60 min. The column was finally washed for 5 min with solvent B. Fractions were collected every minute, and aliquots were taken for
determination of the radioactivity by scintillation counting.
Periodate Oxidation of Oligosaccharides--
Oligosaccharides
were oxidized with 50 mM NaIO4 in 50 mM sodium acetate buffer, pH 4.5, at room temperature, and
oxidized samples were reduced as described previously (43). The
samples, after destroying NaBH4 by acetic acid, were passed
over a small column of Dowex 50 × 8 and dried after the addition
of methanol. The samples were then hydrolyzed in 0.01 M HCl
at 80 °C for 1 h, neutralized with 0.1 M Tris-HCl
buffer, pH 8.5, and applied to a column (1 × 120 cm) of Bio-Gel
P-4 (200-400-mesh) equilibrated with 0.1 M
pyridine-acetate buffer, pH 5.5. Oligosaccharides were also separated
by QAE-Sephadex A-25 column chromatography as described above.
Assays for GlcAT-P and HNK-1ST Activity--
To assay the
activities of GlcAT-P and HNK-1ST, soluble protein A-GlcAT-P and
soluble protein A-HNK-1ST were prepared and adsorbed to IgG-Sepharose
as described previously (33). To assay for GlcAT-P activity, the
reaction mixture (100 µl) consisted of 500 µM
acceptors, 0.1 M HEPES buffer, pH 6.5, 10 mM
MnCl2, 10 mM of galactonic acid-
-lactone, 50 µl of the soluble chimeric enzyme attached to beads, and 50 µM UDP-[14C]GlcA (294 mCi/mmol, PerkinElmer
Life Sciences). The reaction mixture was incubated for 3 h at
37 °C, and the reaction was stopped by adding 50 mM
(final concentration) EDTA.
To assay for HNK-1ST activity, the donor, 3'-phosphate
5'-phospho-[35S]sulfate, and various amounts of an
acceptor were incubated with a solution of the 50% suspension of the
chimeric protein A-HNK-1ST attached to beads under the conditions
described previously (29, 33). The reaction was stopped by 250 mM ammonium formate, pH 4, and labeled products were
obtained by C18 reverse-phase chromatography as described previously
(29). The Km for GlcAT-P and HNK-1ST was determined
using a Lineweaver-Burk plot at various concentrations of the acceptor
(2.5-2000 µM).
Synthesis of Standard Oligosaccharides--
The acceptors
GlcA13Gal14GlcNAc16Man16Man1O(CH2)7CH3
(octyl) (compound 1),
GlcA13Gal14GlcNAc12Man16Man1O(CH2)7CH3 (octyl) (compound 2), and
GlcA13Gal14GlcNAc16(Gal13)GalNAc1O(CH2)7CH3 (octyl) (compound 3) were synthesized from precursors octyl
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-glucopyranosyl(16)-2,3,4-tri-O-benzyl--D-mannopyranosyl(16)-2,3,4-tri-O-benzyl--D-mannopyranoside (compound 4), octyl
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-glucopyranosyl(12)-3,4,6-tri-O-benzyl--D-mannopyranosyl(16)-2,3,4-tri-O-benzyl--D-mannopyranoside (compound 5), and octyl
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-glucopyranosyl(16)[2,3,4,6-tetra-O-benzoyl--D-galactopyranosyl(13)]-2-acetamido-4-O-acetyl-2-deoxy--D-galactopyranoside (compound 6). Each acceptor (compounds 4-6) was glycosylated with the
donor O-(methyl
2,3,4-tri-O-benzoyl--D-glucopyranosuluronate)-(13)-2,4,6-tri-O-benzoyl--D-galactopyranosyl trichloroacetimidate (compound 7) with catalytic trimethylsilyl trifluoromethanesulfonate to give the derivatives of the
pentasaccharides in good to excellent yield. Reduction of the
trichloroacetamydoyl group to the acetimidoyl group (44) followed by
the usual deprotection steps provided the target compounds 1-3.
The products were characterized by 1H and 13C
NMR spectroscopy and high resolution electrospray-time-of-flight mass spectrometry.
Partial 1H NMR (600 MHz,
D2O) (compounds 1 and 2); (300 MHz, D2O)
(compound 3); are as follows. Compound 1: 
4.894 (d,
J1,2 = 1.5 Hz, -Man H-1), 4.678 (d,
J1,2 = 7.8 Hz, GlcA H-1), 4.666 (s,
J1,2 < 1.0 Hz, -Man H-1), 4.586 (d,
J1,2 = 8.3 Hz, GlcNAc H-1), 4.533 (d,
J1,2 = 7.9 Hz, Gal H-1), 2.0 (s, 3 H, NHAc).
Compound 2: 4.930 (d, J1,2 = 1.6 Hz, -Man
H-1), 4.678 (d, J1,2 = 7.8 Hz, GlcA H-1), 4.666 (s, J1,2 < 1.0 Hz, -Man H-1), 4.610 (d, J1,2 = 7.8 Hz, GlcNAc H-1), 4.528 (d,
J1,2 = 7.9 Hz, Gal H-1), 2.0 (s, 3 H, NHAc).
Compound 3: 4.85 (d, J1,2 = 3.5 Hz, GalNAc H-1), 4.65 (d, J1,2 = 7.7 Hz, GlcA H-1), 4.50, 4.49, and 4.48 (3 d, J1,2 = 8.2 Hz,
J1,2 = 8.5 Hz, J1,2 = 7.6 Hz, 2 Gal H-1 and GlcNAc H-1), 1.98 and 1.96 (2s, 6 H, NHAc).
m/z calc. for
C40H69N1Na1O27 (compounds 1 and 2): 1018.3955; found compound 1: 1018.3961; compound 2: 1018.3959. Detailed procedures of the synthesis will be
published elsewhere.2
Gal14GlcNAc1octyl,
Gal14GlcNAc12Man16Man1octyl,
Gal14GlcNAc16Man16Man1octyl, and
Gal14GlcNAc16 (Gal13)GalNAc1octyl were
synthesized as described previously (45-47).
| |
RESULTS |
Expression of HNK-1 Glycan in Mucin-type O-Glycans Attached to
NCAM--
To determine the biosynthetic pathway of HNK-1 glycan in
mucin-type oligosaccharides attached to NCAM, Chinese hamster ovary mutant Lec1 cells were utilized. Lec1 cells contain the deficient N-acetylglucosaminyltransferase I, which is the first key
enzyme to form complex N-glycans (36). Because this enzyme
is absent in Lec1 cells, N-glycans remain as the high
mannose type, and N-acetyllactosamine, which is a precursor
for HNK-1 glycan, is not formed in N-glycans.
Lec1 cells also lack core 2 -1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT) as in
wild-type Chinese hamster ovary cells. Since mucin-type
oligosaccharides can acquire N-acetyllactosamine when core 2 branches are formed by Core2GlcNAcT, Lec1 cells expressing HNK-1 glycan
(Lec1-HNK-1) were further transfected with cDNA encoding Core2GlcNAcT-I (see Fig. 1). As shown in
Fig. 2A, HNK-1 was detected by
Western blotting in NCAM(MSD)·IgG synthesized in Lec1 cells expressing HNK-1ST and GlcAT-P, together with Core2GlcNAcT-I
(lanes 4). When HNK-1ST was absent,
glucuronylated precursor glycan was detected by anti-glucuronic acid
antibody, M6749 (GlcA), but not by anti-HNK-1 antibody (HNK-1)
(lanes 6 in Fig. 2A). Multiple HNK-1-positive bands may represent NCAM(MSD)·IgG containing different numbers of sulfate groups, since glucuronylated molecules correspond mainly to the lowest molecular weight band (Fig. 2A,
lane 6). Moreover, NCAM(MSD)·IgG did not react
with either anti-HNK-1 antibody or M6749 antibody when Lec1 cells were
not transfected with Core2GlcNAcT cDNA (Fig. 2A,
lanes 1-3). These results establish that
Core2GlcNAcT, GlcAT-P, and HNK-1ST are all necessary to form the HNK-1
glycan in NCAM(MSD).
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Fig. 1.
Biosynthetic pathway of HNK-1 glycan on
mucin-type O-glycans. Core 1 O-glycans
can be converted to core 2 O-glycans, followed by
galactosylation by 4GalT-IV. The resultant
N-acetyllactosaminyl core 2 O-glycans can be
converted by 1,3-glucuronyltransferase (1,3-GlcAT) and
HNK-1 sulfotransferase (HNK-1ST) to HNK-1 glycan on core 2 branched O-glycans (right). On the other hand,
core 1 O-glycans can be sialylated by ST3Gal-I followed by
2,6-sialyltransferases, which form disialyl core 1 O-glycans (left), based on the present and
previous studies (59-61).
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Fig. 2.
Western blot analysis of NCAM·IgG chimeric
protein in Lec1 cells transfected with GlcAT-P and HNK-1ST.
A, Lec1 cells expressing HNK-1ST (S), GlcAT-P
(G), or both HNK-1ST and GlcAT-P (SG) were
transiently transfected with NCAM(MSD)·IgG chimeric protein together
with (+) or without ( ) Core2GlcNAcT-I (C2GnT). Released
chimeric NCAM·IgG was purified by protein A column and subjected to
Western blot analysis after separating SDS-polyacrylamide gel
electrophoresis. The blot was reacted with anti-NCAM
(N-CAM), anti-HNK-1 (HNK-1), or anti-glucuronic
acid (GlcA) M6749 antibody. B, Lec1 cells
expressing both HNK-1ST and GlcAT-P were transiently transfected with
NCAM·IgG (devoid of MSD) together with (+) or without ()
Core2GlcNAcT-I (C2GnT). NCAM-IgG was analyzed in the same
manner as in A.
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To determine whether HNK-1 glycans can be attached to
O-glycans in sequences other than MSD, NCAM·IgG lacking
the MSD sequence was also expressed in the same Lec1 cells expressing
Core2GlcNAcT-I and HNK-1ST glycan on the cell surface. As shown in Fig.
2B, NCAM synthesized in these Lec1 cells did not bear the
HNK-1 glycan or glucuronylated precursor regardless of whether
Core2GlcNAcT was present or absent. Minor HNK-1-positive glycoproteins
were detected when Lec1 cells expressed Core2GlcNAcT-I, GlcAT-P, and HNK-1ST (Fig. 2B, lane 4). They were
judged to be contaminating glycoproteins other than NCAM based on their
mobility in SDS-polyacrylamide gel electrophoresis. These results
indicate that the MSD sequence is essential for acquiring the HNK-1
glycan in O-glycans attached to NCAM.
Structural Characterization of the HNK-1 Glycan Attached to the MSD
Sequence in NCAM--
To determine the structure of the HNK-1 glycan
attached to NCAM, NCAM(MSD)·IgG was transiently expressed in
Lec1-core 2·HNK-1 cells expressing HNK-1 glycan and core 2 branched
O-glycans. Twenty-four h after the transfection, the
transfected cells were cultured for an additional 48 h in
sulfate-free medium in the presence of [3H]GlcN,
[3H]Gal, and [35S]sulfate. Similarly, the
transfected cells were cultured in glucose-free medium with
[3H]GlcN or in sulfate-free medium with
[35S]sulfate. NCAM(MSD)·IgG molecules purified from the
cultured medium were then subjected to SDS-polyacrylamide gel
electrophoresis and fluorography. The results showed that
[35S]sulfate, [3H]galactose, and
[3H]glucosamine were incorporated into NCAM(MSD)·IgG
(data not shown).
As a first series of experiments for structural characterization,
NCAM(MSD)·IgG labeled with [3H]galactose,
[3H]glucosamine, and [35S]sulfate was used.
Glycopeptides were prepared and applied to ConA-Sepharose
chromatography. The results shown in Fig.
3 demonstrated that
35S-labeled material was not bound to ConA-Sepharose. The
glycopeptides eluted with 10 mM methyl -glucoside and
250 mM methyl -mannoside represent biantennary and high
mannose type N-glycans, respectively, and they did not
contain sulfate. The glycopeptides unbound to ConA-Sepharose (shown in
the horizontal bar in Fig. 3), representing O-glycans and multiantennary N-glycans, were then
subjected to alkaline borohydride treatment to release
O-linked oligosaccharides.
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Fig. 3.
Separation of glycopeptides derived from
metabolically labeled NCAM(MSD)·IgG by concanavalin A-Sepharose
column. Lec1-core2·HNK-1 cells were transiently transfected with
NCAM(MSD)·IgG in the presence of [35S]sulfate,
[3H]GlcN, and [3H]Gal, and NCAM(MSD)·IgG
was isolated from the cultured medium. Glycopeptides prepared from
radiolabeled NCAM(MSD)·IgG were applied to a column of
ConA-Sepharose. After washing the column, it was eluted with 10 mM methyl -glucoside and 250 mM methyl
-mannoside. Open and closed circles represent
35S and 3H radioactivity, respectively.
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The treated sample was separated into two peaks after Sephadex G-25 gel
filtration. The first peak of higher molecular weight contained both
35S and 3H radioactivity, whereas the second
peak contained only 3H radioactivity (data not shown). Upon
QAE-Sephadex column chromatography, all of the 35S-labeled
material was recovered in the fraction eluted with 1 M NaCl
in the pyridine-acetate buffer (Fig.
4A, Q3). This 35S-
and 3H-labeled material eluted again in this highly anionic
fraction after sialidase treatment (Fig. 4B). Since no
3H radioactivity was released, peak Q3 did not contain
sialic acid, which can be labeled from [3H]glucosamine
precursor. In contrast, the glycopeptides eluted with 2-250
mM NaCl (Q0, Q1, and Q2 in Fig. 4A) were mostly
eluted at the void volume after sialidase treatment (data not shown), consistent with the fact that they were devoid of
[35S]sulfate.
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Fig. 4.
QAE-Sephadex column chromatography and
Bio-Gel P-4 gel filtration of released O-glycans.
A, O-glycans were prepared from glycopeptides
designated by a horizontal bar in Fig. 3 and
separated by QAE-Sephadex column chromatography. B and
C, O-glycans eluted by 1 M NaCl
(shown by a horizontal bar in A) were
subjected to the same QAE-Sephadex column chromatography after
sialidase treatment (B) or removal of sulfate by solvolysis
(C). D, peak c in
panel C was applied for a short column of Bio-Gel
P-4. Peaks d and e denote the elution
positions of intact sulfated HNK-1 glycan and free sulfate,
respectively. Open and closed circles represent
35S and 3H radioactivity, respectively.
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Since no sulfatase isolated to date is known to remove a sulfate group
from HNK-1 glycan, we opted to remove sulfate by solvolysis. The sample
after solvolysis eluted with 70 mM NaCl (Fig.
4C, peak b), where material containing
one acidic group elutes. There was still substantial radioactivity
eluted with 250 mM and 1 M NaCl (Fig.
4C, peak c). The latter material was
mostly free [35S]sulfate (fractions 24-32 in Fig.
4D), while a small amount of remaining HNK-1 glycan existed
(peak d in Fig. 4D). Peak
b in Fig. 4C, desulfated HNK-1 glycan, was
subjected to Bio-Gel P-4 gel filtration before and after
-glucuronidase treatment. The isolated oligosaccharide,
peak b in Fig.
5B, was converted by -glucuronidase digestion to
Gal14GlcNAc16(Gal14)GalNAcOH, peak
c in Fig. 5C. These results established that
HNK-1 glycan, shown as peak a in Fig.
4B, has the structure
sulfateGlcA1Gal14GlcNAc16(Gal13)GalNAcOH.
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Fig. 5.
Bio-Gel P-4 gel filtration of HNK-1 glycans
before and after various treatments. A-C,
peak a in Fig. 4B (A) and
peak b in Fig. 4C before
(B) and after -glucuronidase treatment (C)
were subjected to Bio-Gel P-4 gel filtration. D-F, the
samples shown in A-C were subjected to Smith degradation
and subjected to the same Bio-Gel P-4 gel filtration. Peaks
a-c correspond to
sulfo3GlcA13Gal14GlcNAc16(Gal13)GalNAcOH,
GlcA13Gal14GlcNAc16(Gal13)GalNAcOH,
and Gal1 4GlcNAc16(Gal13)GalNAcOH,
respectively. Peaks d-g are explained under
"Results."
Gal14GlcNAc16(Gal13)GalNAcOH was also
prepared from HL-60 cells as described previously (43).
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Periodate Oxidation of HNK-1 Glycan and Its Desulfated
Form--
The above results establish that the HNK-1 glycan is
attached to core 2 branched O-glycans. The above
experiments, however, did not determine the linkage and attachment
sites of sulfate and glucuronic acid. In order to obtain this
information, the HNK-1 glycan and its nonsulfated form
(peaks a and b in Fig. 5, A
and B, respectively) were subjected to periodate oxidation
followed by reduction and mild acid hydrolysis (Smith degradation).
Periodate oxidation takes place in a cis-glycol group.
As a control experiment, core 2 O-glycan,
Gal14GlcNAc16(Gal13)GalNAc (Fig. 5C,
c) was subjected to Smith degradation. This treatment
produced two peaks: f and g (Fig. 5F).
Peaks f and g represent GlcNAc and
glycerol derived from galactose, respectively. Peak
c derived from HNK-1 glycans yielded the same results. When peak b in Fig. 5B, nonsulfated HNK-1
glycan, was subjected to the same treatment, peaks
e and g were produced (Fig. 5E).
Peak e corresponds to Gal14GlcNAc, which
was confirmed by exoglycosidase digestion. These results show that
glucuronic acid prevented galactose from oxidation, indicating that
glucuronic acid residue was attached to C-3 of galactose. Otherwise,
peak b in Fig. 5B and peak
c in Fig. 5C would produce the same product. When
peak a in Fig. 5A was subjected to the
same treatment, peak d was obtained (Fig. 5D). Peak d was larger than
peak e and was judged to be produced from
sulfated glycans. Peak e in Fig. 5D
was probably produced from nonsulfated glycans that were formed during
various preparation steps. These results thus indicate that sulfate
protected the GlcA residue from oxidation, and peak
d corresponds to sulfo3GlcA13Gal14GlcNAc. The presence of sulfate and glucuronic acid in peak
d was confirmed by the fact that peak
d still had two anionic charges as assessed by QAE-Sephadex
column chromatography (data not shown).
These results, combined together, establish the structure of
HNK-1 glycan as
sulfo3GlcA13Gal14GlcAc1 6(Gal13)GalNAc.
NCAM in Mouse Embryonic Brain and Heart and C2C12 Cells Contain
Negligible Amounts of HNK-1 Glycans Attached to O-Glycans--
We then
examined whether NCAM in mouse embryonic brain and heart contain HNK-1
glycans attached to O-glycans. NCAM present in brain at
embryonic day 12 exhibit broad high molecular weights that were
converted to bands of 200 and 140 kDa after N-glycanase treatment (Fig. 6A,
lanes 1 and 2). When the same blot was incubated with anti-HNK-1 antibody, no band corresponding to
N-glycanase-treated NCAM reacted with the antibody (Fig.
6A, lanes 2 and 6). The
same experiment showed that small amounts of HNK-1 glycan were attached to N-glycans in NCAM, displaying two bands that are slightly
larger than 220 kDa (Fig. 6A, lane 5).
The remaining HNK-1 antigen was mostly associated with molecules larger
than 220 kDa, suggesting that those HNK-1 glycans are associated with
proteoglycans. HNK-1 was barely detected in heart tissue, although NCAM
was detected (Fig. 6A, lanes 3,
4, 7, and 8).
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Fig. 6.
HNK-1 glycan present in mouse fetal brain and
heart and C2C12 myoblast cells. A, lysates derived from
mouse brain and heart at embryonic day 12 were separated by
SDS-polyacrylamide gel electrophoresis and subjected to Western
analysis using anti-NCAM (lanes 1-4) or
anti-HNK-1 (lanes 5-8) antibody. The lysates
were subjected to analysis before () and after (+)
N-glycanase treatment. B, NCAM was
immunoprecipitated from the cell lysates of
[35S]sulfate-labeled C2C12 cells and subjected to
SDS-polyacrylamide gel electrophoresis and fluorography.
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By using similar methods, the myoblast cell line, C2C12, was subjected
to analysis. C2C12 cells were derived from mouse cells and can be
induced to form myotubes (25). Our preliminary results showed that
HNK-1 glycan was barely detected by Western analysis of cell lysates.
We thus use 35S-sulfate incorporation to detect sulfated
glycans. The results shown in Fig. 6B illustrate that after
4 days of induced differentiation, C2C12 cells expressed
[35S]sulfate molecules that were heterogeneous in
molecular sizes. However, none of this polydispersed radioactive band
was converted by N-glycanase treatment to a sharp band (Fig.
6B, lanes 1-4), whereas NCAM in day 0 and 4 culture apparently contained sulfate groups in N-glycans
(Fig. 6B, lanes 1-4). It is possible that NCAM
in days 4 and 7 may contain polysialic acid attached to
O-glycans, or these broad bands may represent contaminating
proteoglycans such as heparan sulfate. These results indicate that NCAM
in C2C12 cells bear minimum amounts of HNK-1 glycan.
HNK-1 Glycan Synthesis Takes Place Preferentially on
N- Glycans, although Both GlcAT-P and HNK-1ST Almost
Equally Act on N- and O-Glycan Oligosaccharide
Acceptors--
The above results could be obtained if one of the
glycosyltransferases responsible for HNK-1 glycans prefers
N-glycans acceptors over O-glycan acceptors. To
test this hypothesis, we first assayed 1,3-glucuronyltransferase
(GlcAT-P) using various synthetic acceptors. The results shown in Fig.
7 illustrate that GlcAT-P utilizes
synthetic acceptor oligosaccharides that mimic both N- and
O-glycans. GlcAT-P utilizes
Gal14GlcNAc16Man16Man1octyl slightly better
than core 2 branched O-glycan, but core 2 branched
O-glycan acceptor is a slightly better acceptor than
Gal14GlcNAc12Man16Man1octyl. We then
compared HNK-1ST activity in three different acceptors. As shown in
Fig. 8, HNK-1ST acts slightly better on
GlcA13Gal14GlcNAc12Man16Man1octyl (Km = 169 µM) than
GlcA13Gal14GlcNAc16Man16Man1octyl (Km = 358 µM). Core 2 branched
O-glycan is an intermediate between these
N-glycan acceptors (Km = 283 µM). These results combined indicate that core 2 branched
O-glycan is as good an acceptor as N-glycans for
both GlcAT-P and HNK-1ST.
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Fig. 7.
Incorporation of 14C-GlcA by
GlcAT-P to various acceptors. A soluble chimeric GlcAT-P was
incubated with UDP-[14C]GlcA and acceptors shown. As a
control, the culture medium derived from mock-transfected cells was
used as an enzyme source.
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Fig. 8.
Dependence of HNK-1ST activity on the
concentrations of various acceptors. A soluble chimeric HNK-1ST
was incubated with [35S]PAPS and various concentrations
of acceptors. B, the Lineweaver-Burk plot of A.
The activity was for
GlcA13Gal1 4GlcNAc12Man16Man1octyl
( ), GlcA13Gal14GlcNAc16Man16Man1octyl
( ), and
GlcA13Gal14GlcNAc16(Gal13)GalNAC1octyl
( ).
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Despite the fact that the enzymes responsible for HNK-1 synthesis can
utilize N-glycan and O-glycan acceptors almost
equally well, HNK-1 was barely detected in NCAM O-glycans in
tissues examined. These results obtained as a whole thus suggest that
HNK-1 may be more efficiently added to N-glycans than
O-glycans when they are attached to NCAM. In order to test
this hypothesis, Lec1 cells expressing GlcAT-P and HNK-1ST were
transiently transfected with cDNA encoding GnT-I. This
transfection allows Lec1 cells to gain complex N-glycans,
which can then become substrates for both GlcAT-P and HNK-1ST. The
amount of HNK-1 glycan was significantly increased in NCAM(MSD) derived
from Lec1 cells expressing GnT-I, compared with NCAM(MSD) derived from
the parent Lec1 cells (Fig.
9A, compare lanes
7 and 9). The large majority of HNK-1 glycans
were removed after N-glycanase treatment (Fig.
9A, lane 10), indicating that the
majority of HNK-1 glycan is associated with newly synthesized N-glycans.
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Fig. 9.
NCAM(MSD)·IgG released from Lec1 and Lec2
cells in the presence or absence of GnT-I. Lec1 and Lec2 cells
stably expressing HNK-1 glycan were transiently transfected with
NCAM(MSD)·IgG in the absence () or presence (+) of GnT-I.
A, NCAM(MSD)·IgG isolated was subjected to Western
analysis before () or after (+) N-glycanase treatment.
B, the transfected cells were metabolically labeled with
[35S]sulfate and released NCAM(MSD)·IgG was purified by
protein A-conjugated resin. The left panel shows
Western blot analysis using anti-NCAM antibodies to adjust the amount
of NCAM(MSD)·IgG for assaying anti-HNK-1 reactivity (A) or
fluorography (B). In lane 9 of
panel A, contaminating glycoproteins released
from the Lec1 cells also contained HNK-1 glycan.
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It is possible that sialylation inhibits HNK-1 glycan formation in core
2 branched O-glycans, since sialylation and glucuronylation compete for the same N-acetyllactosamine. To test this
possibility, Lec2 cells that are deficient in sialylation were used to
express HNK-1 glycan. Transfected Lec2 cells bearing both
N-glycans and O-glycans expressed significant
amounts of HNK-1 glycans, but almost all HNK-1 glycans were removed
after N-glycanase treatment (Fig. 9A,
lanes 11 and 12). Almost identical
results were obtained when NCAM(MSD) were labeled with
[35S]sulfate (Fig. 9B). These results as a
whole indicate that HNK-1 synthesis is shifted from
O-glycans to N-glycans once N-glycan acceptor substrates are available.
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DISCUSSION |
The present study demonstrated that HNK-1 can be synthesized in
O-glycans when three enzymes, GlcAT-P, HNK-1ST, and
Core2GlcNAcT, are transfected into Lec1 cells, which lack the ability
to form the HNK-1 glycan attached to complex and hybrid
N-glycans. Since Lec1 cells lack Core2GlcNAcT, a
simultaneous expression of Core2GlcNAcT is essential to form HNK-1
glycan on O-glycans. Our structural analysis of HNK-1 glycan
indeed demonstrated that HNK-1 glycan is synthesized on
N-acetyllactosamine in core 2 branched O-glycans (Fig. 1). This requirement of Core2GlcNAcT was also demonstrated for
sialyl Lewis x synthesis in mucin-type O-glycans, since
sialyl Lewis x, Sia23Gal14(Fuc13)GlcNAc1R, is
also synthesized on N-acetyllactosamine backbone, which is
formed by Core2GlcNAcT (41, 48).
The present study also demonstrated that mucin-type
O-glycans bearing HNK-1 can be synthesized only when NCAM
contains an MSD. MSD is enriched with threonine, serine, and proline,
which is characteristic for amino acid sequences that attach mucin-type O-glycans (23, 24). A short amino acid sequence that
consists of these amino acids is also present in the other part of
NCAM, but it apparently does not serve for O-glycosylation
attachment sites. MSD is encoded by four different exons, and
differential splicing of NCAM precursor mRNA leads to the formation
or absence of MSD. The expression of MSD-containing NCAM is
temporally and spatially restricted, and myotubes express NCAM
containing MSD (24).
Since the amino acid sequence of MSD is enriched with proline,
threonine, and serine, MSD most likely does not have a unique conformation, such as an -helical or -sheet structure (49). Since
O-glycosylation takes place in the Golgi apparatus,
O-glycosylation sites need to be exposed to the environment
after a protein folds. Non--helical and non--sheet structure
satisfies such a requirement. In addition, it is most likely that such
an amino acid sequence does not take a particular conformation, and its
structure is flexible (49). It is thus possible that MSD may function
as a hinge region where O-glycans are attached. This
situation is similar to the hinge region of IgA, where multiple
O-glycans are attached (50, 51). Lysosomal membrane
glycoproteins (LAMP-1 and LAMP-2) also contain a hingelike
structure, and the hingelike structure of human LAMP-1 has homology to
the IgA hinge region (52, 53). Future studies are of importance to
determine whether MSD in NCAM also functions as a hingelike structure.
Despite the fact that HNK-1 glycan can be added to O-glycans
to NCAM(MSD) upon transfection with GlcAT-A and HNK-1, NCAM isolated from embryonic brain, heart, and C2C12 cells did not bear detectable amounts of HNK-1 epitope in O-glycans. This is most likely
not due to the absence of Core2GlcNAcT, since Core2GlcNAcT-I is
expressed in the brain and heart (54). One possible explanation for
this discrepancy is that the expression levels of GlcAT-P and HNK-1ST are lower in these tissues compared with Lec1 cells or HeLa cells (data
not shown) transfected with GlcAT-P and HNK-1ST. On the other hand, the
same experiment clearly detected HNK-1 glycan in molecules with high
molecular weights. Since those HNK-1-positive molecules still showed a
polydispersity after N-glycanase treatment, it is likely
that these molecules represent proteoglycans. These results indicate
that the method used was sensitive enough to detect HNK-1 glycan. As a
whole, it can be concluded that HNK-1 glycan is only barely, if at all,
added on O-glycans attached to NCAM in the tissues examined.
The above results could be obtained if GlcAT-P and HNK-1 utilize
N-glycans as acceptors better than O-glycans.
However, GlcAT-P and HNK-1ST did not utilize N-glycan
acceptors better than O-glycans when synthetic
oligosaccharides that mimic a portion of N-glycans or
O-glycans were used as acceptors. Moreover, the synthesis of HNK-1 glycans on NCAM was substantially increased when Lec1 cells were
converted to synthesize complex-type N-glycan acceptors for HNK-1 glycan by transfecting with GnT-I. The amount of HNK-1 glycan on
O-glycans is much less than that on N-glycans
(for Lec1 cells bearing complex type N-glycans). These
results indicate that GlcAT-P and HNK-1ST are more accessible to
N-glycan acceptors than to O-glycan acceptors in
glycoproteins such as NCAM. This is probably because
N-glycans are more extended from the polypeptide backbone than O-glycans. On the other hand, PSGL-1 glycoprotein
containing numerous O-glycans has an extended rodlike
structure and has been shown to have sulfated
N-acetyllactosamines, presumably on the O-glycans
(55, 56). These combined results strongly suggest that MSD of NCAM may
not display as preferable a conformation for enzymes that synthesize
O-glycans as the conformation of PSGL-1.
HNK-1 glycan has been shown to bind to laminin (2), whereas sialylated
core 1 O-glycan,
NeuNAc23Gal13(NeuNAc26)GalNAc, was shown to bind
Siglec such as myelin-associated glycoprotein (57). These
results suggest that the addition of a HNK-1-capping structure on NCAM
O-glycans may switch the interaction of NCAM with
myelin-associated glycoprotein to that with laminin. Such switching
probably plays a critical role in neural cell development.
Our studies demonstrated that HNK-1 synthesis may be synthesized in a
complex manner and depends on how much N-glycan acceptors versus O-glycan acceptors are available.
Similarly, it has been demonstrated that
GlcNAc-6-O-sulfotransferase preferentially acts on
N-glycans when glycoprotein acceptors such as CD34 bear both N- and O-glycans (41). By contrast, the same
enzyme can act efficiently on O-glycans when the enzyme acts
on GlyCAM-1, which almost exclusively contains O-glycans
(58). In addition, it has been demonstrated that
N-acetyllactosamine formation in core 2 branched
O-glycans is much less efficient than in
N-glycans (59). These results suggest that the synthesis of
N-acetyllactosamine in core 2 glycans can be a rate-limiting
step for the addition of HNK-1 epitope structure on
O-glycans. These results combined indicate that the
synthesis of terminal, functional oligosaccharide groups in
O-glycans is regulated in a complex manner and that the
availability of acceptors in N- or O-glycans and
their conformation play a critical role in displaying such functional
groups in different glycans.
,
,
TOP
ABSTRACT
INTRODUCTION
Present address: Corvas International Inc., San Diego, CA 92121.
Present address: Deptartment of Neuroscience, Institute
Pasteur, 75724 Paris cedex 15, France.
