From the Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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The HNK-1 carbohydrate is expressed on various
adhesion molecules in the nervous system and is suggested to play a
role in cell-cell and cell-substratum interactions. Here we describe
the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A
mutant Chinese hamster ovary cell line, Lec2, which stably
expresses human neural cell adhesion molecule (N-CAM)
(Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a
human fetal brain cDNA library, a cDNA encoding the rat
glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate,
and a vector encoding the polyoma large T antigen. The transfected
Lec2-NCAM cells expressing the HNK-1 glycan were enriched by
fluorescence-activated cell sorting. Sibling selection of recovered
plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST,
that directs the expression of the HNK-1 carbohydrate epitope on the
cell surface. The deduced amino acid sequence indicates that the enzyme
is a type II membrane protein. Sequence analysis revealed that there is
a short amino acid sequence in the presumed catalytic domain, which is
highly homologous to the corresponding sequence in other
Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST
transcript is high in fetal brain compared with fetal lung, kidney, and
liver. Expression of HNK-1ST resulted in the formation of the HNK-1
epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to
add a sulfate group to a precursor,
GlcA1
3Gal
1
4GlcNAc
1
R, forming sulfo
3GlcA
1
3Gal
1
4GlcNAc
1
R. The results combined
together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the
nervous tissues.
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INTRODUCTION |
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Neural cells express unique carbohydrates that are often shared by
immune cells (1, 2). One of them is the HNK-1 carbohydrate epitope,
originally discovered by a monoclonal antibody raised against
human natural killer cells (3),
although its role in immune cells is not known. The functional
significance of the HNK-1 carbohydrate was first recognized as an
auto-antigen involved in peripheral demyelinative neuropathy. The
structural analysis of glycolipids reacting with these auto-antibodies
led to the discovery that the HNK-1 epitope is
sulfo3GlcA
1
3Gal
1
4GlcNAc
1
R (4, 5).
By using HNK-1-specific antibodies and carbohydrate structural studies, the HNK-1 glycan has been found in a number of neural cell adhesion molecules including N-CAM,1 myelin-associated glycoprotein, L1, contactin, and P0 (2, 6-9). The studies, using either monoclonal antibodies or isolated carbohydrates, demonstrated that the HNK-1 glycan is involved in cell-cell and cell-substratum interactions (10, 11). In one study, the inhibition by HNK-1 oligosaccharide was abolished by desulfation of the HNK-1 glycan indicating the critical role of the sulfate group (11). The expression of the HNK-1 epitope is spatially and developmentally regulated, and is found on migrating neural crest cells, cerebellum, and myelinating Schwann cells in motor neurons but not on those in the sensory neurons (12-14). In addition, the HNK-1 carbohydrate was shown to bind to P- and L-selectins (15), suggesting that the interactions between immune cells and the nervous system may be mediated through the binding of the HNK-1 carbohydrate in neural cells.
The HNK-1 carbohydrate is synthesized in a stepwise manner by the
addition of a -1,3-linked glucuronic acid to a precursor N-acetyllactosamine followed by the addition of a sulfate
group to GlcA
1
3Gal
1
4GlcNAc
R (16, 17). Recently, Terayama
et al. (18) reported the cloning of a
-1,3-glucuronyltransferase, GlcAT-P, that forms an HNK-1 precursor
carbohydrate, GlcA
1
3Gal
1
4GlcNAc
1
R, in glycoproteins
(18). As a part of our systematic studies on neural cell
glycoconjugates (19-22, 45), we describe herein the expression cloning
of HNK-1 sulfotransferase, HNK-1ST. Using the cDNA isolated, the
expression profile of the HNK-1ST transcripts was compared with that of
GlcAT-P transcripts for various fetal and adult tissues. We also
demonstrate the HNK-1ST activity in vivo and in
vitro using N-CAM, synthetic oligosaccharides and glycolipids as
acceptors.
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EXPERIMENTAL PROCEDURES |
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Preparation of Recipient Cells and Plasmids--
A mutant cell
line of Chinese hamster ovary cells, Lec2, was used as recipient cells.
-Glucuronylation of N-acetyllactosamines is extremely
efficient in Lec2 cells (18) because sialylation is absent in this cell
line (23). Lec2 cells were first transfected with
pH
APr-1-neo-NCAM 140 (24) and a stable cell line
expressing human N-CAM, Lec2-NCAM, was selected as described before
(19). For cloning of GlcAT-P, the cDNA was synthesized from
poly(A)+ RNA of rat brain (CLONTECH)
using a reverse transcription-PCR kit (Stratagene). Using the cDNAs
synthesized as templates, PCR was performed to amplify the GlcAT-P
sequence under the conditions described previously (21). The 5'- and
3'-primers correspond to nucleotides
32 to
10 and nucleotides
1047-1027, respectively, of the reported rat GlcAT-P sequence (18).
The 5'- and 3'-primers also contain HindIII and
XhoI sites, respectively. The PCR product was digested with
HindIII and XhoI, then cloned into pcDNA3,
resulting in pcDNA3-GlcAT-P.
Isolation of a Human HNK-1ST cDNA Clone-- Lec2-NCAM cells were found to be negative for the HNK-1 antigen after pcDNA3GlcAT-P was transiently expressed. Lec2-NCAM cells were thus co-transfected with 18 µg of a human fetal brain cDNA library in pcDNAI (19), 6 µg of pcDNA3-GlcAT-P, and 6 µg of pPSVE1-PyE harboring the polyoma large T cDNA (25), using LipofectAMINETM (Life Technologies, Inc.) as described previously (19). After 62 h, the transfected cells were dissociated into monodispersed cells using the enzyme-free cell dissociation solution (Hanks' based, purchased from Cell and Molecular Technologies, Lavellette, NJ), followed by fluorescence-activated cell sorting of the HNK-1-positive cells using anti-HNK-1 monoclonal antibody (Becton Dickinson). Plasmid DNA from the sorted cells was isolated by the Hirt (26) procedure and amplified in the host bacteria Escherichia coli MC1061/P3 in the presence of ampicilin and tetracycline. The pcDNAI vector contains the supF suppressor tRNA, so that MC1061/P3 cells containing pcDNAI are resistant to both ampicillin and tetracycline. In contrast, MC1061/P3 cells harboring pcDNA3GlcAT-P or pPSVE1-PyE are resistant to ampicillin but not to tetracycline. Because of this difference, only plasmids derived from pcDNAI were rescued and amplified by this procedure (19), allowing the isolation of plasmids responsible for the HNK-1 glycan expression.
Bacteria harboring plasmids, which were isolated by the Hirt procedure, were divided into 20 plates. Plasmid DNA was prepared from each plate and separately transfected into Lec2-NCAM cells together with pcDNA3-GlcAT-P. The transfectants were screened by immunofluorescence microscopy using anti-HNK-1 antibody to identify a plasmid pool that directed the expression of the HNK-1 glycan. By narrowing down the plasmid pools using the same procedure, it was possible to isolate a single clone containing the plasmid DNA (pcDNAI-HNK-1ST) encoding a human sulfotransferase that directed the expression of the HNK-1 carbohydrate epitope.Construction of Vectors Harboring Short 5'- and 3'-Untranslated Sequences-- To shorten the long 3'-untranslated sequence of pcDNAI- HNK-1ST, HNK-1ST cDNA was digested utilizing an internal EcoRI site 15 nucleotides downstream of the stop codon and cloned into pcDNA3, resulting in pcDNA3-HNK-1ST (short). A truncated cDNA containing only 9 and 6 nucleotides of 5'- and 3'-untranslated sequences in addition to the coding sequence was prepared by PCR. The 5'- and 3'-primers for the PCR were 5'-GTCAAGCTTTGTGACAAACATGCACCACCAGTGGCT-3' and 5'-GCGCTCGAGTATGCATTAGTTTAGCAAAAAGTC-3'. HindIII and XhoI sites are singly underlined, while HNK-1ST-coding sequences are doubly underlined. After restriction enzyme digestion, the PCR product was cloned into pcDNA3, yielding pcDNA3-HNK-1ST (ORF). Nucleotide sequences were determined in both strands by an automated sequencer (Applied Biosystems 377XL).
Northern Blot Analysis of Various Human Tissues-- Human multiple tissue Northern blots of poly(A)+ RNA (CLONTECH) were hybridized sequentially with gel-purified cDNA inserts of pcDNA3-HNK-1ST (ORF) and pcDNA3-GlcAT-P, after labeling with [32P]dCTP by random oligonucleotide primers (Prime-IT II labeling kit, Stratagene).
Western Blot Analysis of N-CAM Expressing the HNK-1 Carbohydrate-- Lec2-NCAM cells were transiently transfected with pcDNAI- HNK-1ST and pcDNA3-GlcAT-P, pcDNAI-HNK-1ST alone or pcDNA3-GlcAT-P alone. Forty-eight h after transfection, cell lysates were made from the transfected cells and incubated with a mouse anti-human N-CAM monoclonal antibody (ERIC-1, Santa Cruz Biotechnology) (27), followed by protein G-agarose (Pierce). After solubilization, the immunoprecipitates were separated by SDS-polyacrylamide (5%) gel electrophoresis and transferred onto nitrocellulose membrane. The blot was then incubated with the anti-N-CAM antibody, anti-HNK-1 antibody, or M6749 antibody (28) followed by horseradish peroxidase-conjugated sheep anti-mouse immunoglobulins and visualized by an ECL kit (Amersham Corp.).
Expression of the Protein A-HNK-1ST Fusion Protein-- The cDNA fragment encoding the stem region plus catalytic domain of HNK-1ST was prepared by PCR using pcDNA3-HNK-1ST (short) as a template and fused with the cDNA encoding a signal peptide sequence and the IgG binding domain of Staphylococcus aureus protein A (20, 29). The 5'-primer for this PCR is TTAGATCTACCAGATGTGTACAGTGCC, where BglII site is underlined, and the coding sequence of HNK-1ST is doubly underlined. The 3'-primer is SP6 promoter sequence. The PCR product was digested by BglII and XhoI then cloned into BamHI and XhoI sites of pcDNAI-A (20), yielding pcDNAI-A·HNK-1ST. pcDNAI-A·HNK-1ST and pcDNAI-A were separately transfected to COS-1 cells and the enzyme was adsorbed to IgG-Sepharose 6FF (Pharmacia Biotech Inc.) as described previously (20).
Assay of in Vitro Activity of
HNK-1ST--
Gal1
4GlcNAc
1
octyl,
GlcA
1
3Gal
1
4GlcNAc
1
octyl, and
sulfo
3GlcA
1
3Gal
1
4GlcNAc
1
octyl were synthesized
according to the reported procedures (30, 31) with a slight
modification. The detailed procedure for the synthesis of these
oligosaccharides will be published
elsewhere.2 Key
1H NMR (600 MHz, D2O) for
GlcA
1
3Gal
1
4GlcNAc
1
octyl is included;
4.680 (d, J = 8.0 Hz, H-1 GlcA), 4.520 (secondary
order, J1, 2 = 8.1 Hz) and 4.502 (d,
J = 7.9 Hz) (H-1 Gal and H-1 Glc), 4.190 (d,
J = 3.1, H-4 Gal), 2.040 (s,
NCOCH3).
GlcA
1
3Gal
1
4GlcNAc
1
3Gal
1
4Glc
ceramide was
prepared by acid hydrolysis from its sulfated form (4) and kindly
provided by Dr. Firoze Jungalwala.
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RESULTS |
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Isolation of a cDNA Clone That Directs the Expression of the HNK-1 Carbohydrate Epitope-- Lec2-NCAM cells were co-transfected with a human fetal brain cDNA library in pcDNAI, pcDNA3-GlcAT-P, and pPSVE1-PyE (25). The transfected cells were incubated with anti-HNK-1 antibody followed by fluorescein isothiocyanate-conjugated secondary antibody, then subjected to cell sorting. Plasmid DNA, recovered from anti-HNK-1 antibody-positive Lec2-NCAM cells, was immediately subjected to sibling selection with sequentially smaller, active pools, identifying a single clone containing the plasmid, pcDNAI-HNK-1ST, that directed the expression of the HNK-1 glycan (Fig. 1C). Weak staining was observed when only HNK-1ST was present (Fig. 1B) probably due to endogenous expression of a small amount of GlcAT-P. No staining was observed when HNK-1ST cDNA was not expressed (Fig. 1A).
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Predicted Amino Acid Sequence of HNK-1ST-- The cDNA insert encoding HNK-1ST contains an open reading frame predicting a protein of 356 amino acid residues (42,206 Da) (Fig. 2). A hydropathy plot predicts that this protein has a type II membrane topology, and the transmembrane domain (residues 5-22) is flanked by basic amino acids. This topology has been found in almost all mammalian glycosyltransferases so far cloned (32). Although there is no significant similarity between the cloned HNK-1ST and sequences for other proteins deposited in GenBankTM, the amino acid sequence of residues 187-195 (see the doubly underlined sequence in Fig. 2 and Fig. 3) has homology with the sequences found in other Golgi-associated sulfotransferases such as chick chondroitin sulfate 6-O-sulfotransferase, human galactosylceramide sulfotransferase, hamster heparan sulfate 2-O-sulfotransferase, and human heparan sulfate 3-O-sulfotransferase (33-36). In particular, the RDP sequence (residue 189-191) is completely conserved, and hydrophobic amino acids are shared among these amino acid sequences (Fig. 3). Moreover, the amino acid sequence of the rat HNK-1ST reported recently (37) has the identical sequence as the human HNK-1ST in residues 187-195.
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Expression of HNK-1ST mRNA in Human Tissues-- Northern blots of poly(A)+ RNA derived from various human tissues were examined (Fig. 4). An HNK-1ST transcript of ~3 kilobases was prominently detected in fetal brain, moderately in lung, and kidney but barely in liver. The same transcript was strongly detected in adult brain, testis, ovary, and moderately in heart, skeletal muscle, pancreas, spleen, and thymus, but weakly in other tissues. Among various parts of the brain, HNK-1ST transcript is expressed more in the frontal lobe than the other parts.
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Expression of the HNK-1 Epitope on N-CAM-- To determine if HNK-1ST is capable of adding the HNK-1 epitope on N-CAM, Lec2-NCAM cells were transiently transfected with pcDNAI- HNK-1ST and pcDNA3-GlcAT-P, pcDNAI-HNK-1ST alone or pcDNA3-GlcAT-P alone. Western blot analysis of N-CAM derived from those transfected cells demonstrated that the HNK-1 glycan was formed on N-CAM when both HNK-1ST and GlcAT-P were expressed while the HNK-1 glycan was not expressed in the absence of either enzyme (Fig. 5B, lanes 1-3).
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Demonstration of In Vitro Activity of HNK-1ST--
To formally
prove that the cloned cDNA encodes HNK-1ST, a soluble chimeric
HNK-1ST was expressed in COS-1 cells. The enzyme adsorbed to
IgG-Sepharose was then incubated with acceptor oligosaccharides or
glycolipid and [35S]PAPS. As shown in Fig.
6, a substantial amount of
35S-sulfate was incorporated to
GlcA1
3Gal
1
4GlcNAc
1
octyl (lane 4), while no
incorporation was detected using the medium from mock-transfected COS-1
cells (lane 3), or using Gal
1
4GlcNAc
octyl as an
acceptor (lane 2). Similarly, 35S-sulfate was
incorporated into the glycolipid acceptor (lane 6).
Considering that the concentration of the oligosaccharide acceptors is
3.8 times higher than that of the glycolipid acceptor, HNK-1ST added
sulfate to the oligosaccharide as efficiently as to the glycolipid. The
reaction products shown in lanes 2-6 were subjected to thin
layer chromatography followed by autoradiography. The sulfated product
migrated at the same position of a standard synthetic oligosaccharide,
sulfo
3GlcA
1
3Gal
1
4GlcNAc
1
octyl (Fig.
7). These results establish that the
cloned HNK-1ST transfers a sulfate group to C-3 of GlcA attached to
N-acetyllactosamine, forming
sulfo
3GlcA
1
3Gal
1
4GlcNAc
1
R.
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DISCUSSION |
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In this study, we describe the isolation of a cDNA clone
encoding a human sulfotransferase, HNK-1ST, the enzyme responsible for
the formation of a sulfate group attached to the C-3 of glucuronic acid
residue in GlcA1
3Gal
1
4GlcNAc
1
R structure. For
this cloning, Lec2 cells were first stably transfected to express human N-CAM. Those Lec2 cells expressing N-CAM were then transiently co-transfected with a human fetal brain cDNA library in pcDNAI, pcDNA3-GlcAT-P, and pPSVE1-PyE. pcDNAI has the
supF gene while both pcDNA3-GlcAT-P and
pPSVE1-PyE contain only the ampicillin-resistant gene. Only plasmids
derived from pcDNAI can be thus rescued and amplified in bacteria
containing P3 episome such as MC1061/P3 cells in the presence of
ampicilin and tetracycline (38). In theory, N-CAM cDNA could be
also co-transfected transiently since its vector contains only an
ampicillin resistant marker. This procedure represents an improved
method over previous expression cloning strategy where a vector
encoding the polyoma large T antigen was stably expressed (25, 39). By
avoiding the preparation of stable transfectants, which are necessary
for the expression of the acceptor carbohydrates and polyoma large T
antigen, the time necessary for cloning has been shortened
dramatically. The cloning of the HNK-1ST cDNA started right after
the publication of GlcAT-P sequence (18) and took approximately 2.5 months to obtain a single clone.
After the submission of our paper, Bakker et al. (37) reported the cloning of a rat HNK-1ST. Their cloning strategy differs slightly from ours because CHOP2 cells, which are Lec2 cells stably expressing the polyoma large T antigen (40), were used as recipient cells, and the N-CAM cDNA was not co-transfected. Since we were interested in cloning the cDNA that adds the HNK-1 glycan on neural cell adhesion molecules, Lec2 cells stably expressing N-CAM were used as recipient cells in our studies. In the end, however, the cDNAs cloned by us and their group probably encode the same enzyme, since 90.2% of amino acid residues are identical between the human HNK-1ST and the cloned rat enzyme.
As shown in Fig. 3, there appears to be a consensus sequence among various Golgi-associated sulfotransferases. It is possible that the amino acid sequence may be involved in the binding of PAPS or in catalysis. Notably, this sequence is not shared by N-deacetylase/N-sulfotransferase, which has dual functions and is involved in heparan sulfate synthesis (41, 42). It is tempting to speculate that N-deacetylase/N-sulfotransferase is different in evolutionary origin than the other sulfotransferases. Moreover, the consensus sequence shown in Fig. 3 is different from the presumed PAPS binding sequences found among different soluble cytosolic sulfotransferases (43). Further studies are necessary to determine if the consensus sequence found among different Golgi-associated sulfotransferases plays a role in the enzymatic function.
Our studies revealed that the cloned human HNK-1ST adds the HNK-1
carbohydrate epitope on N-CAM. Bakker et al. (37) on the other hand showed that the rat GlcAT-P and rat HNK-1ST add the -glucuronic acid and sulfate into a wide variety of glycoproteins in
CHOP2 cells, although none of them was identified. These results strongly suggest that GlcAT-P can add glucuronic acid to
N-acetyllactosamine present in a wide variety of
glycoproteins and HNK-1ST can use those carbohydrates as acceptor
molecules.
Our studies also demonstrate that the transcript of HNK-1ST is more widely distributed in different tissues compared with that of GlcAT-P (Fig. 4). In the brain, both the HNK-1ST and GlcAT-P transcripts are highly expressed, suggesting that the cloned HNK-1ST and GlcAT-P are most likely responsible for the formation of the HNK-1 glycan in the nervous tissues. It has been shown that there are two glucuronyltransferases specific for forming the HNK-1 precursor structure in glycoproteins and glycolipids, respectively (44). In contrast, HNK-1ST cloned in the present study was shown to add a sulfate group to both glycoproteins and glycolipids. The HNK-1ST present in tissues other than the brain may act on glycolipid acceptors. Alternatively, there is another GlcAT-P acting on glycoprotein acceptors, which may differ in tissue distribution than the one that has been cloned (18). Future studies are important to address this problem.
As described in the Introduction, the HNK-1 carbohydrate is associated with a number of cell adhesion molecules in the nervous tissues. The addition of the HNK-1 glycan to these various adhesion molecules are thought to modulate cell-cell and cell-substratum interactions. The HNK-1 cDNA obtained in the present study will be a powerful molecular tool to manipulate the expression level of the HNK-1 glycan in specific cell types, allowing us to dissect the intricate and complex cell processes of cell-cell interactions during development.
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ACKNOWLEDGEMENTS |
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We thank Drs. Firoze Jungalwala, Toshisuke
Kawasaki, and Frank Walsh for the kind gifts of glucuronylated
paragloboside, M6749 antibody, and pHAPr-1-neo-NCAM, respectively,
Drs. Yoshiaki Miura and Isabelle Franceschini for useful discussion,
and Susan Greaney for organizing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health, NCI Grants PO1 CA71932 and R01 CA33895.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) AF033827.
Recipient of a postdoctoral fellowship from the Natural Sciences
and Engineering Research Council of Canada.
§ To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3144; Fax: 619-646-3193; E-mail: minoru{at}burnham-inst.org.
1 The abbreviations used are: N-CAM, the neural cell adhesion molecule; GlcAT-P, glycoprotein-specific glucuronyltransferase; HNK-1ST, HNK-1 sulfotransferase; PCR, polymerase chain reaction; PAPS, 3'-phosphoadenosine 5'-phosphosulfate.
2 Y. Ding and O. Hindsgaul, manuscript in preparation.
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REFERENCES |
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