From the ¶ Zentrum fuer Molekulare Neurobiologie, Universitaet
Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany and the
Department of Pathology, Washington University School of
Medicine, St. Louis, Missouri 63110
Received for publication, December 21, 2000, and in revised form, January 3, 2001
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ABSTRACT |
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We have identified and characterized an
N-acetylgalactosamine-4-O-sulfotransferase
designated GalNAc-4-ST2 (GenBankTM accession number
AF332472) based on its homology to HNK-1 sulfotransferase (HNK-1 ST).
The cDNA predicts an open reading frame encoding a type II membrane
protein of 443 amino acids with a 12-amino acid cytoplasmic domain, a
23-amino acid transmembrane domain, and a 408-amino acid luminal domain
containing four potential N-linked glycosylation sites.
GalNAc-4-ST2 displays a high degree of amino acid sequence identity
with GalNAc-4-ST1 (46%), HNK-1 ST (23%), chondroitin
4-O-sulfotransferase-1 (C4ST-1) (27%), and chondroitin
4-O-sulfotransferase-2 (C4ST-2) (24%). GalNAc-4-ST2 transfers sulfate to the C-4 hydroxyl of terminal Sulfated carbohydrate structures have been shown to play important
roles in a large number of different molecular interactions including
symbiotic interactions between plants and nitrogen-fixing bacteria (1),
homing of lymphocytes to lymph nodes (2), control of the circulatory
half-life of the glycoprotein hormones LH1 and TSH (3, 4), binding
of growth factors by proteoglycans (5-7), and triggering preferential
neurite outgrowth (8). HNK-1, an example of such a sulfated glycan, has
the structure SO4-3-GlcUA Expression cloning of the rat and human sulfotransferases responsible
for synthesis of the HNK-1 epitope revealed the presence of two
sequence motifs that are associated with
3'-phosphoadenosyl-5'-phosphosulfonate (PAPS) binding (17, 18) and are
common among all sulfotransferases cloned to date (19-21). Despite the
presence of these motifs, HNK-1 sulfotransferase shows only limited
similarity to other sulfotransferases outside of the PAPS-binding
regions. Nonetheless, three additional sulfotransferases, chondroitin
4-O-sulfotransferase 1 (C4ST-1) (22, 23), chondroitin
4-O-sulfotransferase 2 (C4ST-2) (23), and
N-acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24, 25), were identified based on their homology to the
catalytic C-terminal domain of HNK-1 ST. C4ST-1 and C4ST-2 (Fig. 2)
show 25 and 22% identity to HNK-1 ST, respectively, whereas
GalNAc-4-ST1 is 23% identical to HNK-1 ST. In addition to the motifs
associated with binding PAPS, HNK-1 ST, C4ST-1, C4ST-2, and
GalNAc-4-ST1 have three additional motifs of unknown function that are
located C-terminal to the PAPS-binding motifs. While homologous,
these sulfotransferases have distinct saccharide specificities. HNK-1 ST transfers sulfate to the C-3 hydroxyl of terminal We have identified and characterized a fourth sulfotransferase that is
homologous to HNK-1 ST with 23% identical amino acids. N-Acetylgalactosamine-4-O-sulfotransferase 2 (GalNAc-4-ST2) transfers sulfate to the C-4 hydroxyl of terminal
Materials--
[ Molecular Cloning of a Human cDNA Homologous to HNK-1
Sulfotransferase--
A human EST derived from human infant
brain (GenBankTM accession number H15485) with
high sequence similarity to rat HNK-1 ST (17) was identified using
BLASTN and TBLASTN algorithms (26) against the data bases of
GenBankTM, EMBL, and DDBJ EST Divisions at the National
Center for Biotechnology Information (National Institutes of Health,
Bethesda). The insert of the corresponding I.M.A.G.E. Consortium
(LLNL) cDNA clone, ID 49547 (IMAGp951B2016 obtained from RZPD,
Berlin, Germany) (27), was sequenced and found to contain a complete
open reading frame encoding for a 358-amino acid protein showing
significant sequence similarity to rat and human HNK-1
sulfotransferases. This sequence was designated as GalNAc-4-ST2(Met-86)
(GenBankTM accession number AF332473).
The forward primer 5'-GGG AGA GTG GAG AAG AGA AGA GAA C-3' and the
reverse primer 5'-AAG CCA ATC CAT TTA GTA CCA TCA GA-3' were used to
amplify a 595-bp fragment (nucleotides at position 211-805 in the
GalNAc-4-ST2 cDNA) including the putative start codon from
first-strand cDNA produced from 100 ng of total RNA obtained from a
human glioma and the human kidney 293 cell line using Omniscript®
reverse transcriptase (Qiagen). An additional 38 bps (designated exon 4 in Fig. 3) were present in this product when compared with
GalNAc-4-ST2(Met-86) and shifted the stop codons located 5' out of the
reading frame of the putative translation start site. Similarity
searches identified a bovine EST (GenBankTM accession
number BE724107) with sequence homology to the 5' end of the amplified
fragment, allowing us to predict a chimeric sequence from the human and
bovine cDNAs that would contain an open reading frame encoding for
a putative 443-amino acid protein. Human genomic clones
(GenBankTM accession numbers AC009872, AC023575, and
AC010854) were analyzed with FGENESH (28), and the predicted exons were evaluated using the chimeric GalNAc-4-ST2 cDNA. Primer pairs
derived from exonic sequences were designed and used to amplify the
full-length GalNAc-4 ST2 sequence shown in Fig. 1A
(GenBankTM accession number AF332472) from both human
embryonic kidney 293 cell line and glioma cDNA.
In Silico Analysis of the Human GalNAc-4-ST2 Locus--
A BLASTN
search with the GalNAc-4-ST2 and GalNAc-4-ST2(Met-86) sequences against
the High Throughput Genomic Sequences (htgs) data base retrieved
several entries (GenBankTM accession numbers AC012269,
AP001272, AC032032, AC009872, and AC023575) annotated to be derived
from chromosome 18q11.2. The genomic clone with the
GenBankTM accession number AC010854 was retrieved with a
BLASTN search against the nonredundant data base. The organization of
the GalNAc-4-ST2 gene shown in Fig. 3 was deduced by
comparing the cDNA of GalNAc-4-ST2 with these genomic sequences.
Construction of pcDNA3.1-GalNAc-4-ST2(Met-86) and
pcDNA3.1- GalNAc-4-ST2--
The complete open reading frame of
GalNAc-4-ST2 was amplified from cDNA prepared from human kidney 293 cells by polymerase chain reaction (PCR) using the following: 1) the
5'-specific primer 5'-ATA TGG ATC CGC CAC CAT GCA GCC ATC TGA AAT G-3'
containing a BamHI site, the consensus Kozak sequence GCCACC
(29) and a start codon; and 2) the 3'-specific primer 5'-ATA TTC TAG
ACT ACA AAA ATG GAG TTG TAT AAT TAA-3' containing a stop codon and an
XbaI site. The open reading frame of GalNAc-4-ST2(Met-86)
was amplified by PCR from the human I.M.A.G.E. IMAGE clone ID 49547 clone ID 49547 using the 5'-specific primer 5'-GCG GAT CCG CCA CCA TGC
CTG AGG ATG TAC GAG AA-3' (contains an BamHI site, consensus Kozak sequence GCCACC, and a start codon) and the same 3' primer that
was used to obtain GalNAc-4-ST2, above. PCRs were carried out using
PfuTurbo® polymerase (Stratagene) with 35 cycles of a
reaction consisting of 45 s denaturation at 95 °C, 60 s
annealing at 55 °C, and 60 s of elongation at 72 °C. The PCR
fragments had the expected lengths of 1332 and 1077 bp, respectively,
and were directionally subcloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen).
Transient Expression of GalNAc-4-ST2 and
GalNAc-4-ST2(Met-86)--
CHO/Tag cells were transfected with 13 µg
of pcDNA3.1-GalNAc-4-ST2, pcDNA3.1-C4ST-1,
pcDNA3.1-GalNAc-4-ST2(Met-86), or pcDNA3.1 and 35 µg of
LipofectAMINE (Life Technologies, Inc.) in serum-free medium for 6 h per the manufacturer's protocol. Sixty hours after transfection, the
cells and medium were collected separately for analysis. Cells were
lysed with 200 µl of 20 mM HEPES buffer, pH 7.4, 5 mM MgCl2, 175 mM KCl, 2% Triton
X-100, protease inhibitors (23 millitrypsin inhibitor units of
aprotinin and 4 µg each of leupeptin, antipain, pepstatin, and
chymostatin) per 100-mm diameter culture plate. The homogenate was
mixed by rotation for 1 h and sedimented at 12,000 × g for 20 min. The supernatant was designated as the cell
extract. The culture medium was pooled, sedimented at 12,000 × g for 20 min, and the supernatant adjusted to a final concentration of 20 mM HEPES, pH 7.4, and protease
inhibitors were added as noted above.
Sulfotransferase Assays--
GalNAc-4-sulfotransferase
reactions (50 µl) were carried out as described (30) at 28 °C for
15 h. Each reaction contained 15 mM HEPES, pH 7.4, 1%
Triton X-100, 40 mM 2-mercaptoethanol, 10 mM
NaF, 1 mM ATP, 4 mM magnesium acetate, 13%
glycerol, protease inhibitors, 2 µM unlabeled PAPS,
1 × 106 cpm [35S]PAPS, 20 µM GalNAc Product
Characterization--
[35S]SO4-GalNAc
The [35S]SO4-labeled chondroitin and dermatan
products were digested with 30 milliunits of chondroitinase ABC
(Seikagaku America, Inc.) in 100 mM Tris-HCl, pH 8.0, for
16 h at 37 °C. The completeness of digestion was determined by
gel filtration on Sephadex G-25 in 100 mM
NH4HCO3. The released
[35S]SO4-labeled saccharides were
characterized by HPLC using a 4.6 × 250 mm MicroPak AX-5 column
(Varian Associates). Saccharides were separated using a linear gradient
of 10 mM KH2PO4 to 450 mM KH2PO4 over 40 min at 1.0 ml per
min (32). Standards were detected by their absorbance at 210 or 234 nm,
and fractions were collected at 0.5-min intervals for determination of
radioactivity. In excess of 90% of the
[35S]SO4 label was recovered.
Sulfation of Glycoprotein Acceptors--
Glycoproteins were
tested as substrates for GalNAc-4-ST2 using the conditions described
above for oligosaccharide acceptors with the following modifications.
No unlabeled PAPS was added to the [35S]PAPS. Each
50-µl reaction contained 3 µg of purified bovine parotid carbonic
anhydrase VI (CA-VI) (33), bovine LH, enzymatically desulfated bovine
LH, or asialo human chorionic gonadotrophin (hCG). After 16 h at
28 °C, duplicate reactions were either stopped by addition of an
equal volume of sample buffer (10% glycerol, 5% 2-mercaptoethanol,
2% SDS, 0.003% bromphenol blue, and 62.5 mM Tris, pH 6.8)
or were digested with 34 microunits of peptide:N-glycosidase F (PNGase F) as described (3) prior to addition of sample buffer. The
[35S]SO4-labeled proteins were separated by
SDS-polyacrylamide gel electrophoresis (12% acrylamide) and detected
by autoradiography.
Northern Blot and Expression Array Analysis--
Human Multiple
Tissue Northern (MTN®) blots and Human Multiple Tissue Expression
(MTETM) arrays were purchased from
CLONTECH Laboratories, Inc. They were hybridized
with 5-15 × 106 cpm of a GalNAc-4-ST2 specific
Identification of a Human cDNA Related to HNK-1 ST and
GalNAc-4-ST1--
We recently identified and characterized a human
GalNAc-4-O-sulfotransferase, GalNAc-4-ST1 (24), based on its
homology to the HNK-1 ST that transfers sulfate to
GlcUA
Transcripts derived from a human embryonic kidney 293 cell line and
glioma cDNA contained an additional 38-bp sequence, later identified as exon 4 (see below), that was not present in
GalNAc-4-ST2(Met-86). A virtual cDNA consisting of this amplified
sequence and an EST (GenBankTM accession number BE724107)
from the 5' end of the coding region of the bovine orthologue was
deduced and used to analyze genomic clones representing the human
GalNAc-4-ST2 locus (see below). Primer pairs were designed for the
predicted open reading frame and used to amplify the sequence
designated GalNAc-4-ST2 in Fig. 1A (GenBankTM
accession number AF332472) from first strand cDNAs derived from
both 293 cells and a glioma. The open reading frame of GalNAc-4-ST2 is
preceded at its 5' end by a stop codon in all three reading frames and
predicts a protein of 443 amino acids with four potential glycosylation
sites and a molecular mass of 52.1 kDa. GalNAc-4-ST2 is predicted to
have a membrane-spanning domain by the TMHMM algorithm (34) and a type
II topology. If exon 4 is absent due to alternative splicing,
GalNAc-4-ST2(Met-86) would be initiated from the alternative ATG start
codon located at the position of the Met that is equivalent to amino
acid 86 in full-length GalNAc-4-ST2 (Fig. 1).
Multiple alignment of the protein sequence of GalNAc-4-ST2 with other
members of the HNK-1 sulfotransferase family using the ClustalW
algorithm implemented in the BIOEDIT suite (35) (Fig. 2) revealed that GalNAc-4-ST2 is 46%
identical to GalNAc-4-ST1. GalNAc-4-ST2 also has a high percentage of
identical amino acids when compared with other HNK-1 ST-related
sulfotransferases as follows: 23% to human HNK-1 ST, 27% to human
chondroitin 4-O-sulfotransferase-1 (C4ST-1), and 24% to
human chondroitin 4-O-sulfotransferase-2 (C4ST-2) (Fig. 2).
Regions with the highest degree of identity include the putative
5'-phosphosulfonate-binding site (5'-PSB), the putative
3'-phosphate-binding site (3'-PB), and three regions designated III,
IV, and V (23) that do not have an identified function.
Genomic Organization and Chromosome Localization of
GalNAc-4-ST2--
BLAST similarity searches of the htgs and the
nonredundant data set were performed using the GalNAc-4-ST2 cDNA
sequence and retrieved multiple matching sequences (see "Experimental
Procedures"). Among the genomic clones retrieved
GenBankTM accession numbers AP001272 and AP001087 were both
annotated to be mapped on chromosome 18q11.2. Comparisons between the
cDNA and the genomic sequences AC009872, AC023575, and AC010854 showed that the coding region of GalNAc-4-ST2 is distributed over five
exons (Fig. 3A). The sizes of
the untranslated regions of exon 1 and exon 5 have not yet been
determined. As noted above, GalNAc-4-ST2(Met-86) does not contain exon
4. Synthesis of GalNAc-4-ST2(Met-86) is therefore initiated from the
alternative start codon located at amino acid position 86 of
GalNAc-4-ST2 producing a protein that does not have a transmembrane
domain (Fig. 3B). Each of the exons identified, including
exon 4, was found to have conserved donor and acceptor splice sites
(Fig. 3C).
GalNAc-4-ST2 Transfers Sulfate to Terminal
The sulfated product was characterized as described previously (30) to
establish the location of the sulfate. The
[35S]SO4-labeled GalNAc
We also examined GalNAc-4-ST2 for its ability to transfer sulfate to
glycoproteins bearing N-linked oligosaccharides terminating with the sequence GalNAc GalNAc-4-ST2 Transfers Sulfate to Internal
When pcDNA3.1-GalNAc-4-ST2(Met-86) is expressed in CHO/Tag cells,
virtually all of the activity directed at either GalNAc
The location of the sulfate in the
[35S]SO4-chondroitin and in
[35S]SO4-dermatan products was determined by
digestion with chondroitinase ABC and HPLC analysis (Fig.
7). Both chondroitin and dermatan yielded
predominantly the sulfated disaccharide Expression Pattern of GalNAc-4-ST2--
Array and Northern blot
analyses were used to determine the expression pattern for GalNAc-4-ST2
in human tissues (Fig. 8 and Fig.
9, respectively). A strong signal was
obtained for the trachea with the human Multiple Tissue Expression
(MTETM) array system when probing with radiolabeled
GalNAc-4-ST2 cDNA (Fig. 8, 7H). Significantly weaker
signals were detected in the following tissues listed in order of
decreasing intensity of hybridization signal: fetal lung
(G11), adult pancreas (B9), testis
(F8), and salivary gland (E9) stronger than
pituitary gland (D3), apex of the heart (H4), lung
(A8), prostate (E8), and mammary gland
(F9) stronger than heart (A4), liver
(A9), and the spinal cord (E3). Even though the
signals did not reproduce well and could not be quantitated by
densitometry, they are considered specific since the negative controls
showed no visual signal (Fig. 8, 12A-H). In addition, a
specific species of mRNA could be detected by Northern blot
analysis in a number of the tissues that provided a weak signal by
array analysis (Fig. 9). A transcript of ~2.1 kilobase pairs was
detected by Northern blot analysis in heart, liver, and pancreas, with
a significantly lower signal in lung.
We and others recently reported the cloning and characterization
of a GalNAc-4-O-sulfotransferase, GalNAc-4-ST1, based on its
homology to HNK-1 ST (24) and C4ST (25), respectively. GalNAc-4-ST1 is
highly expressed in the pituitary and other regions of the brain.
GalNAc-4-ST1 accounts for the addition of sulfate to terminal
GalNAc-4-ST2 is the fifth member of a family of structurally related
sulfotransferases that thus far include HNK-1 ST, C4ST-1, C4ST-2,
GalNAc-4-ST1, and GalNAc-4-ST2. Since HNK-1 ST was the first member to
be cloned, we and others (24, 25) have used the term HNK-1 ST
family to describe this family. Like all other sulfotransferases the
members of HNK-1 family of sulfotransferases have two motifs that are
hypothesized to mediate binding of the 5'-phosphosulfonate (5'-PSB in
Fig. 2) and 3'-phosphate (3'-PB in Fig. 2) group of the high energy
donor PAPS (3'-phospho-adenosine-5'-phosphosulfonate). Three additional
regions (III, IV, and V in Fig. 2) located C-terminal to the
5'-phosphosulfonate and 3'-phosphate-binding regions also have a high
percentage of identical amino acids, whereas the cytosolic, transmembrane, and stem regions have few identical amino acids. GalNAc-4-ST2 and GalNAc-4-ST1 have the highest percentage of identical amino acids with 46%. In contrast, C4ST-1 and C4ST-2 have 29% identical amino acid residues. The multiple sequence alignment in Fig.
2 indicates that GalNAc-4-ST2 is 23% identical to HNK-1 ST, 27% to
C4ST-1, and 24% to C4ST-2.
The specificities of the members of the HNK-1 sulfotransferase family
are summarized in Table II. HNK-1 ST is
the only family member that transfers sulfate to the C-3 hydroxyl of
terminal 1,4-linked GalNAc
in the sequence GalNAc-
1,4GlcNAc
-R found on N-linked oligosaccharides and nonterminal
1,4-linked GalNAc in chondroitin and dermatan. The translated region of GalNAc-4-ST2 is encoded by five
exons located on human chromosome 18q11.2. Northern blot analysis
reveals a 2.1-kilobase transcript. GalNAc-4-ST2 message is most highly
expressed in trachea and to a lesser extent in heart, liver, pancreas,
salivary gland, and testis. The I.M.A.G.E. cDNA clone 49547 contains a putative GalNAc-4-ST2 splice form with an open reading frame
encoding a protein of 358 amino acids that lacks the transmembrane
domain and the stem region. This form of GalNAc-4-ST2 is not retained
by transfected cells and is active against chondroitin but not terminal
1,4-linked GalNAc. Thus, as with GalNAc-4-ST1, sequences N-terminal
to the catalytic domain contribute to the specificity of GalNAc-4-ST2
toward terminal
1,4-linked GalNAc.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3Gal
1,4GlcNAc-R and is
found at the nonreducing termini of glycoprotein and glycolipid
oligosaccharides (9, 10). The structure was first identified with
monoclonal antibodies as an epitope on human natural killer cells (11).
The HNK-1 epitope is a hallmark of many neural recognition molecules
and displays phylogenetic conservation (12), highlighting its
functional importance. It is expressed in the central and peripheral
nervous systems during development and regeneration (13, 14) and has recently been recognized to be a crucial player in synaptic plasticity involving inhibitory interneurons in the hippocampus (15). HNK-1 is
also a predominant autoantigen in demyelinating diseases of the
peripheral nervous system (16).
1,3-linked GlcUA in the sequence GlcUA
1,3Gal
1,4GlcNAc
-R (17, 18). C4ST-1 and C4ST-2 transfer sulfate to the C-4 hydroxyl of nonterminal
1,4-linked GalNAc in chondroitin and dermatan (22, 23). GalNAc-4-ST1 transfers sulfate to the C-4 hydroxyl of terminal
1,4-linked GalNAc
in the sequence GalNAc-
1,4GlcNAc
-R that is found on
N-linked oligosaccharides (24, 25).
1,4-linked GalNAc in the sequence GalNAc-
1,4GlcNAc
-R and to
the C-4 hydroxyl of internal
1,4-linked GalNAc moieties in
chondroitin and dermatan. We describe the properties of this novel
sulfotransferase that differ in its specificity and expression from
other GalNAc-specific sulfotransferases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP and Megaprime
labeling kit were purchased from Amersham Pharmacia Biotech and human
Multiple Tissue Northern blots (MTN®) and human Multiple Tissue
Expression (MTETM) arrays from CLONTECH
Laboratories, Inc. Bovine LH was provided by the NIDDK National Hormone
and Pituitary Program, Dr. A. F. Parlow.
1,4 GlcNAc
1,2Man
-MCO, and enzyme.
[35S]SO4-GalNAc
1,4 GlcNAc
1,2Man
-MCO
was separated from [35S]PAPS and labeled endogenous
acceptors by passage over a Sep-Pak C18 cartridge (Waters)
(30). Control reactions were done in the absence of substrate or
enzyme. Chondroitin and dermatan GalNAc-4-sulfotransferase activities
were determined using a previously described assay (23) and 50 µg of
desulfated chondroitin (Seikagaku America, Inc.) or desulfated dermatan
(31) as acceptor. HNK-1-sulfotransferase activity was assayed using the
acceptor
GlcUA
1,3Gal
1,4GlcNAc
1,3Gal
1,4Glc
1,C2H4NHCOCF3 as described previously (17) except that the reactions were carried out
for 15 h.
1,4GlcNAc
1,2Man
-MCO
obtained by incubation with GalNAc-4-ST2 or GalNAc-4-ST2(Met-86) for
16 h at 28 °C was isolated on a Sep-Pak C18
(Waters) and characterized as described previously with a CarboPak
PA1 column (Dionex) (30). Sulfated monosaccharides were released from
the
[35S]SO4-GalNAc
1,4GlcNAc
1,2Man
-MCO
product by mild acid hydrolysis and separated from intact
oligosaccharides and other degradation products by gel filtration on
Sephadex G-10 in 100 mM NH4HCO3 prior to analysis by HPLC on CarboPak PA1 column (Dionex) as described (30).
-32P-labeled cDNA probe and washed according to the
manufacturer's specifications. The membranes were exposed to Biomax MS
films (Eastman Kodak Co.) for 2-6 days at
80 °C with intensifying
screens. The 503-bp probe used for the labeling reactions corresponds
to nucleotide 745-1247 of the GalNac-4-ST2 cDNA
(GenBankTM accession number AF332472).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3Gal
1,4GlcNAc-R to produce the HNK-1 epitope
SO4-3-GlcUA
1,3Gal
1,4GlcNAc-R. In contrast to HNK-1
ST, GalNAc-4-ST1 transfers sulfate to the C-4 hydroxyl of
1,4-linked
GalNAc on N-linked oligosaccharides such as those found on
the glycoprotein hormones LH and TSH (24, 25). Further screening of the
EST database at GenBankTM, National Center for
Biotechnology Information, using the deduced amino acid sequence of the
catalytic domain of rat HNK-1 ST (GenBankTM accession
number AF022729) identified the human EST H15485. The insert of the
corresponding I.M.A.G.E. Consortium (LLNL) cDNA clone ID 49547 contains an open reading frame of 1077 bp with a single in-frame ATG
codon at the 5' end preceded by two in-frame stop codons. The open
reading frame predicts a protein of 358 amino acid residues with a
molecular mass of 42.1 kDa and four potential N-linked
glycosylation sites. The predicted protein sequence is closely related
to GalNAc-4-ST1 with 44% identical residues. However, no
membrane-spanning regions are predicted to be present by hidden Markov
model based algorithm TMHMM (34). This suggests the presumptive
sulfotransferase, designated as GalNAc-4-ST2(Met-86)
(GenBankTM accession number AF332473), is synthesized as a
soluble glycoprotein.
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Fig. 1.
Nucleotide and deduced amino acid sequence of
human GalNAc-4-ST2 cDNA (GenBankTM accession
number AF332472). A, the predicted amino acid
sequence of GalNAc-4-ST2 is denoted by capital letters below
the nucleotide sequence. The single membrane-spanning domain predicted
by the hidden Markov model-based algorithm TMHMM (34) and four
potential N-linked glycosylation sites are indicated by the
bold underline and by the underline with a below the
glycosylated Asn, respectively. The truncated form of GalNAc-4-ST2,
GalNAc-4-ST2(Met-86), is initiated at the AUG (double
underline) encoding Met-86 (double underline) of
GalNAc-4-ST2. The nucleotide sequence derived from exon 4 is shown in
lowercase letters. B, shows the hydrophobicity
plot for GalNAc-4-ST2.
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Fig. 2.
Comparison of amino acid sequences of human
GalNAc-4-ST2, GalNAc-4-ST1, C4ST-1, C4ST-2, and HNK-1 ST.
Alignment was performed using the ClustalW program implemented in the
BioEdit suite (35). Introduced gaps are shown as hyphens,
and aligned amino acids are boxed (black for
identical residues and dark gray for similar residues).
Putative binding sites for the 5'-phosphosulfonate group (5'-PSB) and
3'-phosphate group (3'-PB) of PAPS, and three additional highly
conserved domains (III-V) are marked.
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Fig. 3.
Structure of the human
GalNAc-4 ST2 gene. A, exons that
contribute to the 5'- and 3'-untranslated region are boxed.
Exonic sequences that are transcribed into the coding sequence are
boxed and shaded in black. Exons are
numbered from 1 (E1) to 5 (E5). Relevant genomic
clones are denoted with their GenBankTM accession numbers
below the schematic. The splicing pattern for GalNAc-4-ST2
is shown above and for GalNAc-4-ST2(Met-86) below
the schematic. Note that I.M.A.G.E. cDNA clone 49547 only contains
exonic sequences from the position marked a to the position
marked b. B, mRNA and the translated
GalNAc-4-ST2 protein are shown. Nucleotide positions are denoted
below the mRNA scheme. TM denotes the
transmembrane domain. The, AUG in brackets marks the start codon for
GalNAc-4-ST2(Met-86) that would be generated if exon 4 is absent.
C, the intron-exon boundaries are shown for the human
GalNAc-4-ST2 gene. Exon-derived sequences are capitalized. Note that
all boundaries have an invariant intron derived GT and AG at the donor
and acceptor splice sites.
1,4-Linked GalNAc on
N-Linked Oligosaccharides--
Since GalNAc-4-ST2 is closely related
to GalNAc-4-ST1, the specificity of GalNAc-4-ST2 for saccharides
terminating with
1,4-linked GalNAc was examined using the substrates
shown in Table I.
pcDNA3.1-GalNAc-4-ST2 was transfected into CHO/Tag cells, and cell
extracts were prepared for analysis. Like GalNAc-4-ST1,
GalNAc-4-ST2 transfers sulfate to GalNAc
1,4 GlcNAc
1,2Man
-MCO
and to GalNAc
1,4 GlcNAc
-MCO. GalNAc-4-ST2 does not transfer
sulfate to either Gal
1,4GlcNAc
-MCO or GlcNAc
1,2Man
-MCO
(Table I) indicating that the
1,4-linked GalNAc is required and
likely the sugar was modified with sulfate. The oligosaccharide
acceptors in Table I were tested at 20 and 100 µM,
concentrations that are equal to and 5-fold greater than the
Km of 15 µM that we previously
reported for GalNAc
1,4 GlcNAc
1,2Man
-MCO with the
GalNAc-4-sulfotransferase found in the pituitary gland (36). As we have
observed with the GalNAc-4-sulfotransferase from the pituitary
gland (30, 36) and with GalNAc-4-ST1 (24), GalNAc-4-ST2 transfers
sulfate to GalNAc
1,4GlcNAc
1,2Man
-MCO more efficiently than to
GalNAc
1,4GlcNAc
-MCO.
The GalNAc-4-ST2 transfers sulfate to terminal 1,4-linked GalNAc
1,4
GlcNAc
1,2Man
-MCO product comigrated with authentic
SO4-4-GalNAc
1,4 GlcNAc
1,2Man
-MCO when analyzed by
HPLC (Fig. 4A). Following mild
acid hydrolysis, GalNAc-4-SO4 was the only product obtained
(Fig. 4B). Thus, GalNAc-4-ST2 transfers sulfate exclusively
to the C-4 hydroxyl of the terminal GalNAc in GalNAc
1,4
GlcNAc
1,2Man
-MCO.
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Fig. 4.
GalNAc-4-ST2 transfers sulfate exclusively to
C-4 of the 1,4-linked GalNAc.
GalNAc
1,4 GlcNAc
1,2Man
-MCO was incubated with
[35S]PAPS and GalNAc-4-ST2 and the
[35S]SO4-labeled product isolated by passage
over a Sep-Pak C18. A, analysis of the
[35S]SO4-GalNAc
1,4 GlcNAc
1,2Man
-MCO
product on a CarboPak PA1 column. B, analysis of the
[35S]SO4-GalNAc
1,4 GlcNAc
1,2Man
-MCO
product on CarboPak PA1 following mild acid hydrolysis. The elution
positions of standards are indicated by the numbers: 1, GlcNAc-3-SO4; 2, SO4; 3, GalNAc-3-SO4; 4, SO4-3-GalNAc
1,4
GlcNAc
1,2Man
-MCO; 5, GalNAc-4-SO4;
6, SO4-4-GalNAc
1,4
GlcNAc
1,2Man
-MCO; and 7, GalNAc-6-SO4
1,4 GlcNAc
1,2Man
such as are found on
the glycoprotein hormones LH and TSH (37) and carbonic anhydrase VI
(CA-VI) (33). GalNAc-4-ST2 transfers sulfate to bovine CA-VI isolated
from parotid gland and to bovine LH (Fig.
5). The N-linked oligosaccharides on CA-VI isolated from parotid glands bear terminal
1,4-linked GalNAc due to the absence of sulfotransferase in the parotid gland (33). In contrast, >90% of the GalNAc on the
N-linked oligosaccharides of LH is substituted with sulfate
(38-40). Following removal of terminal sulfate moieties from the
N-linked oligosaccharides on LH by digestion with
GalNAc-4-sulfatase, there is a marked increase in the amount of sulfate
transferred to LH. This indicates the sulfate is added to the terminal
1,4-linked GalNAc that has been exposed by the digestion (Fig. 5).
The sulfate label is released from CA-VI and LH by digestion with
PNGase F further confirming its location on N-linked
oligosaccharides. Glycoproteins bearing oligosaccharides with terminal
1,4-linked Gal, such as asialo-hCG, were not modified with sulfate
(not shown). Thus, GalNAc-4-ST2, like GalNAc-4-ST1, is capable of
specifically modifying oligosaccharides on glycoproteins bearing
terminal
1,4-linked GalNAc. We did note that GalNAc-4-ST1 shows a
preference for LH while GalNAc-4-ST2 prefers CA-VI, suggesting the
peptide portion of these glycoproteins may contribute to recognition by
one or the other GalNAc-4-ST.
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Fig. 5.
GalNAc-4-ST2 transfers sulfate to
N-linked oligosaccharides terminating with the
sequence GalNAc 1,4GlcNAc
1,2Man
on glycoproteins.
GalNAc-4-ST2 was incubated with 3 µg each of bovine CA-VI (from
parotid gland), LH, or desulfated LH (treated with GalNAc-4-sulfatase)
and [35S]PAPS. An equal amount of each reaction was
digested with PNGase F to release N-linked oligosaccharides.
No sulfated products were seen in control lanes containing hCG,
asialo-hCG, or media from cells transfected with pcDNA3.1 with no
cDNA (not shown). Lane 1, CA-VI; lane 2, CA-VI + PNGase F; lane 3, LH; lane 4, LH + PNGase
F; lane 5, desulfated LH; and lane 6, desulfated
LH + PNGase F.
1,4-Linked
GalNAc Moieties in Chondroitin and Dermatan--
Even though
GalNAc-4-ST2 displays the highest degree of identity with GalNAc-4-ST1,
it is also homologous to C4ST-1, C4ST-2, and HNK-1 ST. We therefore
examined extracts and medium from CHO/Tag cells transfected with
pcDNA3.1-GalNAc-4-ST2 and pcDNA3.1-GalNAc-4-ST2(Met-86) for
transfer of sulfate to chondroitin, dermatan, and the HNK-1 precursor
GlcUA
1,3Gal
1,4GlcNAc
1,3Gal
1,4Glc
1,C2H4NHCOCF3. No evidence of transfer to
GlcUA
1,3Gal
1,4GlcNAc
1,3Gal
1,4Glc
1,C2H4NHCOCF3 was seen (not shown). Following transfection of
pcDNA3.1-GalNAc-4-ST2 into CHO/Tag cells, 15% of the
sulfotransferase activity directed at GalNAc
1,4
GlcNAc
1,2Man
-MCO was found in the cell extract and 85% in the
medium (Fig. 6). Release of GalNAc-4-ST2
into the medium most likely results from proteolytic cleavage as has
been seen for a number of transferases (41) including GalNAc-4-ST1 (24). Whereas GalNAc-4-ST2 in the cell extract is able to transfer sulfate to chondroitin, there is a 3-fold increase in the relative rate
of transfer of sulfate to chondroitin versus GalNAc
1,4
GlcNAc
1,2Man
-MCO for GalNAc-4-ST2 that has been released into the
medium as compared with GalNAc-4-ST2 retained by the cell (Fig. 6).
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Fig. 6.
GalNAc-4-ST2 transfers sulfate to
GalNAc 1,4GlcNAc
1,2Man
, chondroitin, and dermatan.
CHO/Tag cells were transfected with pcDNA3.1-GalNAc-4-ST2,
pcDNA3.1-GalNAc-4-ST2(Met-86), or pcDNA3.1. After 60 h in
culture cells were harvested and solubilized in 200 µl of 1% Triton
X-100. The medium was collected and concentrated 10-fold to 2.0 ml by
ultrafiltration. Each assay utilized 10 µl of either the cell extract
or concentrated medium. Incorporation is reported as pmol/h/100-mm
plate. Basal levels of incorporation have been subtracted. The
transferase reactions were carried out with 20 µM
GalNAc
1,4GlcNAc
1,2Man
-MCO (gray bar), 50 µg of
chondroitin (striped bar), or 50 µg of dermatan
(dark bar) in a 50-µl reaction containing
[35S]PAPS (1 × 106 cpm/nmol) for
16 h. the products were separated as described under
"Experimental Procedures."
1,4 GlcNAc
1,2Man
-MCO or chondroitin is present in the medium
indicating little or no retention in the Golgi in the absence of the
N-terminal transmembrane domain and stem region. Notably the
GalNAc-4-ST2(Met-86) released into the medium transfers sulfate to
chondroitin while showing little transfer to GalNAc
1,4
GlcNAc
1,2Man
-MCO (Fig. 6). Furthermore, GalNAc-4-ST2 and
GalNAc-4- ST2(Met-86) that have been released into the medium transfer
sulfate to dermatan as well as to chondroitin. Thus the relative rates
of transfer to GalNAc
1,4 GlcNAc
1,2Man
-MCO, chondroitin, and
dermatan differ significantly for GalNAc-4-ST2 in the cell (0.4:0.1:0),
GalNAc-4-ST2 released into the medium (2.3:1.7:1.0), and
GalNAc-4-ST2(Met-86) released into the medium (0.2:4.5:1.0). This
suggests that the transmembrane domain and stem region dictates the
specificity for terminal GalNAc and also has an impact on recognition
of chondroitin versus dermatan.
Di-4S
(D-gluco-4-enepyranoside
1,3GalNAc-4-SO4).The same result was obtained with the product produced by human C4ST-1 expressed in CHO/Tag cells (not shown). Thus GalNAc-4-ST2 transfers sulfate predominantly to the C-4 hydroxyl of internal GalNAc moieties in both chondroitin and dermatan.
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Fig. 7.
GalNAc-4-ST2 transfers sulfate to the
C-4 hydroxyl of internal 1,4-linked GalNAc
residues in chondroitin. Chondroitin (50 µg) (A) and
dermatan (50 µg) (B) were incubated with GalNAc-4-ST2 and
[35S]PAPS. The labeled product was separated from free
label by gel filtration and digested with 30 milliunits of
chondroitinase ABC in 100 mM Tris acetate buffer, pH 8.0, for 6 h at 37 °C. The product was analyzed on a Micropak AX-5
column (Varian) developed with a gradient of 10-450 mM
KH2PO4 over 40 min at a flow rate of 1.0 ml/min. The elution times for for authentic standards are indicated by
the following numbers: 1, GalNAc-4-SO4;
2,
Di6
(D-gluco-4-enepyranoside
1,3 GalNAc-6-SO4);
3,
Di4
(D-gluco-4-enepyranoside
1,3GalNAc-4-SO4);
4,
Di-diSE
(D-gluco-4-enepyranoside
1,3GalNAc-4,6-diSO4);
and 5, free SO4.
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Fig. 8.
RNA dot blot analysis of GalNAc-4-ST2
transcripts. The human Multiple Tissue Expression
(MTETM) array shown was hybridized with a
32P-labeled human GalNAc-4-ST2-specific cDNA probe (see
"Experimental Procedures"). Tissue sources for the RNA are
indicated below the blot. Asterisks indicate the
following: *, paracentral gyrus of cerebral cortex; **, peripheral
blood leukocytes; ***, Burkitt's lymphoma Raji; ****, Burkitt's
lymphoma Daudi; *****, colorectal adenocarcinoma, SW280.
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Fig. 9.
Northern blot analysis of GalNAc-4-ST2
transcripts. Each lane of the MTN® Northern blot
contains 2 µg of poly(A)+ RNA and was hybridized with a
32P-labeled cDNA probe specific for human GalNAc-4-ST2
(see "Experimental Procedures"). Tissues used to prepare the RNA
are indicated above each lane. Migration positions of standards are
indicated at the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-linked GalNAc on the N-linked oligosaccharides of LH
and other pituitary glycoproteins (24). We have now cloned a second,
novel GalNAc-4-O-sulfotransferase, GalNAc-4-ST2, utilizing the same strategy. Differences in the specificity and expression GalNAc-4-ST2 as compared with GalNAc-4-ST1 indicate it has a distinct biologic role in vivo.
1,3-linked glucuronic acid. Each of the other family
members transfers sulfate to the C-4 hydroxyl of
1,4-linked GalNAc.
In the case of C4ST-1 and C4ST-2 the GalNAc is found within the
repeating disaccharide sequences of chondroitin and dermatan,
i.e. it is not located at the nonreducing terminus. In
contrast, GalNAc-4-ST1 and GalNAc-4-ST2 transfer sulfate to GalNAc
moieties located at the nonreducing termini of oligosaccharide
acceptors. Thus, like GalNAc-4-ST1 and GalNAc-4-ST2, HNK-1 ST transfers
sulfate to a terminal
-linked sugar. The specificities of
GalNAc-4-ST1 and GalNAc-4-ST2 are indicative of a structural
relationship with C4ST-1 and C4ST-2. Whereas the native,
membrane-associated form of GalNAc-4-ST1 does not transfer sulfate to
chondroitin, the native, membrane-associated form of GalNAc-4-ST2
transfers sulfate to chondroitin as well as to GalNAc
1,4
GlcNAc
1,2Man
-MCO but at a lower rate. The truncated forms of
GalNAc-4-ST1 and GalNAc-4-ST2, i.e. GalNAc-4-ST1(Met119) and
GalNAc-4-ST2(Met-86), both transfer sulfate to nonterminal
1,4-linked GalNAc residues in chondroitin. GalNAc-4-ST1(Met-119)
retains its ability to transfer sulfate to the terminal GalNAc of
GalNAc
1,4 GlcNAc
1,2Man
-MCO, whereas GalNAc-4-ST2(Met-86) no
longer transfers sulfate to GalNAc
1,4 GlcNAc
1,2Man
-MCO.
Specificities of the HNK-1 ST family members
, or +/
for active, inactive, or weakly active, respectively. The
saccharide that is modified with sulfate is in each case shown in bold
letters. GalNAc-4-ST1 and GalNAc-4-ST2 indicate the native,
membrane-associated, intracellular forms.
Thus, for both GalNAc-4-ST1 and GalNAc-4-ST2 the transmembrane and stem
region have a major impact on the specificity for terminal
versus internal 1,4-linked GalNAc. The presence of the stem and transmembrane domains markedly reduce but do not abolish transfer of sulfate to chondroitin and dermatan for GalNAc-4-ST2, whereas these regions completely abolish sulfate transfer to
chondroitin by GalNAc-4-ST1. The different rates of sulfate transfer to
chondroitin and dermatan seen with GalNAc-4-ST2 proteolytically
released into the medium and truncated GalNAc-4-ST2(Met-86) suggest
that the stem region may also have an impact on the specificity for the
1,4-linked GalNAc and the adjacent uronic acid. However, more detailed analyses will be required to address these issues fully.
The five members of the HNK-1 family of sulfotransferases thus far
defined are localized to five different chromosomes as follows: HNK-1
ST to chromosome 2 (GenBankTM accession number AC012493),
C4ST-1 to chromosome 12q23, C4ST-2 to chromosome 7p22 (23),
GalNAc-4-ST1 to chromosome 19q13.1 (24), and GalNAc-4-ST2 to chromosome
18q11.2. The coding sequence for C4ST-2 (GenBankTM
accession number AC004840) is found within a single exon. In contrast
the coding sequence of GalNAc-4-ST1 is found in 3 exons whereas that
for GalNAc-4-ST2 is found in 5 exons. For both GalNAc-4-ST1 and
GalNAc-4-ST2 the entire catalytic domain including the
5'-phosphosulfonate-binding site, the 3'-phosphate-binding site, and
regions III-V are encoded by a single exon. The additional exons
encode the cytosolic domain, the transmembrane domain, and the majority
of the stem region. It is these regions that contribute to shifting the
specificity away from chondroitin and dermatan to terminal
GalNAc1,4GlcNAc
. Thus, GalNAc-4-ST1 and GalNAc-4-ST2 may have
evolved from C4ST-1 and C4ST-2 by the introduction of these additional
exons that may have markedly altered the properties of the stem region.
The presence of these five homologous sulfotransferases on five
different chromosomes suggests that they diverged quite some time ago.
Even though GalNAc-4-ST1 and GalNAc-4-ST2 are the most closely related
HNK-1 ST family members in terms of their genomic organization and
protein sequence identity, the differences in their enzymatic properties and their patterns of expression indicate they have distinct
functions in vivo. The high level of expression of
GalNAc-4-ST1 in pituitary is consistent with its role in adding sulfate
to the GalNAc1,4GlcNAc
termini found on N-linked
oligosaccharides of LH, TSH, pro-opiomelanocortin, and other hormones
(24). Expression of GalNAc-4-ST1 in other regions of the brain
indicates that the same structures are present on other glycoproteins
produced in these regions. We have, for example, found that
GalNAc-4-ST1 and glycoproteins bearing sulfated N-linked
oligosaccharides are abundant in the
cerebellum.2 In contrast,
Northern blots indicate that GalNAc-4-ST2 is not highly expressed in
brain but is highly expressed in trachea and to lesser extent in heart,
liver, pancreas, salivary gland, testis, and lung. Since
GalNAc-4-ST2 can add sulfate to chondroitin as well as to
GalNAc
1,4GlcNAc
, the actual products produced in these tissues
remain to be established. In addition, it is possible that the
truncated form of GalNAc-4-ST2, GalNAc-4-ST2(Met-86), is expressed in
specific cells and/or tissues. GalNAc-4-ST2(Met-86) is missing
exon 4 (see Fig. 3) and does not contain a transmembrane domain or the
stem region due to initiation from the alternative start codon that
encodes Met-86 in GalNAc-4-ST2. As a result GalNAc-4-ST2(Met-86) is
synthesized and released as a soluble chondroitin- and
dermatan-specific GalNAc-4-sulfotransferase. Whether the truncated form
of GalNAc-4-ST2 is synthesized in vivo remains to be
established; however, GalNAc-4-ST2(Met-86) and GalNAc-4-ST2 would
clearly have different functions.
GalNAc-4-ST1 and GalNAc-4-ST2 appear to have different expression
patterns in vivo; however, we have found that two cell lines known to produce N-linked oligosaccharides terminating with
1,4-linked GalNAc-4-SO4, human kidney 293 cells, and
human SH-SY5Y neuroblastoma cells3 contain messages for
both sulfotransferases. Clearly understanding the relationship of these
two closely related sulfotransferases will be important for assessing
the role of the sulfated saccharides they produce in vivo.
The cloning and characterization of a second GalNAc-4-sulfotransferase,
GalNAc-4-ST2, that is closely related to GalNAc-4-ST1 but clearly has
distinct properties adds further strength to the view that the sulfated
saccharides produced have critical biologic roles.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Westphal (Hamburg University School of Medicine, Germany) for human glioma tissue and Dr. J. J. Hopwood, Women's and Children's Hospital, Adelaide, Australia, for purified recombinant GalNAc-4-sulfatase. We also thank Jeremy Keusch for helpful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01-DK41738 (to J. U. B.), by Deutsche Forschungsgemeinschaft Grant SCHAI85/15-1 (to M. S.), and by a German Academic Exchange Service (DAAD) postdoctoral fellowship (to G. X.).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) AF332472 and AF332473.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Zentrum fuer
Molekulare Neurobiologie, Universitaet Hamburg, Martinistrasse 52, 20246 Hamburg, Germany. Tel.: 49-40-42803-6246; Fax: 49-40-42803-6248; E-mail: melitta.schachner@zmnh.uni-hamburg.de.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M011560200
2 A. Woodworth, Y. L. Mi, and J. U. Baenziger, unpublished observation.
3 J. J. Keusch and J. U. Baenziger, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LH, lutropin;
TSH, thyrotropin;
HNK-1 ST, HNK-1 sulfotransferase;
, C4ST, chondroitin
4-O-sulfotransferase;
GalNAc-4-ST, N-acetylgalactosamine-4-O-sulfotransferase;
MCO, (CH2)8-COOCH3;
bp, base pair;
PAPS, 3'-phospho-adenosine-5'-phosphosulfonate;
GlcNAc-R, R is the
underlying saccharide structure;
ORF, open reading frame;
htgs, high
throughput genomic sequences;
CHO, Chinese hamster ovary;
hCG, human
chorionic gonadotrophin;
PNGase F, peptide:N-glycosidase F;
Di-4S, D-gluco-4-enepyranoside
1,3GalNAc-4-SO4;
HPLC, high pressure liquid chromatography;
PSB, phosphosulfonate-binding site;
PB, phosphate-binding site.
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