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INTRODUCTION |
Keratan sulfate proteoglycan, the most abundant carbohydrate in
the cornea, plays an important role in maintenance of corneal transparency (1, 2). Three acceptor proteins, lumican, keratocan, and
mimecan, have been reported to carry keratan sulfate via
N-linked oligosaccharide in the cornea. The expression of
these carrier proteins is regulated during eye development, suggesting
the importance of keratan sulfate proteoglycans in corneal tissue
(3-8).
Keratan sulfate consists of a linear
poly-N-acetyllactosamine chain that carries sulfate residues
on C-6 of GlcNAc and Gal. Because the sulfation of carbohydrates
affects their biochemical characteristics, such as water solubility and
electrical charge, this modification appears to be important for the
function of keratan sulfate proteoglycans in the cornea. The importance
of keratan sulfate sulfation in the cornea has been also suggested that
lack of sulfation on keratan sulfate is a major cause of the hereditary
eye disorder, macular corneal dystrophy
(MCD)1 (1, 9, 10).
MCD patients show spotted opacity in the cornea, especially in the
extracellular matrix of the stroma. The size of the opaque area
increases progressively, and the patients require keratoplasty. By
genetic linkage analysis, the critical region for MCD has been mapped
to chromosome 16q22 (11-13). Previous reports indicated that the
cornea of MCD patients synthesizes normal levels of
poly-N-acetyllactosamine but does not contain keratan
sulfate, suggesting that the sulfation step of keratan sulfate is
impaired in MCD (9). One of the carbohydrate sulfotransferases, keratan
sulfate Gal-6 sulfotransferase, has shown that the enzyme transfers
sulfate to the Gal residue of poly-N-acetyllactosamine and
keratan sulfate, but the gene encoding this sulfotransferase does not
map to the MCD candidate region (14).
Several carbohydrate sulfotransferases have been identified through
biochemical and functional genomic approaches (14-19). These
sulfotransferases are highly homologous to each other, especially in
the binding domains to the sulfate donor, PAPS (20, 21). We previously
identified a carbohydrate sulfotransferase that maps to the critical
MCD region by EST data base searches, and we designated it corneal
GlcNAc 6-O-sulfotransferase (22). This protein, which is
homologous to other carbohydrate sulfotransferases, is expressed in
corneal cells. Several types of mutations, including deletion and
missense mutations, were found in this gene in genomic DNAs derived
from MCD patients, leading to identification of the causative gene of
MCD. Here, we analyze the enzymatic activity of human corneal GlcNAc
6-O-sulfotransferase (hCGn6ST) using transfected HeLa cells,
and we show that the enzyme transfers sulfate onto the C-6 of GlcNAc in
a synthetic substrate and poly-N-acetyllactosamine. We also
confirm that missense mutations in hCGn6ST found in MCD patients result
in a failure of synthesizing highly sulfated keratan sulfate in the
transfected cells. Moreover, our results indicate that mouse intestine
GlcNAc 6-O-sulfotransferase (mIGn6ST), which has been
identified to be a homologue of human intestine GlcNAc 6-O-sulfotransferase (hIGn6ST) (18), has the same activity
as hCGn6ST, suggesting that mIGn6ST is the mouse orthologue of hCGn6ST and functions to produce keratan sulfate in the mouse cornea.
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EXPERIMENTAL PROCEDURES |
Construction of Sulfotransferase Expression Vectors--
A
nucleotide fragment of the hCGn6ST ORF was obtained from previously
reported hCGn6ST cDNA (22) by PCR using the following primers:
5'-AGCCCTGGACAGTGGCCCCC-3' and 5'-CTCCCGGGCCTAGCGCCT-3'. ORFs of
hKSG6ST and hIGn6ST were also amplified from human genomic DNA by PCR
using the following primers: for hKSG6ST, 5'-TTGGAGAAGCAGCCCCATGAAG-3' and 5'-CACCGCCCGGGTCACGAG-3'; and for hIGn6ST,
5'-TGGGAAGTCCAATGGGTAGGGT-3' and 5'-CCCCAGCTCCCTCTCCACCA-3'. Obtained
amplicons were cloned into pcDNA3 (Invitrogen, Carlsbad, CA)
by the TA cloning method, resulting in pcDNA3-hCGn6ST,
pcDNA3-hKSG6ST, and pcDNA3-hIGn6ST.
An EST containing mIGn6ST cDNA was purchased from Genome Systems
(St. Louis, MO). Based on the EST sequence, full-length cDNA was
amplified by the rapid amplification of cDNA ends technique using
mouse brain Marathon-ready cDNA (CLONTECH, Palo
Alto, CA). The obtained amplicon, which encodes a peptide sequence
identical to mIGn6ST (18), was inserted into pcDNA3, resulting in
pcDNA3-mIGn6ST.
An expression vector encoding soluble hCGn6ST was prepared as follows.
A DNA fragment coding catalytic domain of hCGn6ST was amplified from
pcDNA-hCGn6ST vector by PCR using the following primers:
5'-GGTAGATCTGCCAGGGCCCTCGTCCCCA-3' and 5'-GATTTAGGTGACACTATAG-3' (SP6 primer, Invitrogen). After digestion with BglII
and XbaI (New England Biolabs, Beverly, MA), this amplicon
was inserted into the BamHI-XbaI sites of
pcDNAHSH, which encodes the signal sequence of human
colony-stimulating factor and multicloning site of EpiTagTM
pcDNA3.1/His B (Invitrogen) in pcDNA3.1/Hygro vector. The
resultant expression vector, pcDNAHSH-hCGn6ST, encodes a cleavable
signal sequence (23) following polyhistidine, enterokinase cleavage site, and catalytic domain of hCGn6ST.
An expression vector encoding R50C mutant hCGn6ST was prepared as
follows. A DNA fragment of hCGn6ST, which contains R50C mutation, was
amplified from an MCD patient (22) by PCR using the following primers:
5'-AGACCTTCCTCCTCCTCTTTCTGGTT-3' and 5'-GCGCACCACGCGCAGGC-3'. After
digestion with ApaI and SfiI (New England
Biolabs), this amplicon was inserted to the pcDNA3-hCGn6ST that was
digested with ApaI and SfiI. Five expression
vectors, each of which encoded a hCGn6ST mutant, K174R, D203E, R211W,
A217T, and E274K, were prepared with the same method described above
except PCR primers (5'-GACGTGTTTGATGCCTATCTGCCTTG-3' and
5'-CGGCGCGCACCAGGTCCA-3') and the restriction enzymes for replacement
(SfiI and XhoI).
In Vitro Analysis of Sulfotransferase Activity--
An
expression vector for intact hCGn6ST was transfected into HeLa cells
using LipofectAMINE PLUS reagent (Life Technologies, Inc.). After
incubation for 48 h in Dulbecco's modified Eagle's medium
containing 10% of fetal bovine serum (DMEM, 10% FBS), the cells were
washed with PBS, scraped, and collected into a 1.5-ml microcentrifuge tube. The cells were again washed with PBS and suspended into cell lysis buffer (20 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 0.5% Triton
X-100, and 1 mM phenylmethylsulfonyl fluoride). After
incubation on ice for 30 min, the sample was centrifuged at 9,000 × g for 10 min at 4 °C, and the supernatant was used as
an enzyme fraction.
Soluble hCGn6ST was prepared as follows. The expression vector
pcDNAHSH-hCGn6ST was transfected into HeLa cells as described above. Following 24 h of incubation in DMEM, 10% FBS, the medium was replaced with Opti-MEM (Life Technologies, Inc.). After incubation for 24 h, the medium was collected and concentrated by Microcon YM-30 (Millipore Corp., Bedford, MA) and used as an enzyme fraction.
Protein concentration of each enzyme fraction was determined by BCA
protein assay kit (Pierce) using bovine serum albumin as a standard.
The condition for sulfotransferase reaction was as described previously
(24). One µg of protein from enzyme fraction was incubated with 15 µl of reaction mixture containing 50 mM imidazole HCl, pH
6.8, 10 mM MnCl2, 2 mM 5'-AMP, 20 mM NaF, 50 nCi of [35S]PAPS (PerkinElmer Life
Sciences) and 0.5 mM substrate
(GlcNAc
1-6Man
1-6Man
-octyl) (25) at 27 °C for 40 (intact
hCGn6ST) or 1 h (soluble hCGn6ST). After adding 1 ml of water to
stop the reaction, the product was purified by High Load C18 column
(Alltech Associates, Deerfield, IL) (26). A portion of the purified
product was subjected to scintillation counting, and the remainder was
lyophilized and used for further analysis.
Preparation of 35S-Labeled Carbohydrate from
Transfected Cells--
Expression vectors encoding intact hCGn6ST
and/or hKSG6ST were transfected into HeLa cells as described above.
Following 24 h of incubation in DMEM, 10% FBS, the medium was
replaced with S-MEM (Life Technologies, Inc.) containing 10%
dialyzed fetal bovine serum and [35S]sodium sulfate
(PerkinElmer Life Sciences) at a concentration of 100 µCi/ml. After a
24-h incubation, cells were washed with PBS, scraped, and collected
into a 1.5-ml microcentrifuge tube. The cells were washed again
with PBS and extracted with 500 µl of chloroform/methanol (2:1). The
cell pellets were washed with 200 µl of methanol and digested in 200 µl of 0.1 M Tris-HCl, pH 8.0, 1 mM
CaCl2 with 20 µl of 1 mg/ml Pronase (Calbiochem). After an overnight incubation at 37 °C, 20 µl of freshly prepared
Pronase (1 mg/ml) were added and incubated overnight at 37 °C. The
digested mixture was boiled for 5 min to stop the reaction. After
phenol and chloroform extraction, the sample in the aqueous phase was subjected to Sephadex G-50 column chromatography (1 × 45 cm,
equilibrated with 0.1 M NH4HCO3).
The carbohydrate fraction that eluted in the void volume was collected,
desalted by Sephadex G-25 gel filtration (1 × 30 cm, equilibrated
with 7% 1-propanol/water), and lyophilized. The sample was dissolved
in 150 µl of water and used for further analyses.
Enzymatic and Chemical Cleavages of Carbohydrates--
Purified
oligosaccharides (each 5000 cpm) produced by in vitro
sulfotransferase reaction were digested with 10 milliunits of
-N-acetylglucosaminidase A from human placenta (Sigma),
which cleaves and release both GlcNAc and GlcNAc(6S) from
non-reducing terminal of carbohydrate, in 20 µl of 25 mM
sodium citrate buffer, pH 3.5 and 100 mM galactose for
overnight at 37 °C. The digested samples were boiled for 5 min and
were analyzed by HPLC.
Metabolically labeled carbohydrate samples (each 5 × 106 cpm) from transfected HeLa cells were digested with 250 milliunits of keratanase from Pseudomonas sp. (Calbiochem)
in 90 µl of 50 mM Tris-HCl, pH 7.4. After overnight
incubation at 37 °C, the samples were boiled for 5 min to stop the
reaction and applied to a column (1 × 45 cm) of Sephadex G-50
equilibrated with 0.1 M NH4HCO3.
Each 400 µl of fraction was collected in a tube. Ten µl of each
fraction were used to count 35S radioactivity, and the
remainder of the fraction was desalted and lyophilized.
To remove sialic acid, 0.1 volume of 0.1 N HCl was added to
the sample and incubated at 90 °C for 1 h. The reaction was
stopped by cooling on ice and addition of 0.1 volume of 2 M
NH4HCO3. The sample was desalted and
lyophilized. After dissolving in water, the sample was subjected to
column chromatography.
Exo-
-galactosidase treatment was carried out as follows. The
desialylated sample was digested with 0.375 units of jack bean
-galactosidase (Seikagaku Co., Tokyo, Japan) in 0.1 M
sodium phosphate buffer, pH 4.0, overnight at 37 °C. After boiling
for 5 min, the sample was desalted, lyophilized, and analyzed by column chromatography.
Column Chromatography--
A Sephadex G-50 column (1 × 45 cm) was used for gel filtration chromatography. The column was
equilibrated and eluted with 0.1 M
NH4HCO3. Fractions of 300 µl were collected,
and 35S radioactivity was determined by scintillation counting.
A Whatman Partisil SAX-10 column (4.6 mm × 25 cm) was used for
HPLC analysis. This column was equilibrated with 5 mM
KH2PO4. The elution conditions were as
described previously (14, 27). In brief, the column was eluted with 5 mM KH2PO4 isocratically for the
samples produced by
-N-acetylglucosaminidase A treatment. For the samples produced by keratanase treatment, the column was eluted
with 5 mM KH2PO4 for 5 min followed
by a 20-min gradient from 5 to 250 mM
KH2PO4. The flow rate was 1 min/ml. Fractions of 0.5 min were collected, and 35S radioactivity was
determined by scintillation counting.
Western Blot Analysis--
Transfected cells were transferred to
a 1.5-ml tube by scraping and washed in PBS. The cells were suspended
into 800 µl of TKMS buffer (20 mM Tris-HCl, pH 7.6, 25 mM KCl, 2.5 mM MgCl2, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride)
and lysed by 5 times freeze/thaw cycles. After centrifugation at
9,000 × g for 10 min at 4 °C, the precipitate was
washed with TKMS buffer and suspended in 200 µl of TKMS buffer
containing 1% Triton X-100. After 10 min of incubation on ice, the
sample was centrifuged at 9,000 × g for 10 min at
4 °C, and the supernatant was collected as a membrane fraction.
Proteins in the membrane fraction were precipitated by cold acetone and
dissolved in 1% SDS. Fifty µg of membrane proteins from each
transfectant were separated by SDS-polyacrylamide gel electrophoresis
and blotted onto a nitrocellulose membrane by electroblotting. The
blotted filter was soaked in 10% skim milk in PBST5.3 (0.05% Tween 20 in PBS, pH 5.3) overnight at 4 °C. The filter was washed once with
PBST5.3 and incubated with PBST5.3 containing 1% bovine serum albumin
and diluted monoclonal antibody 5D4 (Seikagaku Co.) for 1 h at
room temperature. After washing with PBST5.3 three times, the filter
was incubated with PBST5.3 containing 1% bovine serum albumin and
diluted horseradish peroxidase-conjugated goat (anti-mouse IgG)
antibody for 1 h at room temperature. After washing with PBST5.3
three times, peroxidase activity was detected by an ECL Plus kit
(Amersham Pharmacia Biotech).
Immunocytochemistry by Anti-keratan Sulfate
Antibody--
Transfected cells were fixed in cold methanol at
20 °C for 30 min and treated with 0.3%
H2O2 in methanol overnight at 4 °C to
destroy endogenous peroxidase activities. After washing with PBS5.3
(PBS, pH 5.3), the cells were incubated in 10% goat serum in PBS5.3
for 45 min at room temperature. The cells were then incubated with
diluted 5D4 monoclonal antibody in PBS5.3 containing 10% goat serum
for 45 min at room temperature. After washing with PBS5.3 three times,
the cells were incubated with horseradish peroxidase-conjugated goat
(anti-mouse IgG) antibody in PBS5.3 for 30 min at room temperature. The
cells were washed three times with PBS5.3 and reacted with
aminoethylcarbazole (Zymed Laboratories Inc.
Laboratories, South San Francisco, CA) for detection of peroxidase activity.
In Situ Hybridization and Immunohistochemistry--
A specific
sequence of mIGn6ST mRNA was amplified by PCR using the following
primers: 5'-CTCAGCGACCCTGCGCTCAAC-3' and
5'-CGCACATGGCTGCGGCATAC-3'. The amplified fragment was cloned
into the SmaI site of pGEM3Zf(+) (Promega, Madison, WI) and
used to prepare RNA probes with a DIG RNA Labeling Kit (Roche Molecular
Biochemicals). In situ hybridization was performed as
described previously (28). Immunohistochemical detection of keratan
sulfate was performed using the monoclonal antibody 5D4 by the indirect
method as described (29).
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RESULTS |
Enzymatic Activity of hCGn6ST--
hCGn6ST has high homology to
human and mouse intestinal GlcNAc 6-O-sulfotransferases and
belongs to the carbohydrate sulfotransferase family (22) (Fig.
1). All sulfotransferases in this family
have an activity that transfers sulfate to C-6 of GlcNAc, Gal, or
GalNAc. Furthermore, mutations in the gene encoding hCGn6ST cause MCD, a hereditary eye disease in which patients lack sulfated keratan sulfate in the cornea and serum (22). Therefore, we hypothesized that
hCGn6ST is a sulfotransferase that catalyzes sulfation of the C-6 of
GlcNAc in keratan sulfate. To examine the enzymatic activity of
hCGn6ST, HeLa cells were transfected with expression vectors harboring
cDNAs encoding intact or soluble hCGn6ST, and enzyme fractions were
prepared for analyzing of sulfotransferase activity. In
vitro analysis showed that the enzyme fraction from intact hCGn6ST
cDNA transfectant has sulfotransferase activity that transfers
sulfate from PAPS to a substrate, GlcNAc
1-6Man
1-6Man
-octyl (Fig. 2A).
-N-Acetylglucosaminidase A treatment revealed that the
35S-labeled oligosaccharides produced by in
vitro sulfotransferase reaction have sulfate on C-6 of GlcNAc
(Fig. 2B). This enzyme fraction also had sulfotransferase
activity for a synthetic substrate, GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-6Man
1-6Man
-octyl, in vitro (data not shown). Since the enzyme fraction
prepared from HeLa cells transfected with the pcDNA3 vector alone
has no sulfotransferase activity, and the secreted soluble hCGn6ST
fraction collected from the culture medium of transfectant cells with
pcDNAHSH-hCGn6ST also has the same activity as the intact hCGn6ST
fraction (Fig. 2), we concluded that hCGn6ST has sulfotransferase
activity that transfers sulfate to C-6 of GlcNAc.

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Fig. 1.
Relationships of carbohydrate
sulfotransferases. A, the amino acid sequences of human
and mouse sulfotransferases were compared using the ClustalW method,
and the results are shown here as a phylogenic tree. The
following sequences were compared: hCGn6ST, human corneal GlcNAc
6-O-sulfotransferase (22); hIGn6ST, human intestinal GlcNAc
6-O-sulfotransferase (18); mIGn6ST, mouse intestinal GlcNAc
6-O-sulfotransferase (18); hLSST/HECGn6ST, human
L-selectin ligand sulfotransferase or high
endothelial cell GlcNAc 6-O-sulfotransferase (17, 36);
mLSST/HECGn6ST, mouse L-selectin ligand sulfotransferase or high
endothelial cell GlcNAc 6-O-sulfotransferase (36);
hCh6ST, human chondroitin 6-O-sulfotransferase (43,
44); mCh6ST, mouse chondroitin 6-O-sulfotransferase (45);
hKSG6ST, human keratan sulfate Gal
6-O-sulfotransferase (14); hGlcNAc6ST-1, human GlcNAc
6-O-sulfotransferase-1 (27, 35); mGlcNAc6ST-1, mouse GlcNAc
6-O-sulfotransferase-1 (16, 27); and hGlcNAc6ST-4/Ch6ST-2,
human GlcNAc 6-O-sulfotransferase-4 or
chondroitin 6-O-sulfotransferase-2 (19, 27). B,
aligned amino acid sequences of hCGn6ST, hIGn6ST, and mIGn6ST.
Conserved amino acids are indicated by a black
background. Positions of missense mutation found on
CHST6 in MCD patients are marked by
asterisks.
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Fig. 2.
In vitro analysis of
sulfotransferase activity of hCGn6ST. A, cell lysate
from pcDNA3 or pcDNA3-hCGn6ST transfected cells, or concentrated
culture medium from pcDNAHSH or pcDNAHSH-hCGn6ST
transfected cells was used as an enzyme source. These enzymes were
incubated with [35S]PAPS and a synthetic
carbohydrate substrate, GlcNAc 1-6Man 1-6Man -octyl, and
incorporation of 35S radioactivity to the substrate was
counted. B, radiolabeled substrates produced by in
vitro sulfotransferase reaction with an enzyme fraction from
pcDNA3-hCGn6ST transfectant (circles) or from
pcDNAHSH-hCGn6ST transfectant (triangles) were digested with
-N-acetylglucosaminidase A and were subjected to SAX-10
HPLC. Arrows indicate the elution positions of
standards: 3S, GlcNAc(3S); 6S,
GlcNAc(6S).
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We next analyzed the structure of sulfated carbohydrates produced by
transfectant cells with sulfotransferases. HeLa cells were transfected
with expression vectors harboring cDNAs encoding hCGn6ST and a
sulfotransferase, hKSG6ST, that transfers sulfate onto C-6 of Gal in
keratan sulfate (14). The transfected cells were metabolically labeled
with [35S]sulfate, and carbohydrates isolated from those
cells were analyzed by keratanase treatment. Keratanase recognizes the
disaccharide repeat of keratan sulfate that consists of unsulfated Gal
connected to sulfated GlcNAc and cleaves the Gal
1-4GlcNAc(6S)
linkage (30). A carbohydrate with the same backbone as keratan sulfate
but with sulfate on C-6 of Gal or no sulfate on GlcNAc cannot be
digested by keratanase. Carbohydrates isolated from HeLa cells
transfected with the pcDNA3 vector alone or with pcDNA3-hKSG6ST
were resistant to keratanase treatment (Fig.
3). This finding is consistent with the
substrate specificity of keratanase and indicates that HeLa cells
express no endogenous sulfotransferases that produce keratan sulfate.
On the other hand, keratanase treatment of samples from HeLa cells
transfected with pcDNA3-hCGn6ST produced fragments that eluted in
fractions 57-72 from a Sephadex G-50 column (Fig. 3). Carbohydrates
from the hCGn6ST-expressing cells digested with keratanase were
separated into two populations, fractions 63-66 and 68-72, by
Sephadex G-50 chromatography. Keratanase-sensitive carbohydrates were
also found in cells co-transfected with pcDNA3-hCGn6ST and
pcDNA3-hKSG6ST. However, the resultant carbohydrate fragments by
keratanase digestion were seen as one broad peak, in contrast to the
two peaks observed in hCGn6ST-expressing cells following the same
chromatography (Fig. 3). These results suggest that hCGn6ST has
activity that transfers sulfate to C-6 of GlcNAc on
poly-N-acetyllactosamine chains resulting in production of
keratan sulfate. The data also suggest that co-expression of hCGn6ST
and hKSG6ST in HeLa cells produces highly sulfated keratan sulfate with
sulfate residues not only on GlcNAc but also on C-6 of Gal, making it
less sensitive to keratanase.

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Fig. 3.
Sephadex G-50 gel filtration chromatography
of keratanase digests from transfected HeLa cells. Cells were
transfected with the pcDNA3 expression vector with no insert
(circles), pcDNA3-hCGn6ST (triangles),
pcDNA3-hKSG6ST (squares), or co-transfected with
pcDNA3-hCGn6ST and pcDNA3-hKSG6ST (diamonds).
Metabolically 35S-labeled carbohydrate from each
transfectant was digested with keratanase and analyzed by Sephadex G-50
column chromatography. Regions designated by horizontal bars
(I and II) were collected for further
analyses.
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To confirm that hCGn6ST produces keratan sulfate, we analyzed the
carbohydrate structure of keratanase-digested fragments obtained from
cells transfected with pcDNA3-hCGn6ST. Two carbohydrate fractions
(pools I and II in Fig. 3) were analyzed by
Sephadex G-50 gel filtration and SAX-10 anion exchange HPLC. By HPLC
analysis, the carbohydrate in pool I was eluted at the 12.5-min
retention position (Fig. 4A).
This retention time was identical to a carbohydrate standard,
GlcNAc(6S)
1-3Gal, that was prepared from bovine corneal keratan
sulfate by keratanase treatment (30). In contrast, carbohydrate in pool
II did not elute at the position of known standards. We further
analyzed its carbohydrate structure by mild acid and
exo-
-galactosidase treatment. The carbohydrate in pool II was
cleaved by mild acid treatment that releases sialic acid from
carbohydrate chains (Fig. 5B).
The de-sialylated carbohydrate was further digested by
exo-
-galactosidase (Fig. 5C). This product, which was
derived from pool II by de-sialylation and de-galactosylation, eluted
at the 12.5-min retention position that was identical to the
carbohydrate in pool I and the standard monosulfated disaccharide by
SAX-10 HPLC (Fig. 4C). From these results, we conclude
that the carbohydrates in pool I and II shown in Fig. 3 originated from
sulfated poly-N-acetyllactosamine chains, which have sulfate
residue on C-6 of GlcNAc, with a sialylgalactose on their non-reducing
terminal. These findings indicate that hCGn6ST has sulfotransferase
activity that transfers sulfate to C-6 of GlcNAc in
poly-N-acetyllactosamine and results in production of keratan sulfate.

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Fig. 4.
SAX-10 HPLC of oligosaccharides I and
II. Carbohydrate fractions in pools I and II (Fig. 3) were
analyzed by SAX-10 HPLC column chromatography. Each panel shows the
elution pattern of carbohydrate from pool I (A), pool II
(B), and de-sialylated and exo- -galactosidase-treated
carbohydrate from pool II (C). Arrows indicate
the elution positions of standards: a, GlcNAc(6S) 1-3Gal;
b, GlcNAc(6S) 1-3Gal(6S) 1-4GlcNAc(6S) 1-3Gal;
c, 35SO . The
dotted lines indicate the KH2PO4
elution gradient.
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Fig. 5.
Sephadex G-50 column chromatography of
oligosaccharides I and II. Carbohydrates from pools I and II were
analyzed by Sephadex G-50 gel filtration. A shows the
chromatogram of pool I (closed circles) and pool II
(open circles). Carbohydrate in pool II was de-sialylated by
mild acid treatment (B), further digested with
exo- -galactosidase (C), and analyzed by Sephadex G-50
chromatography.
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Co-expression of hCGn6ST and hKSG6ST Produces Highly Sulfated
Keratan Sulfate--
hKSG6ST transfers sulfate to the C-6 of Gal in
keratan sulfate (14), and HeLa cells expressing both hCGn6ST and
hKSG6ST produced sulfated carbohydrate less sensitive to keratanase
treatment than carbohydrate produced in cells expressing hCGn6ST alone
(Fig. 3). Therefore, we assumed that co-expression of both hKSG6ST and hCGn6ST results in highly sulfated keratan sulfate. To confirm this
hypothesis, we compared the reactivity of carbohydrates from transfected cells to the mouse monoclonal antibody, 5D4, that detects
keratan sulfate in a variety of tissues (29, 31). The minimum epitope
recognized by this antibody is a linear pentasulfated hexasaccharide
(32). A longer epitope reacts with this antibody more effectively than
the shorter one (32), and 5D4 does not recognize desulfated keratan
sulfate (33, 34). First, we performed Western blot analysis against
proteoglycans prepared from HeLa cells transfected with the expression
vectors. The SDS-PAGE pattern of proteins stained by Coomassie Blue
showed no differences among these transfected HeLa cells (Fig.
6A, lanes a-d). However, the 5D4 antibody detected proteoglycans extracted only from hCGn6ST/hKSG6ST co-expressing cells (Fig. 6A, lane h). Furthermore,
immunostaining of transfected HeLa cells by the 5D4 antibody showed
positive signals only on pcDNA3-hCGn6ST/pcDNA3-hKSG6ST
co-transfectants (Fig. 6B, d) but not on the HeLa cells
transfected with only one expression vector (Fig. 6B, b and
c). These results clearly indicate that co-expression of hCGn6ST
and hKSG6ST efficiently produces highly sulfated keratan sulfate in
cells and suggests, since the two enzymes are expressed in the cornea
(14, 22), that both are involved in the corneal keratan sulfate
production.

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Fig. 6.
Western blot analysis and immunocytochemistry
of transfected HeLa cells with anti-keratan sulfate antibody.
Keratan sulfate production in transfected HeLa cells was analyzed by
Western blotting (A) and immunostaining (B).
A, HeLa cells were transfected with pcDNA3 (lanes
a and e), pcDNA3-hCGn6ST (lanes b and
f), pcDNA3-hKSG6ST (lanes c and
g), and both pcDNA3-hCGn6ST and pcDNA3-hKSG6ST
vectors (lanes d and h). Membrane protein
fractions were prepared from those transfectants and separated by
SDS-PAGE followed by Coomassie Blue staining (lanes a-d) or
Western blotting using an anti-keratan sulfate antibody, 5D4
(lanes e-h). B, transfected cells were analyzed
by immunocytochemistry using the 5D4 antibody. Each panel shows
HeLa cells transfected with pcDNA3 (lane a),
pcDNA3-hCGn6ST (lane b), pcDNA3-hKSG6ST (lane
c), and both pcDNA3-hCGn6ST, and pcDNA3-hKSG6ST
(lane d). Bar in lane a is 20 µm.
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Missense Mutations Found in MCD Abolish hCGn6ST Activity--
In a
previous report (22), we found missense mutations in the ORF of
CHST6, which encodes hCGn6ST protein, in the genome of MCD
patients. All of those reported mutations, plus a newly identified one
(A217T, data not shown), were located in regions encoding conserved
residues among carbohydrate sulfotransferases reported to date (Fig.
1B). Such regions are likely to affect enzymatic activity.
By using immunostaining, we analyzed the activity of mutant hCGn6STs
for processing keratan sulfate. Mutant cDNA sequences were
amplified by PCR from the genomic DNAs of the patients and were cloned
into the pcDNA3 expression vector. Each mutant hCGn6ST cDNA was
co-transfected with hKSG6ST cDNA into HeLa cells. Immunostaining
with the anti-keratan sulfate antibody gave positive signals when wild
type hCGn6ST and hKSG6ST were co-expressed but not when mutant forms of
hCGn6ST plus hKSG6ST were co-expressed (Fig.
7). These indicate that missense
mutations of hCGn6ST found in MCD patients result in a failure of
synthesizing highly sulfated keratan sulfate and suggest that lack of
sulfation on GlcNAc in keratan sulfate leads to the MCD phenotype.

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Fig. 7.
Immunocytochemistry of HeLa cells transfected
with mutant hCGn6STs. A vector expressing wild type (a)
or missense mutants of hCGn6ST (b, R50C; c,
K174R; d, D203E; e, R211W; f, A217T;
and g, E274K) was transfected together with
pcDNA3-hKSG6ST into HeLa cells. After transfection, cells were
stained with the 5D4 antibody. Bar in a is 20 µm.
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mIGn6ST Produces Keratan Sulfate in the Mouse Cornea--
Since
hCGn6ST is highly homologous to hIGn6ST and mIGn6ST (Fig.
1B), we tested whether both hIGn6ST and mIGn6ST have the
same activity as hCGn6ST. To do so we transfected expressible cDNAs encoding these proteins plus and minus hKSG6ST into HeLa cells, and we
examined keratan sulfate production by 5D4 antibody staining (Fig.
8). Unexpectedly,
pcDNA3-mIGn6ST/pcDNA3-hKSG6ST co-transfectants produced keratan
sulfate (Fig. 8h), but cells expressing both hIGn6ST and
hKSG6ST showed no 5D4-positive staining (Fig. 8g). Since
HeLa cells transfected with pcDNA3-mIGn6ST alone produced no
reactivity to the 5D4 antibody (Fig. 8d), we concluded that mIGn6ST transfers sulfate onto C-6 of GlcNAc in the
poly-N-acetyllactosamine chain, which is identical to the
activity of hCGn6ST and results in highly sulfated keratan sulfate
production in cooperation with hKSG6ST. Although hIGn6ST is highly
homologous to hCGn6ST in amino acid sequence, hIGn6ST lacks activity
necessary for keratan sulfate production in transfected HeLa cells. The
substrate specificity of hIGn6ST is unknown.

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Fig. 8.
Immunocytochemistry of HeLa cells transfected
with human and mouse IGn6STs. The expression vector pcDNA3
(a and e), pcDNA3-hCGn6ST (b and
f), pcDNA3-hIGn6ST (c and g), and
pcDNA3-mIGn6ST (d and h) were transfected
together with (e-h) or without (a-d)
pcDNA3-hKSG6ST into HeLa cells. Transfectants were stained with the
anti-keratan sulfated antibody 5D4. Bar in a is
20 µm.
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We also analyzed the distribution of keratan sulfate and mIGn6ST
mRNA in the mouse cornea. Immunohistochemistry using the 5D4
antibody showed positive staining in mouse corneal tissues (Fig.
9a), indicative of the
presence of keratan sulfate. In situ hybridization signals
were also detected mainly in corneal epithelial cells and in stromal
and endothelial cells (Fig. 9c). This observation is similar
to those obtained for hCGn6ST in the human cornea (22), suggesting that
the mIGn6ST plays the same role as hCGn6ST does, and is involved in the
production of keratan sulfate in the mouse cornea.

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Fig. 9.
Immunohistochemistry and in situ
hybridization of keratan sulfate and mIGn6ST mRNA in the
mouse cornea. Semi-serial sections of corneal tissues were
sequentially analyzed by immunohistochemistry (a and
b) and in situ hybridization (c and
d). The clefts in the stroma are artifacts produced during
tissue processing. Specimens were stained with (a) or
without (b) the 5D4 antibody for immunohistochemistry and
were stained with mIGn6ST antisense (c), or a sense
(d) probe for in situ hybridization.
ep, epithelium; st, stroma; en,
endothelium. Bar in d is 50 µ m.
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DISCUSSION |
In this report, we demonstrate that hCGn6ST transfers sulfate onto
C-6 of GlcNAc in poly-N-acetyllactosamine chain catalyzing the synthesis of keratan sulfate. So far, eight GlcNAc
6-O-sulfotransferases have been cloned and characterized
(16-18, 22, 27, 35, 36). Prior to the molecular characterizations of
these sulfotransferases, biochemical analyses suggested that a GlcNAc
6-O-sulfotransferase adds sulfate only to the non-reducing
terminal GlcNAc of carbohydrate chains (37). Uchimura et al.
(16, 27) reported that both GlcNAc 6-O-sulfotransferase-1
and -4 (also named chondroitin 6-O-sulfotransferase-2) transfer sulfate to the non-reducing terminal but not to internal GlcNAc. Therefore, it is possible that hCGn6ST also transfers sulfate
onto C-6 of non-reducing terminal GlcNAc during keratan sulfate chain
synthesis. Previous reports suggested that sulfation of GlcNAc residues
in keratan sulfate is coupled to the elongation step of carbohydrate
chain synthesis (38-40), supporting our hypothesis.
Another keratan sulfate sulfotransferase, hKSG6ST, transfers sulfate
onto the C-6 of Gal in keratan sulfate. Since hKSG6ST preferentially
adds sulfate to a Gal residue adjacent to the sulfated GlcNAc in
vitro (14), and reduction in GlcNAc
6-O-sulfotransferase activity is the major cause of
decreases in keratan sulfate production in cultured chick corneal
stromal cell (41), GlcNAc 6-O-sulfotransferase is thought to
be a critical enzyme in keratan sulfate biosynthesis. Indeed, we found
mutations in CHST6, the gene encoding hCGn6ST, in MCD
patients who produce no keratan sulfate in their cornea and serum (22).
It is therefore likely that the sulfation of GlcNAc in keratan sulfate
takes place during the elongation of the
poly-N-acetyllactosamine chains. The sulfation of GlcNAc may be required for the sulfation of Gal by hKSG6ST.
By structural analysis, we found that HeLa cells expressing hCGn6ST
produced sulfated poly-N-acetyllactosamine that has sulfate on GlcNAc and a sialylgalactose on its non-reducing terminal. Huckerby
et al. (42) found a keratan sulfate structure in which the
non-reducing terminal is capped by sialic acid in bovine cartilage. This is identical structure to that we found. Oeben et al.
(40), however, reported a biantennary complex type structure that has a
sialylgalactosyl N-acetyllactosamine and sulfated
poly-N-acetyllactosamine without sialylation on its
non-reducing terminal in pig corneal keratan sulfate. This discrepancy
may be due to tissue differences because corneal cells produce
N-linked keratan sulfate proteoglycans rather than the
O-linked type found in cell types such as cartilage. HeLa
cells transfected with hCGn6ST cDNA may produce O-linked keratan sulfate proteoglycan, similar to cartilage tissue.
In the present study, we confirmed that missense mutations in
CHST6 found in MCD patients abolish the sulfotransferase
activity of the encoded protein (Fig. 7). It is possible that the
mutations cause rapid degradation or intracellular mislocalization of
the protein instead of functional inactivation. However, since all of
the missense mutations examined, except for A217T, substituted the
amino acids conserved among carbohydrate sulfotransferases (Fig. 1), it
is likely that these residues are necessary for hCGn6ST activity. These
amino acids may also be important for other carbohydrate sulfotransferases. Structure analysis (20, 21) suggested such conserved
motifs form binding domains for a sulfate donor, PAPS, and
site-directed mutagenesis of the PAPS-binding motifs of HNK-1 sulfotransferase result in marked decreases in its enzymatic activity (26). Therefore, the mutations found in MCD patients are likely to
cause inactivation of the enzyme rather than protein degradation or mislocalization.
The A217T missense mutation does not occur in motifs conserved among
sulfotransferases (Fig. 1B). Because hCGn6ST with this mutation had no activity for keratan sulfate production (Fig. 7), and
mIGn6ST, which has the same enzymatic activity as hCGn6ST, conserves
the alanine residue at the identical position, this amino acid must be
important for sulfotransferase activity. A217 may be required for
recognition of specific carbohydrate structure for a substrate.
mIGn6ST is highly homologous to hIGn6ST, whose substrate specificity is
not known (18). In this report, we demonstrate that mIGn6ST, but not
hIGn6ST, has the same enzymatic activity as hCGn6ST (Fig. 8). The
expression pattern of mIGn6ST mRNA (Fig. 9c) is also
similar to that of hCGn6ST in corneal cells (22), suggesting that
mIGn6ST is the mouse orthologue of hCGn6ST. In humans, genes encoding
hCGn6ST and hIGn6ST are homologous not only in the coding region but
also in the 5'- and 3'-flanking sequences (22), suggesting that the two
genes may have been produced by gene duplication. We found no other
mouse EST with homology to hCGn6ST higher than that of hIGn6ST in the
GenBank EST data base (release 120). It is possible that the mouse
genome encodes only one GlcNAc 6-O-sulfotransferase gene
involved in keratan sulfate production and that during evolution the
gene was duplicated to produce CHST5 and CHST6,
each of which encodes hIGn6ST and hCGn6ST, respectively, in the human
genome. Sequence information of the flanking regions of
Chst5, which encodes mIGn6ST protein, is required to confirm
this hypothesis. Because the specific carbohydrate substrate of hIGn6ST
has not been identified, it is not known whether mIGn6ST has an
additional enzymatic activity that is similar to hIGn6ST activity. It
is possible that the mouse sulfotransferase has dual enzymatic
activities and the human homologues recognize each specific substrate.
In this report, we demonstrate that hCGn6ST and mIGn6ST have enzymatic
activity that produces keratan sulfate in cooperation with hKSG6ST. The
Chst5 knockout mouse may show a phenotype similar to MCD
patients and is likely to be a useful animal model for further studies
of the corneal dystrophy.