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INTRODUCTION |
Keratan sulfate proteoglycan is a major component of the corneal
stroma. Keratan sulfate proteoglycan is thought to play an important
role in maintaining corneal transparency by organizing and providing
proper hydration of the extracellular matrix of the corneal
stroma. Lumican, keratocan, and minecan have been identified as
carriers of keratan sulfate glycosaminoglycans
(GAGs)1 in an
N-linked manner (1-3). The expression of these core
proteins is regulated during developmental stages and wound healing of the cornea (4-7), suggesting an involvement of keratan sulfate proteoglycans in the reconstruction of the cornea. Studies of the
lumican gene knockout mouse revealed the importance of this proteoglycan in organizing regular spacing and fibrillogenesis of
collagen in the corneal stroma (8-10). Biochemical and electron microscopic analyses indicated that the protein moiety of lumican interacts with collagen, whereas keratan sulfate GAGs attached to
lumican provide the water retention capability necessary for interfibrillar spacing (11, 12).
Keratan sulfate GAG is composed of repeating disaccharide units of
(-3Gal
1-4GlcNAc1-), which is called
poly-N-acetyllactosamine, with sulfate residues at the
6-O-position of GlcNAc and Gal (13, 14). Keratan sulfate GAG
chain is also modified with fucose and sialic acid at GlcNAc and at the
nonreducing terminus, respectively (15-18). Besides the cornea,
keratan sulfate carbohydrate is also found in other tissues such as
cartilage. Corneal keratan sulfates are highly sulfated long
carbohydrates, which link to their core proteins via
N-linkages. More than half of the total sulfated disaccharide units of keratan sulfate GAG are monosulfated disaccharide (-3Gal1-4(SO
-)GlcNAc1-), and a
majority of others are disulfated disaccharide units
(-3(SO-)Gal1-4(SO-)GlcNAc1-) (18, 19). Distribution of sulfate residues in a corneal keratan sulfate
chain is not uniform. On the basis of analysis of keratanase digestion
products, it was postulated (20) that chains are composed of a region
of nonsulfated disaccharides near the linkage to protein, a central
region of monosulfated disaccharides, and a stretch of disulfated
disaccharides near the nonreducing terminus. Variety of the capping
structure at the nonreducing terminus of corneal keratan sulfate is
also reported (17, 19). By contrast, cartilage keratan sulfates have a
shorter carbohydrate backbone than corneal keratan sulfate (13, 14).
Cartilage keratan sulfates link to their core protein via
O-linkages at serine or threonine residues. Fucosylation and
sialylation ratios are higher in cartilage keratan sulfate than in
corneal keratan sulfate (18). Because sulfation of carbohydrate greatly
contributes to its hydrophilicity, the degree of sulfation of corneal
GAGs is likely to affect corneal transparency by changing the water
retention capacity of the corneal stroma. Indeed, several studies have
reported the relationship between sulfation of keratan sulfate and the
acquisition of corneal transparency during development (6, 21-24).
Four enzymes are responsible for production of keratan
sulfate carbohydrate:
1,3-N-acetylglucosaminyltransferase,
1,4-galactosyltransferase, GlcNAc 6-O-sulfotransferase
and Gal 6-O-sulfotransferase. Two glycosyltransferases, both
of which transfer GlcNAc or Gal to a nonreducing terminus of a
carbohydrate core structure connected to carrier proteins such as
lumican, are involved in elongation of the
poly-N-acetyllactosamine backbone of keratan sulfate. The other two enzymes, which transfer sulfate to the
6-O-position of GlcNAc or Gal, are involved in the
modification of poly-N-acetyllactosamine required to form
keratan sulfate composed of both mono- and disulfated disaccharides.
Previous reports have proposed a putative biosynthetic pathway of
keratan sulfate carbohydrate (14, 20, 25, 26); however, no studies have
firmly established such a pathway.
Previously, we reported that human corneal GlcNAc
6-O-sulfotransferase (hCGn6ST, also known as GlcNAc6ST-5 and
GST4), which is encoded by CHST6, is involved in
production of corneal keratan sulfate and that lack of hCGn6ST activity
in corneal cells results in a hereditary eye disease, macular corneal
dystrophy (MCD) (27, 28). In human, there is a second homologous
sulfotransferase called human intestinal GlcNAc
6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4
),
which is encoded by CHST5 (29). CHST5 is highly
homologous to CHST6 in both coding and noncoding regions and
is located next to CHST6 on human chromosome 16q22 (27, 30).
Due to their homology, these two genes are probably the result of gene
duplication during evolution. Unlike CHST5 and CHST6 in humans, the mouse genome only has one
sulfotransferase gene, designated Chst5, which encodes mouse
intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse
GlcNAc6ST-3/GST4) and is an orthologue of human CHST5 and
CHST6 (30). The fact that all three enzymes, hCGn6ST,
hIGn6ST, and mIGn6ST, are highly homologous to each other suggests that
they have similar biological function (30). However, in previous
studies, we expressed each of these sulfotransferases in mammalian
cells and found that cells expressing hCGn6ST and mIGn6ST, but not
hIGn6ST, produce a carbohydrate structure that is recognized by an
anti-keratan sulfate antibody (28). These observations strongly suggest
that despite of the apparent structural homology of these
sulfotransferases, hCGn6ST and mIGn6ST exhibit activities functionally
distinct from that of hIGn6ST.
In this study, we analyzed the substrate specificity of hCGn6ST,
hIGn6ST, and mIGn6ST in vitro using synthetic
oligosaccharide substrates and found that hCGn6ST and mIGn6ST can
transfer sulfate to longer oligosaccharide substrates with a
poly-N-acetyllactosamine backbone. We also found that the
keratan sulfate disulfate disaccharide unit can be synthesized by
sequential enzymatic reactions of 1,4-galactosyltransferase-I (4Gal-TI), 1,3-N-acetylglucosaminyltransferase-2
(3Gn-T2), hCGn6ST, and keratan sulfate Gal
6-O-sulfotransferase (KSG6ST) in vitro. These
results establish a biosynthetic pathway for the mono- and disulfated
disaccharide sequences in corneal keratan sulfate.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors for Sulfotransferases and a
Glycosyltransferase--
Mammalian expression vectors each encoding a
full-length hCGn6ST, hIGn6ST, and mIGn6ST have been described
previously (28). Expression vectors for CD34-IgG chimeric protein
(pcDM8-CD34-IgG) (31) and for core-2 GlcNAc transferase-I
(pcDNAI-C2GnTI) (32) were kindly provided by Dr. Minoru Fukuda (The
Burnham Institute).
An expression vector designated pcDNA3.1-HSH, which consists of a
DNA sequence encoding a cleavable signal sequence, polyhistidine, and
enterokinase cleavage sequence at the multicloning site of pcDNA3.1/Hygro (Invitrogen) (33), was used to prepare soluble enzymes
for this study. A DNA fragment encoding the catalytic domain of each
sulfotransferase was amplified by PCR using a specific primer (see
below) and the SP6 primer (Invitrogen) from the expression vector
encoding the full-length cDNA (28). The amplified DNA fragment was
digested by BglII and XbaI and inserted into the BamHI-XbaI site of pcDNA3.1-HSH. Primer sequences
used for amplifying the catalytic domain of each
sulfotransferase are as follows: for hCGn6ST,
5'-GGTAGATCTGCCAGGGCCCTCGTCCCCA-3'; for hIGn6ST, 5'-CATAGATCTGCCAGGGCCCTCATCCCCA-3'; for mIGn6ST,
5'-GGTAGATCTGCAAGTGCCATCGTCCCCA-3'.
An expression vector encoding soluble 3Gn-T2 was prepared as
follows. A DNA fragment encoding the catalytic domain of
3Gn-T2 (34) was amplified by PCR using the primer pair
5'-CAAGGATCCGTTCTGGAAGATATCTACCCCTCCCG-3' and
5'-GAAGTCGACATGGGAACTATTCAAAATACACCTTTCT-3' from human cornea cDNA (provided by Dr. Kohji Nishida, Department of Ophthalmology, Osaka
University School of Medicine, Japan) and digested by BamHI and SalI. The digested product was cloned into the
BamHI-XhoI site of pcDNA3.1-HSH.
An expression vector encoding soluble KSG6ST was prepared as follows. A
DNA fragment encoding the catalytic domain of human KSG6ST was
amplified from human genomic DNA by the specific primers, 5'-TGCCCCGGGCTGGCAGA-3' and 5'-CACCGCCCGGGTCACGAG-3'. The
amplified fragment was inserted by the TA cloning method (35) into the blunted EcoRI site of pcDNA3.1-A, which encodes a
cleavable signal sequence followed by the IgG binding domain of Protein
A (36).
A bacterial expression vector encoding hCGn6ST was prepared as follows.
A DNA fragment that encodes a polyhistidine tag and the
enterokinase-cleavable sequence followed by the catalytic domain of
hCGn6ST was prepared from pcDNA3.1-HSH-hCGn6ST (28) by
NcoI-DraI digestion. The digested fragment was
purified and inserted into the NcoI-blunted XhoI
site of pET28a(+) (Novagen, Madison, WI).
Metabolic Labeling and Carbohydrate Analysis of CHO Cells
Transfected with Expression Vectors--
An expression vector encoding
each full-length sulfotransferase was co-transfected with
pcDM8-CD34-IgG and with or without pcDNAI-C2GnTI into CHO cells
using the LipofectAmine PLUS reagent (BD Biosciences
CLONTECH, Palo Alto, CA). After culturing the cells
for 24 h in -minimal essential medium (Irvine Scientific, Santa
Ana, CA) with 10% fetal calf serum, the medium was replaced with
S-minimal essential medium (Invitrogen) including
10% dialyzed fetal calf serum and 0.1 mCi/ml [35S]sodium
sulfate (PerkinElmer Life Sciences). Following incubation for 24 h, secreted CD34-IgG chimeric protein was isolated from the medium by
Protein A-Sepharose beads (Sigma). The amount of isolated CD34-IgG from
each transfectant was determined by Western blot analysis using
horseradish peroxidase-conjugated anti-human IgG antibody. Equal
amounts of [35S]sulfate-labeled CD34-IgG, which were
produced by transfected cells, were subjected to SDS-PAGE followed by fluorography.
To remove N-linked glycans from isolated CD34-IgG, the
protein was precipitated once with acetone and dissolved in 1× sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 1.4 mM EDTA, pH 8.0, 40 mM dithiothreitol, 7%
sucrose). After adjusting the concentration of CD34-IgG, 1.25 µl of
the solution was treated with 1 unit of N-glycosidase-F
(Roche Diagnostics) in 25-µl reactions including 50 mM
NaPO4, pH 7.5, and 0.75% Triton X-100. Following
incubation for 3 h at 37 °C, the sample was subjected to
SDS-PAGE followed by fluorography.
Preparation of Soluble Enzymes--
Concentrated cultured medium
from transfected HeLa cells was used as an enzyme source of
sulfotransferases and N-acetylglucosaminyltransferase. An
expression vector for soluble enzyme was transfected into HeLa cells as
described above. After culturing transfected cells for 48 h in
-minimal essential medium with 10% fetal calf serum, the medium was
replaced with OPTI-MEM (Invitrogen) and incubated for 24 h at
37 °C. The medium was recovered and concentrated by Microcon YM-30
(Millipore Corp., Bedford, MA). After the addition of an equal volume
of glycerol, the concentrated medium was stored at 
20 °C.
To measure the quantity of soluble polyhistidine-tagged
sulfotransferase in the concentrated medium, polyhistidine-tagged hCGn6ST was prepared in a large quantity from a bacterial culture, purified, and used as a standard. Briefly, competent BL21(DE3) bacteria
(Novagen) were transformed with the expression vector, pET28a-hCGn6ST,
and the resulting transformants were cultured in Luria broth
medium, as recommended by the manufacturer. Polyhistidine-tagged hCGn6ST was purified from an inclusion body fraction of the bacterial lysate by a nickel column (Ni2+-nitrilotriacetic acid;
Qiagen, Valencia, CA). SDS-PAGE and Coomassie Blue staining indicated
that 75.2% of protein components in the bound fraction were hCGn6ST,
which was used as a standard. For calculation of the amount of
sulfotransferase in the enzyme fraction expressed by HeLa cells, an
aliquot of each enzyme source and several aliquots of the hCGn6ST
standard were subjected to SDS-PAGE, and the separated proteins were
blotted onto a polyvinylidene difluoride membrane. Using horseradish
peroxidase-labeled anti-HisG antibody (Invitrogen), bands of
polyhistidine-tagged sulfotransferase protein were visualized, and the
amount of sulfotransferase in the enzyme source was determined by
comparing the intensity of the sulfotransferase band with that of the standards.
Analysis of Substrate Specificity of Sulfotransferases for
Various Substrates--
Synthetic carbohydrate substrates were
described previously (37-39). Each substrate was incubated in a
15-µl reaction mixture containing 50 mM imidazole-HCl, pH
6.8, 10 mM MnCl2, 2 mM 5'-AMP, 20 mM NaF, 100 nCi of [35S]PAPS (PerkinElmer
Life Sciences), 2.25 nmol of substrate, and 1 µl of enzyme fraction
at 37 °C for 1 h. After adding 1 ml of water to stop the
reaction, the product was purified by a reverse phase C18 column (High
Load C18 column, Alltech Associates, Deerfield, IL), and incorporation
of [35S]sulfate by the substrate was determined by
scintillation counting.
Sequential Enzymatic Reactions for Production of Keratan Sulfate
in Vitro--
A synthetic carbohydrate substrate
GlcNAc1-6Man1-6Man1-octyl was labeled with
[35S]sulfate by incubating 11.25 nmol of the substrate in
a 75-µl reaction mixture containing 50 mM imidazole-HCl,
pH 6.8, 10 mM MnCl2, 2 mM 5'-AMP,
20 mM NaF, 1.76 µCi of [35S]PAPS
(PerkinElmer Life Sciences), and 5 µl of the enzyme fraction of
hCGn6ST at 37 °C overnight. The product was purified by passage through a C18 column and then lyophilized and subjected to HPLC. A
Whatman Partisil SAX-10 column (4.6 mm × 25 cm) was used for HPLC
analysis (40). This column was equilibrated with 75% acetonitrile in
H2O. The samples were eluted under the following
conditions: 75% acetonitrile/H2O for 5 min; 1 mM KH2PO4, 75% acetonitrile for 30 min; and a gradient from 5 mM
KH2PO4, 75% acetonitrile to 10 mM
KH2PO4, 75% acetonitrile over 25 min followed
by 10 mM KH2PO4, 75% acetonitrile
for 15 min. The flow rate was 1 ml/min, and 1-ml fractions were
collected. 35S radioactivity of eluates was monitored by
scintillation counting. The identified reaction product was purified by
a C18 column, lyophilized, and dissolved in 100 µl of water.
Next, the sulfated substrate was treated with 4Gal-TI as follows.
The reaction condition basically followed a previous description (37).
In brief, 100 µl of a reaction mixture including 50 mM HEPES-KOH, pH 7.5, 14 mM MnCl2, 1 mM UDP-Gal (Sigma), 10 units of calf intestine alkaline
phosphatase (New England Biolabs, Beverly, MA), 50 µl (1.18 × 106 cpm) of the purified product (product I in Fig. 4), and
10 milliunits of bovine milk 4Gal-TI (Sigma) was incubated at
37 °C overnight. The product was purified by a C18 column,
lyophilized, and subjected to HPLC analysis in the manner described above.
The obtained product was subsequently treated with an enzyme fraction
of human 3Gn-T2. The reaction mixture (150 µl) containing 100 mM HEPES-KOH, pH 7.5, 20 mM MnCl2,
5 mM UDP-GlcNAc (Sigma), 45 µl (3.35 × 105 cpm) of the purified product (product II in Fig. 4),
and 43.2 µl of human 3Gn-T2 was incubated at 37 °C overnight.
The product was purified by a C18 column, lyophilized, and subjected to
HPLC analysis as described above.
The product was again treated with an enzyme fraction of hCGn6ST. A
50-µl reaction mixture containing 50 mM imidazole-HCl, pH
6.8, 10 mM MnCl2, 2 mM 5'-AMP, 20 mM NaF, 1 mM PAPS (Sigma), 30 µl (61,000 cpm)
of the purified product (product III in Fig. 4), and 5 µl of enzyme
fraction of hCGn6ST, was incubated at 37 °C overnight. The product
was purified by a C18 column, lyophilized, and subjected to HPLC as described.
The product was further treated with an enzyme fraction of KSG6ST. A
100-µl reaction mixture containing 50 mM imidazole-HCl, pH 6.8, 10 mM MnCl2, 2 mM 5'-AMP,
20 mM NaF, 1 mM PAPS (Sigma), 60 µl (10080 cpm) of the purified product (product IV in Fig. 4), and 10 µl of
enzyme fraction of KSG6ST was incubated at 37 °C overnight. The
reaction product was purified by a C18 column, lyophilized, and
subjected to HPLC analysis as described.
Keratanase Treatment of Reaction Products--
To determine the
carbohydrate structure, reaction products (products IV and V in Fig. 4)
were treated with keratanase, which recognizes
(SO-)GlcNAc1-3Gal1-4GlcNAc structure and digests Gal1-4GlcNAc linkages (41). The carbohydrate product (3000 cpm of product IV and 1500 cpm of product V) was incubated in a 30-µl reaction mixture containing 50 mM
Tris-HCl, pH 7.4, and 125 milliunits of keratanase from
Pseudomonas sp. (Calbiochem) at 37 °C for 4 h. The
digested product was purified by a C18 column and subjected to HPLC
followed by scintillation counting.
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RESULTS |
hCGn6ST Produces Sulfated N- and O-Glycans in Cultured
Cells--
In a previous study, we found that hCGn6ST and mIGn6ST, but
not hIGn6ST, are required for the production of keratan sulfate (28),
although the amino acid sequences of all three enzymes are highly
homologous to each other. To elucidate the functional similarities and
differences in those sulfotransferases, we analyzed the enzymatic
activity of recombinant sulfotransferases in vitro and
in vivo. First, we expressed the sulfotransferases in CHO cells and analyzed the production of sulfated carbohydrates. We used
CD34-IgG as an acceptor protein because this protein carries both
N- and O-glycans (31), and we used CHO cells as
hosts because they have no core-2
N-acetylglucosaminyltransferase activity and produce
predominantly N-glycans (32). When an expression vector carrying C2GnTI is transfected into CHO cells, the cells produce both
N- and O-glycans (32). Mock-transfected CHO cells
showed a negligible level of sulfated carbohydrate on CD34-IgG, which is produced by the endogenous sulfotransferase (Fig.
1A, lanes 1 and 2). On the other hand, cells transfected
with any of the sulfotransferase expression vector produced significant
amounts of CD34-IgG with sulfated glycans (Fig. 1A,
lanes 3-8). The transfected cells expressing
hCGn6ST produced sulfated N-glycans on CD34-IgG (Fig.
1A, lane 3), and these sulfated
carbohydrates were released from the protein by N-glycanase
treatment (Fig. 1B, lane 3). We also
found that hCGn6ST-expressing CHO cells produced large amounts of
sulfated O-glycans resistant to N-glycanase
digestion (Fig. 1, A and B, lane
4). These results indicate that hCGn6ST can transfer sulfate
on both N- and O-glycans in vivo.
Almost identical results were obtained for mIGn6ST (Fig. 1,
A and B, lanes 7 and
8), suggesting that mIGn6ST has a similar preference for
carbohydrate substrates as hCGn6ST. Unlike these two enzymes,
hIGn6ST-expressing CHO cells produced sulfated O-glycans but
almost no sulfated N-glycans on CD34-IgG (lanes
5 and 6), suggesting that hIGn6ST mostly
functions in the production of sulfated O-glycans in
vivo.
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Fig. 1.
Sulfation of CD34-IgG by hCGn6ST, hIGn6ST,
and mIGn6ST in CHO cells. CHO cells were co-transfected with
expression vectors for CD34-IgG (lanes 1-8), for
sulfotransferases (mock, lanes 1 and
2; hCGn6ST, lanes 3 and 4;
hIGn6ST, lanes 5 and 6; mIGn6ST,
lanes 7 and 8), and for C2GnTI
(lanes 2, 4, 6, and
8). The transfected cells were metabolically labeled with
[35S]sulfate, and the CD34-IgG produced was isolated and
analyzed by fluorography. Fluorograms represent the pattern of intact
(A) and N-glycanase-treated (B)
CD34-IgG. The arrows indicate the migration position of
CD34-IgG.
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Specific Activity of Sulfotransferases on Synthetic
Substrates--
Next we analyzed substrate specificity of all three
sulfotransferases using synthetic carbohydrates (Fig.
2, bottom). As a source of
sulfotransferase, we used concentrated culture medium of HeLa cells
expressing soluble forms of sulfotransferases. The results of the
sulfation analysis and the calculated relative enzymatic activity for
substrates are shown in Fig. 2 and Table I, respectively. We found that all three
soluble sulfotransferases have activity for synthetic carbohydrates and
that hCGn6ST has the strongest activity among the sulfotransferases
tested for all of the reactive substrates (Fig. 2 and Table I). We also calculated the specific activity of each sulfotransferase for a
carbohydrate substrate. Purified hCGn6ST protein from bacterial cells
was used to determine the quantity of sulfotransferase in the reaction
mixture (see "Experimental Procedures"). By this analysis, we
calculated the specific activity of hCGn6ST, hIGn6ST, and mIGn6ST for
the trisaccharide substrate, GlcNAc1-6Man1-6Man1-octyl, as
73.5, 1.69, and 20.2 pmol/h/µg enzyme, respectively (Table I).
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Fig. 2.
Sulfation of synthetic carbohydrate
substrates by hCGn6ST, hIGn6ST, and mIGn6ST in
vitro. Each bar indicates incorporation of
[35S]sulfate per 1 h per 1-µl enzyme fraction of
each sulfotransferase into synthetic substrates at 37 °C. Data
presented are the average of duplicated experiments. Open
and hatched bars indicate the results using
enzyme fractions derived from mock-transfected cells and transfected
cells expressing soluble hCGn6ST (A), hIGn6ST
(B), and mIGn6ST (C). The carbohydrate structure
of each substrate is shown at the bottom.
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All of the soluble sulfotransferases have activity for short
carbohydrate substrates, which have GlcNAc on their nonreducing terminus, such as GlcNAc1-6Man1-6Man1-octyl (Fig. 2,
substrate 1) and GlcNAc1-6(Gal1-3)GalNAc1-octyl (substrate
11). These sulfotransferases have no activity for carbohydrates that
have internal GlcNAc but no nonreducing terminal GlcNAc in the
structure (substrates 2, 6, and 10), indicating that these
sulfotransferases only transfer sulfate on GlcNAc at the nonreducing
terminus of carbohydrate.
A trisaccharide substrate with a core-2 O-glycan structure
is the most suitable substrate for all the sulfotransferases (substrate 11). This finding is consistent with results obtained in
vivo (Fig. 1), which indicates that all of the sulfotransferases
have higher enzymatic activity for O-glycans than for
N-glycans on CD34-IgG. Although core-2 carbohydrate is the
preferable substrate for hCGn6ST and mIGn6ST, all of the carbohydrates
with nonreducing terminal GlcNAc can be utilized as substrates for
hCGn6ST and mIGn6ST. A nonreducing terminal GlcNAc connected to mannose
by 1-2 linkage was a poorer acceptor for these two enzymes than one
connected to mannose by 1-6 linkage (substrates 1 and 5). Interestingly, a biantennary substrate, which has two nonreducing terminal GlcNAc residues by 1-2 and 1-6 linkages (substrate 7),
has much less substrate activity for hCGn6ST and mIGn6ST than a
monoantennary substrate with a nonreducing terminal GlcNAc by 1-6
linkage (substrate 1). We also found that a biantennary substrate that
has a GlcNAc by 1-6 linkage and a Gal1-4GlcNAc by 1-2 linkage (substrate 9) has less substrate activity for the
sulfotransferases than a monoantennary substrate,
GlcNAc1-6Man1-6Man1-octyl (substrate 1). These results
indicate that the carbohydrate on the 1-2 branch hinders sulfation
of GlcNAc on the 1-6 branch by the sulfotransferases. On the other
hand, the substrate activities of a monoantennary GlcNAc1-2Man1-6Man1-octyl (substrate 5) and a biantennary
GlcNAc1-2(Gal1-4GlcNAc1-6)Man1-6Man1-octyl (substrate 8) for both hCGn6ST and mIGn6ST are almost equivalent, suggesting that the sulfation of a GlcNAc residue linked to the mannose
core via 1-2 linkage is not affected by the presence of another
carbohydrate residue on 1-6 linkage. Notably, hCGn6ST and mIGn6ST
utilized carbohydrates with extended GlcNAc1-3Gal repeats
(substrates 3 and 4) as their substrates, and the length of the
GlcNAc1-3Gal repeat did not affect the activity of the enzymatic
reaction. These results suggest that hCGn6ST and mIGn6ST can transfer
sulfate onto the nonreducing terminal GlcNAc of a poly-N-acetyllactosamine structure.
The substrate specificity of hIGn6ST is remarkably different from that
of the other two sulfotransferases (Fig. 2B). hIGn6ST utilized shorter carbohydrates as preferable substrates (substrates 1, 5, 7, 8, and 11) but had very little activity on longer carbohydrates that have more than one unit of GlcNAc1-3Gal disaccharide
(substrates 3, 4, 13, and 14). This result suggests that hIGn6ST does
not play a role for the production of keratan sulfate because keratan sulfate consists of GlcNAc1-3Gal repeats. Interestingly, hIGn6ST has higher enzymatic activity for GlcNAc1-2Man1-6Man1-octyl (substrate 5) than for GlcNAc1-6Man1-6Man1-octyl (substrate 1), whereas hCGn6ST and mIGn6ST have the opposite preference for these
substrates. We also found that hIGn6ST has almost the same sulfotransferase activity for a monoantennary
GlcNAc1-2Man1-6Man1-octyl (substrate 5) and two biantennary
substrates, GlcNAc1-2(GlcNAc1-6)Man1-6Man1-octyl (substrate 7) and
GlcNAc1-2(Gal1-4GlcNAc1-6)Man1-6Man1-octyl (substrate 8). This finding provides further evidence that the presence
of carbohydrate on 1-6 branch does not affect sulfation of GlcNAc
on 1-2 branch by hIGn6ST.
Based on these results, we concluded that hCGn6ST and mIGn6ST can
produce sulfated poly-N-acetyllactosamine carbohydrate that will be processed to keratan sulfate, on both N- and
O-glycans, whereas hIGn6ST is only active on short
carbohydrates such as core-2 trisaccharide.
In Vitro Synthesis of Keratan Sulfate Disaccharide by Sequential
Treatment by Glycosyltransferases and Sulfotransferases--
Since
hCGn6ST functions in the production of keratan sulfate (28) and
transfers sulfate to nonreducing terminal GlcNAc (Fig. 2A), we hypothesized that sulfation of the GlcNAc residue of
keratan sulfate by hCGn6ST is coupled to elongation of the
poly-N-acetyllactosamine chain by
1,3-N-acetylglucosaminyltransferase and
1,4-galactosyltransferase (Fig. 3). To
test this hypothesis, we determined whether keratan sulfate is produced
in vitro using sulfotransferases and glycosyltransferases. For sulfotransferases, we used hCGn6ST and human keratan sulfate Gal
6-O-sulfotransferase (KSG6ST). For a
1,4-galactosyltransferase, we used bovine milk 4Gal-TI, which is
the orthologue of human 4Gal-TI. As a
1,3-N-acetylglucosaminyltransferase, we chose human
3Gn-T2 because it is ubiquitously expressed in human tissues and has
the highest N-acetylglucosaminyltransferase activity among enzymes tested (34). First we produced a radiolabeled
monosulfated trisaccharide,
[35S]SO-GlcNAc1-6Man1-6Man1-octyl by treating substrate 1 in Fig. 2 with hCGn6ST and a sulfate donor, [35S]PAPS. The reaction mixture was then subjected to
SAX-10 HPLC (Fig. 4A). We
pooled the fractions of the product (product I in Fig. 4A),
which were used as a substrate for the next step.
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Fig. 3.
Proposed biosynthetic pathway of corneal
keratan sulfate. Since hCGn6ST only transfers sulfate onto the
nonreducing terminal GlcNAc of a substrate carbohydrate, sulfation of
GlcNAc may be coupled to elongation of the
poly-N-acetyllactosamine backbone. GlcNAc-sulfated
poly-N-acetyllactosamine is a completed product that
represents about 50% of the keratan sulfate disaccharide content in
cornea (18). The remaining structure is further sulfated on the Gal
residues by KSG6ST to produce highly sulfated keratan sulfate
disaccharides. Sulfation of Gal residues and fucosylation of GlcNAc
residues are not coupled but may occur during and/or after GAG chain
elongation. The nonreducing terminus of keratan sulfate may be capped
with a variety of saccharides including sialic acid. In the mouse,
mIGn6ST plays a role corresponding to that of hCGn6ST.
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Fig. 4.
Sequential treatments of a synthetic
substrate by glycosyltransferases and sulfotransferases produce keratan
sulfate. A, HPLC pattern of a sulfation product of
GlcNAc1-6Man1-6Man1-octyl with hCGn6ST and
[35S]PAPS. The sulfated product indicated by a
horizontal bar (product I) was recovered and used
as a substrate for the next reaction. B, HPLC pattern of a
reaction product of product I with bovine milk 4Gal-TI and UDP-Gal.
The product of this reaction (product II) was used as the substrate for
next step. C, HPLC pattern of a reaction mixture of product
II with human 3Gn-T2 and UDP-GlcNAc. The first peak is residual
product II, which remained unmodified by this enzyme treatment. The
second peak (product III) was collected and used as a substrate for the
next reaction. D, HPLC pattern of a reaction product of
product III with hCGn6ST and PAPS. A product (product IV) was collected
and used as a substrate for the next reaction and for keratanase
treatment (Fig. 5A). E, HPLC pattern of a
reaction product of product IV with KSG6ST and PAPS. A product (product
V) was subjected to keratanase treatment (Fig. 5B). The
arrows indicate retention positions of standard
carbohydrates,
[35S]SO-GlcNAc1-6Man1-6Man1-octyl
(a),
[35S]SO-Gal1-4GlcNAc1-6Man1-6Man1-octyl
(b), and
[35S]SO-GlcNAc1-3Gal1-4GlcNAc1-6Man1-6Man1-octyl
(c). Dashed lines represent the
concentration of KH2PO4 in the elution
solution.
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Next we treated product I with 4Gal-TI and a Gal donor, UDP-Gal, and
analyzed the carbohydrate structure by HPLC (Fig. 4B). The
product (product II in Fig. 4B) was eluted later than that of starting material (product I in Fig. 4A) and was slightly
later than the position of a monosulfated tetrasaccharide marker (Fig. 4, arrow b), suggesting that product II is an
isomer of
[35S]SO-Gal1-4GlcNAc1-6Man1-6Man1-octyl. This product II was treated with 3Gn-T2 and a GlcNAc donor,
UDP-GlcNAc, and the reaction product was subjected to HPLC (Fig.
4C). In the HPLC profile, we found that half of the
substrate is modified to a product (product III) that has a later
retention position (fractions 32-35 in Fig. 4C) by
3Gn-T2 treatment. We again treated product III with hCGn6ST and a
sulfate donor, PAPS, and separated the reaction product by HPLC (Fig.
4D). The retention position of the product (product IV in
Fig. 4D) was greatly delayed relative to the starting
material (product III in Fig. 4C), suggesting that product
IV is a sulfated version of product III. To determine the carbohydrate
structure of product IV, we tested the susceptibility of product IV to
keratanase. Keratanase is an endoglycosidase that recognizes
SO-GlcNAc1-3Gal1-4GlcNAc and
digests Gal1-4GlcNAc linkage (41). Additional sulfate on Gal and/or
lack of sulfate on GlcNAc impairs the sensitivity to keratanase. HPLC
analysis showed an identical retention time for the digestion product
(Fig. 5A, closed
square) as a carbohydrate marker,
[35S]SO-GlcNAc1-6Man1-6Man1-octyl (Fig. 5A, marker a), indicating that
keratanase treatment released one
SO-GlcNAc1-3Gal unit from product IV
and produced a carbohydrate structure identical to product I. Thus, we concluded that the carbohydrate structure of product IV is
SO-GlcNAc1-3Gal1-4([35S]SO-)GlcNAc1-6Man1-6Man1-octyl. Since the structure of product IV has an elongated GlcNAc1-3Gal unit on product I and this elongation step has been processed by
4Gal-TI and 3Gn-T2, we concluded that the carbohydrate structures of products II and III are
Gal1-4([35S]SO-)GlcNAc1-6Man1-6Man1-octyl and
GlcNAc1-3Gal1-4([35S]SO-)GlcNAc1-6Man1-6Man1-octyl, respectively.
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Fig. 5.
Keratanase treatment of sulfated
carbohydrates produced in vitro by
sulfotransferases. Products IV and V (in Fig. 4) were treated with
keratanase, and the reaction products were subjected to SAX-10 HPLC
analysis. A, HPLC pattern of product IV before
(open circles) and after (closed
squares) keratanase treatment. B, HPLC pattern of
product V before (open circles) and after
(closed squares) keratanase treatment. The
arrows indicate the elution positions of standard
carbohydrates shown in Fig. 4.
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We further treated product IV with KSG6ST and PAPS to test
whether this enzyme transfers sulfate onto product IV. The retention position of the product following KSG6ST treatment was greatly shifted
to a later fraction (Fig. 4E), indicating that the product received a sulfate residue by the enzyme treatment. We also treated the
carbohydrate component of product V with keratanase; however, keratanase did not change the retention position of the product (Fig.
5B), indicating that product V is resistant to keratanase. Since the addition of sulfate on Gal makes the carbohydrate
resistant to keratanase, we concluded that the carbohydrate
structure of product V is a trisulfated pentasaccharide,
SO-GlcNAc1-3(SO-)Gal1-4([35S]SO-)GlcNAc 1-6Man-1-6Man1-octyl.
The results of sequential enzymatic reactions shown in Fig. 4 indicate
that the sulfated carbohydrate can be processed to keratan sulfate
disaccharide in vitro by four enzymes: 4Gal-TI, 3Gn-T2, hCGn6ST, and KSG6ST. Since the nonreducing terminal
carbohydrate structure of product IV is identical to that of product I,
it is very likely that the sulfation and elongation steps are
repeatable by the three enzymes, hCGn6ST, 1,4-galactosyltransferase,
and 1,3-N-acetylglucosaminyltransferase. This idea is
consistent with the proposed biosynthetic pathway of keratan sulfate,
which is shown in Fig. 3. The sulfated
poly-N-acetyllactosamine chain may be produced by two
glycosyltransferases and GlcNAc 6-O-sulfotransferase in a
cooperative manner. Sulfation of Gal residues by Gal
6-O-sulfotransferase, which may take place later than the
production of the GlcNAc-sulfated poly-N-acetyllactosamine,
completes the keratan sulfate GAG synthesis.
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DISCUSSION |
In previous studies, we found that the loss of enzymatic activity
of hCGn6ST in corneal cells causes a hereditary eye disease, MCD (27),
and that hCGn6ST is required for the production of keratan sulfate GAGs
(28). We also found that mIGn6ST, which is an orthologue of both
hCGn6ST and hIGn6ST, has similar activity as hCGn6ST, but not hIGn6ST,
in the production of keratan sulfate GAGs in the mouse cornea (28).
Keratan sulfate GAGs are found in the cornea as well as in the other
tissues such as cartilage. Corneal keratan sulfate is linked to carrier
proteins in N-linked manner, whereas cartilage keratan
sulfate is attached to proteins largely via O-linkages (13,
14). By metabolic labeling of a glycoprotein produced by
sulfotransferase-expressing CHO cells, we found that both hCGn6ST and
mIGn6ST contribute to the processing of sulfated N- and
O-glycans (Fig. 1). This result is consistent with our
previous hypothesis that hCGn6ST and mIGn6ST function in the production
of keratan sulfate in both the cornea and cartilage (27, 28). By
contrast, hIGn6ST activity resulted only in the production of sulfated
O-glycans on a carrier protein (Fig. 1). Experiments to test
the substrate specificity of hIGn6ST in vitro also showed
that it has the highest activity in the presence of core-2
oligosaccharide, whereas it shows minimal activity for short
N-linked type oligosaccharides (Fig. 2B). Several
studies have reported that hIGn6ST has sulfotransferase activity for
core-2 oligosaccharide and produces L-selectin ligand carbohydrate
structures (42, 43). Our results support this possibility. On the other
hand, Uchimura et al. (43) reported that hIGn6ST has no
activity for GlcNAc1-2Man and GlcNAc1-6ManOMe substrates. By
contrast, we found that hIGn6ST has activity for GlcNAc, which linked
to Man1-6Man1-octyl structures via 1-2 and 1-6 linkage
(Fig. 2B). It is possible that hIGn6ST recognizes trisaccharide but not disaccharide structures as substrates.
The present study also revealed that hIGn6ST exhibits very low
sulfotransferase activity on longer carbohydrate substrates (Fig.
2B and Table I). Thus, hIGn6ST showed significantly reduced sulfotransferase activity toward substrates with one additional unit of
GlcNAc1-3Gal (substrates 3 and 13 in Fig. 2) less than short
carbohydrate substrates (substrates 1 and 11), supporting our
hypothesis that hIGn6ST is not required for production of keratan
sulfate GAGs (28). Based on currently available data, hIGn6ST may only
contribute to L-selectin ligand production (42, 43) (Fig. 2B
and Table I). We have also found that an MCD patient lacking both the
hIGn6ST coding region and the putative gene regulatory region of
hCGn6ST shows no phenotype other than corneal opacity typical of MCD
(27), suggesting that the biological function of hIGn6ST can be
compensated by other sulfotransferases. Since sulfotransferases such as
GlcNAc6ST-1 (also known as GST2) and L-selectin ligand
sulfotransferase/high-endothelial cell specific GlcNAc 6-O
sulfotransferase (also known as GlcNAc6ST-2 and GST3) can
produce L-selectin ligand structures (31, 42-45), it is likely that
lack of hIGn6ST is compensated by sulfotransferases with similar
activity to hIGn6ST.
By analyzing the substrate specificity of sulfotransferases, we found
that mIGn6ST has remarkably similar substrate specificity to hCGn6ST
but not to hIGn6ST (Fig. 2). Previous reports suggest that a mouse gene
called Chst5, which encodes mIGn6ST, is an orthologue of two
human genes, CHST5 and CHST6, which encode
hIGn6ST and hCGn6ST, respectively (27, 30). In vivo
expression experiments indicated that mIGn6ST is the orthologue of
hCGn6ST, since both enzymes have been involved in production of keratan
sulfate (28). However, Uchimura et al. (43) found that
hIGn6ST, another orthologue of mIGn6ST, has no sulfotransferase
activity on poly-N-acetyllactosamine carbohydrate and
questioned the functional relationship of these three enzymes. In the
present study, it is clear that hCGn6ST and mIGn6ST are functionally
related in production of keratan sulfate, since hCGn6ST and mIGn6ST
have activity for poly-N-acetyllactosamine carbohydrates
(Fig. 2, substrates 3, 4, 13, and 14), whereas hIGn6ST has no activity
on those substrates. hIGn6ST may have acquired a different substrate
specificity, such as preference for a nonreducing terminal GlcNAc
linked to mannose via 1-2 linkage (Fig. 2B, substrates 5, 7, and 8) and has probably lost enzymatic activity for
poly-N-acetyllactosamine chains.
In this and previous studies, we hypothesize that the sulfation of
GlcNAc residues on keratan sulfate is coupled to elongation of the
poly-N-acetyllactosamine backbone (28) (Fig. 3). In this study, we tested sequential treatment of a carbohydrate substrate with
soluble candidate enzymes for production of keratan sulfate GAGs (Fig.
4). Sulfated carbohydrate has been utilized for elongation of the
N-acetyllactosamine backbone by a
1,4-galactosyltransferase and a
1,3-N-acetylglucosaminyltransferase (Fig. 4, B
and C), indicating that the presence of a sulfate residue on
the 6-O-position of GlcNAc does not affect elongation of
poly-N-acetyllactosamine chains. The chain length of
poly-N-acetyllactosamine also does not affect sulfation of
nonreducing terminal GlcNAc by hCGn6ST (Fig. 2A, substrates
3 and 4). Because the carbohydrate structure of the nonreducing
terminus of product IV is identical to that of product I (Fig. 4), the
enzymatic reactions of extension and sulfation of
poly-N-acetyllactosamine can be repeated by these three
enzymes, as illustrated in Fig. 3. KSG6ST has sulfotransferase activity
for both the nonreducing terminus and internal Gal residues of keratan
sulfate, and that also has preference for Gal adjacent to
sulfated GlcNAc (40, 46). We also confirmed that sulfation of internal
Gal in a carbohydrate chain is accomplished by KSG6ST (Fig.
4E). Thus, we conclude that sulfation of Gal in keratan sulfate is not coupled with the elongation step but occurs during and/or after production of GlcNAc-sulfated
poly-N-acetyllactosamine carbohydrate (Fig. 3). Since the
sulfation degree of Gal is lower than that of GlcNAc in a keratan
sulfate GAG chain, the Gal sulfation step seems to be independent from
the GlcNAc sulfation step in keratan sulfate biosynthesis (13, 14, 40,
47), and this is consistent with our hypothesis. We also found that an
N-acetylglucosaminyltransferase cannot transfer GlcNAc to
sulfated Gal at the nonreducing
terminus,2 indicating that
sulfation of Gal residues cannot be coupled with the chain elongation
of keratan sulfate GAGs. It is possible that sulfation of the terminal
Gal blocks further elongation of keratan sulfate GAG chain, as
suggested by an earlier study (20).
Previously, we found that mRNA encoding hCGn6ST and mIGn6ST is
present in human and mouse corneal tissues (27, 28). Since hCGn6ST has
been involved in production of keratan sulfate GAGs in vivo
and in vitro (28) (Figs. 2 and 4 and Table I), it is evident
that hCGn6ST is the sulfotransferase that transfers sulfate to GlcNAc
of poly-N-acetyllactosamine in the cornea. KSG6ST may be
responsible for sulfation of Gal residues of corneal keratan sulfate,
because mRNA encoding the enzyme is expressed in chick cornea (40).
Since 3Gn-T2 has the highest GlcNAc transferase activity for
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc, which has
an elongated poly-N-acetyllactosamine structure, among
enzymes tested in the previous study (34), we speculate that 3Gn-T2 functions in the synthesis of corneal keratan sulfate. 3Gn-T2 mRNA is ubiquitously expressed in human tissues and is therefore probably expressed in the cornea (34). There is no information on a
candidate 1,4-galactosyltransferase involved in production of
corneal keratan sulfate to date. Since human genome sequence is
available and several studies have reported nucleotide sequences and
substrate specificities of human 1,4-galactosyltransferases (39,
48-54), analyses of tissue distribution and biological function of
1,4-galactosyltransferases in vivo and in
vitro should identify the 1,4-galactosyltransferases
responsible for keratan sulfate biosynthesis in the cornea.
In the present study, we established a role for hCGn6ST in sulfation of
GlcNAc in keratan sulfate GAGs. We also demonstrated the production of
highly sulfated keratan sulfate in vitro. Keratan sulfate
proteoglycan is important for maintaining both extracellular matrix
organization and corneal transparency. Further studies defining the
corneal keratan sulfate biosynthetic pathway are necessary to determine
how corneal transparency develops and is maintained.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Glycobiology Program,
The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3100; Fax: 858-646-3193; E-mail: takama@burnham-inst.org.
