Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate.

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.

cal 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)(12)(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 trans-fected 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.
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: 5Ј-GGTAGATCTGCCAGGGCCCTCGTCCCCA-3Ј and 5Ј-GA-TTTAGGTGACACTATAG-3Ј (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 EpiTag TM 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: 5Ј-AGACCTTCCTCCTCCTCTTTCTGGTT-3Ј and 5Ј-GCG-CACCACGCGCAGGC-3Ј. 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 (5Ј-GACGTGTTTGATGCCTATCTGCCTTG-3Ј and 5Ј-CGGCGCGCACCAGGTCCA-3Ј) 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 Lipo-fectAMINE 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 MgCl 2 , 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, the sample was centrifuged at 9,000 ϫ 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 g of protein from enzyme fraction was incubated with 15 l of reaction mixture containing 50 mM imidazole HCl, pH 6.8, 10 mM MnCl 2 , 2 mM 5Ј-AMP, 20 mM NaF, 50 nCi of [ 35 S]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 35 S-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 [ 35 S]sodium sulfate (PerkinElmer Life Sciences) at a concentration of 100 Ci/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 l of chloroform/methanol (2:1). The cell pellets were washed with 200 l of methanol and digested in 200 l of 0.1 M Tris-HCl, pH 8.0, 1 mM CaCl 2 with 20 l of 1 mg/ml Pronase (Calbiochem). After an overnight incubation at 37°C, 20 l 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 ϫ 45 cm, equilibrated with 0.1 M NH 4 HCO 3 ). The carbohydrate fraction that eluted in the void volume was collected, desalted by Sephadex G-25 gel filtration (1 ϫ 30 cm, equilibrated with 7% 1-propanol/water), and lyophilized. The sample was dissolved in 150 l 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 l 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 ϫ 10 6 cpm) from transfected HeLa cells were digested with 250 milliunits of keratanase from Pseudomonas sp. (Calbiochem) in 90 l 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 ϫ 45 cm) of Sephadex G-50 equilibrated with 0.1 M NH 4 HCO 3 . Each 400 l of fraction was collected in a tube. Ten l of each fraction were used to count 35 S 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 NH 4 HCO 3 . 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 ϫ 45 cm) was used for gel filtration chromatography. The column was equilibrated and eluted with 0.1 M NH 4 HCO 3 . Fractions of 300 l were collected, and 35 S radioactivity was determined by scintillation counting. A Whatman Partisil SAX-10 column (4.6 mm ϫ 25 cm) was used for HPLC analysis. This column was equilibrated with 5 mM KH 2 PO 4 . The elution conditions were as described previously (14,27). In brief, the column was eluted with 5 mM KH 2 PO 4 isocratically for the samples produced by ␤-N-acetylglucosaminidase A treatment. For the samples produced by keratanase treatment, the column was eluted with 5 mM KH 2 PO 4 for 5 min followed by a 20-min gradient from 5 to 250 mM KH 2 PO 4 . The flow rate was 1 min/ml. Fractions of 0.5 min were collected, and 35 S 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 l of TKMS buffer (20 mM Tris-HCl, pH 7.6, 25 mM KCl, 2.5 mM MgCl 2 , 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride) and lysed by 5 times freeze/thaw cycles. After centrifugation at 9,000 ϫ g for 10 min at 4°C, the precipitate was washed with TKMS buffer and suspended in 200 l of TKMS buffer containing 1% Triton X-100. After 10 min of incubation on ice, the sample was centrifuged at 9,000 ϫ 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 g 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 Ϫ20°C for 30 min and treated with 0.3% H 2 O 2 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 (antimouse 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: 5Ј-CTCAGCGACCCTGCGCTCAAC-3Ј and 5Ј-CGCACATGG-CTGCGGCATAC-3Ј. The amplified fragment was cloned into the SmaI site of pGEM3Zf(ϩ) (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).

RESULTS
Enzymatic Activity of hCGn6ST-hCGn6ST has high homology to human and mouse intestinal GlcNAc 6-O-sulfotrans-ferases 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 35 S-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.
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 [ 35 S]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 hCGn6STexpressing 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.
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-Nacetyllactosamine and results in production of keratan sulfate.
Co-expression of hCGn6ST and hKSG6ST Produces Highly Sulfated Keratan Sulfate-hKSG6ST transfers sulfate to the 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 [ 35 S]PAPS and a synthetic carbohydrate substrate, Glc-NAc␤1-6Man␣1-6Man␤-octyl, and incorporation of 35 S 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).

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 35 Slabeled 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. 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 cotransfectants (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.
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.
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 kera- tan 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.
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. 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 Olinked 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 sitedirected 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 sul-fotransferase 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 5Ј-and 3Ј-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.
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 m.
FIG. 9. Immunohistochemistry and in situ hybridization of keratan sulfate and mIGn6ST mRNA in the mouse cornea. Semiserial 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 m.