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Mouse chondroitin 6-sulfotransferase: molecular cloning, characterization and chromosomal mapping
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Mouse chondroitin 6-sulfotransferase: molecular cloning, characterization and chromosomal mapping
Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3[prime]-phosphoadenosine 5[prime]-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. Using chick C6ST cDNA as a probe, we cloned the cDNA of mouse C6ST. The mouse enzyme was predicted to be composed of 472 amino acids, and exhibited 71% sequence identity with the chicken enzyme. The mouse and chicken catalytic domains exposed to the luminal side exhibited 81% identity, while the homology of the remaining regions was less. Transfection and expression of the mouse cDNA in COS-7 cells yielded C6ST activity. Keratan sulfate sulfotransferase activity, which was simultaneously expressed, amounted to 3% of the C6ST activity, this value being significantly lower than that observed in the case of the chicken enzyme. Mouse C6ST mRNA was strongly expressed in the spleen, lung, and eye. In situ hybridization revealed that the transcript was localized in stromal cells in the marginal zone and red pulp of the spleen, and stromal cells in the bone marrow. Fluorescence in situ hybridization analysis revealed the gene is located in mouse chromosome 9. Key words: chondroitin sulfate/chromosomal mapping/in situ hybridization/keratan sulfate/sulfotransferase
Introduction
Chondroitin sulfates, a class of glycosaminoglycan chains located in proteoglycans, are involved in extracellular interactions such as cell adhesion and cell migration (Kjellén and Lindahl, 1991).
Although chondroitin sulfate proteoglycans (CSPGs) in the cartilage have been best studied, they are also found in many other tissues, including neural tissues and hematopoietic organs. Recently, the possible roles of CSPGs in neural development and neurodegenerative diseases (Faissner et al., 1994; Milev et al., 1994; Thinakaran et al., 1995; Emerling and Lander, 1996; Maeda and Noda, 1996), and in the immune responses and the hematopoietic microenvironment (Kirby and Bentley, 1987; Okayama et al., 1988; Naujokas et al., 1993; Uhlin-Hansen et al., 1993) have gained much attention.
Chondroitin sulfates in mammals and birds are mainly sulfated at the 4 or 6 position of the N-acetylgalactosamine residue. The 6-0-sulfate/4-0-sulfate ratio changes during development, tumor progression and atherosclerosis (Kimata et al., 1973; Edwards and Wagner, 1988; Adany et al., 1990). Furthermore, N-acetylgalactosamine residues sulfated at both the 4 and 6 positions are present in mast cells, the glomeruli and chondrocytes (Kim and Conrad, 1982; Razin et al., 1982; Kobayashi et al., 1985). The diversity of the sulfation patterns of chondroitin sulfates must influence the physiological functions of the proteoglycans carrying them.
The sulfation at a specific position of a sugar residue in glycosaminoglycans is catalyzed by a specific sulfotransferase, whose analysis is essential for investigations of the physiological functions of glycosaminoglycans. Among various sulfotransferases involved in glycosaminoglycan biosynthesis, heparan sulfate N-sulfotransferase/N-deacetylase (Brandan and Hirschberg, 1988; Wei et al., 1993), chondroitin 6-sulfotransferase (Habuchi et al., 1993), heparan sulfate 6-sulfotransferase (Habuchi et al., 1995), heparan sulfate 2-sulfotransferase (Kobayashi et al., 1996), and heparan sulfate 3-sulfotransferase (Liu et al., 1996) have been purified to homogeneity; among them, heparin N-sulfotransferase/N-deacetylase (Eriksson et al., 1994; Orellana and Hirschberg, 1994), heparan sulfate N-sulfotransferase/N-deacetylase (Hashimoto et al., 1992), chondroitin 6-sulfotransferase (Fukuta et al., 1995), and heparan sulfate 2-sulfotransferase (Kobayashi et al., 1997) have been reported to be cloned. Chondroitin 6-sulfotransferase was cloned in the chicken by us, and exhibited keratan sulfate sulfotransferase activity, which amounted to about 10% of the principle activity (Habuchi et al., 1993, 1996; Fukuta et al., 1995). Since the mouse is a preferable system for studying the physiological functions of the products of sulfotransferase by gene manipulation including knockout, we cloned and analyzed the mouse C6ST cDNA, as well as the mode of expression.
The nucleotide sequence for mouse C6ST reported in this paper has been submitted to the GenBank/EMBL data bank with accession numbers AB008937 and AB008938 for a combined sequence of mRACE1 and mRT1, and a sequence of mL1, respectively.
Results
Cloning and structure of mouse C6ST cDNA
We used 32P-labeled chick C6ST cDNA as a probe for screening a cDNA library constructed from ATDC5 cells, which is a cell line derived from a differentiating culture of mouse AT805 teratocarcinoma cells (Atsumi et al., 1990). One positive clone, mL1, was obtained, which contained a 1283 bp cDNA (Figure 1C), out of the 2 × 106 clones screened. Then, RT-PCR and 5[prime]-RACE analyses were performed as described under Materials and Methods and resulted in isolating the mRT1 (1419 bp in Figure 1C) and the mRACE1 (397 bp in Figure 1C) cDNA fragment, respectively. The finally obtained cDNA is 2162 bp (Figure 1A). The nucleotide sequence of the mouse C6ST cDNA and the predicted amino acid sequence are shown in Figure 1A. A single open reading frame beginning at the first ATG codon predicts a protein of 472 amino acid residues with a molecular mass of 54 kDa. The sequence around the first ATG codon fits Kozak's rule (Kozak, 1986, 1997), and the upstream region contains an in-frame stop codon. There is one prominent hydrophobic segment in the amino-terminal region, 15 residues in length, that comprises amino acid residues Tyr24-Ile38 and probably encodes the transmembrane domain (Figure 1A). The mouse C6ST exhibits 71% overall sequence identity with the chicken C6ST (Figure 1B).
Figure 1. (A) Nucleotide and deduced amino acid sequences of mouse C6ST cDNA. Mouse C6ST is predicted to be composed of 472 amino acid residues. A putative transmembrane domain is underlined, and potential N-linked glycosylation sites are boxed. (B) Comparison of the amino acid sequences of mouse and chick C6ST. Identical residues are shown by asterisks, and the transmembrane domain is underlined. (C) A schematic presentation of the corresponding protein. The cytoplasmic domain, transmembrane domain and catalytic domain are shown by shaded, solid and open box, respectively.
The homology is not high in the cytoplasmic region, transmembrane region or stem region, the latter being on the luminal side next to the transmembrane region. Except for these sequences, in the majority of the luminal region (Arg132-C-terminal), 81% identity was found between the mouse and chicken enzymes. This is consistent with the view that the catalytic domain is located in this sequence.
Expression of mouse C6ST activity
The mouse C6ST cDNA was placed in the pcDNA3 (Invitrogen) expression vector and then transfected into COS-7 cells. The transfected cells expressed C6ST activity, and keratan sulfate sulfotransferase (KSST) activity was also slightly expressed (Table I). The ratio of KSST activity to C6ST activity was 0.03:1, this value being significantly lower than that observed in the cases of both the chicken and human enzymes, in which cases the ratios are 0.1:1 (Fukuta et al., 1995) and 0.3:1 (M. Fukuta and O. Habuchi, unpublished observations), respectively. In this respect, mouse C6ST appeared to exhibit low affinity to keratan sulfate.
Expression of mouse C6ST mRNA
Upon Northern blot analysis, mouse C6ST mRNA migrated as a band corresponding to 8.0 kb (Figure 2A-C). The size of the mRNA was considerably greater than that of chicken C6ST, which ranged from 2.5-5.8 kb in chondrocytes (Fukuta et al., 1995). Surprisingly, mouse C6ST mRNA expression was strongest in the spleen, followed by in the lung, eye, and stomach (Figure 2A). C6ST was constitutively expressed at low level during the mid- to late-gestation period (Figure 2B). On the other hand, C6ST was expressed in the brain in a temporally controlled manner: the expression peaked at 2 weeks after birth in the cerebellum, but at 3 weeks in the cerebrum (Figure 2C). On in situ hybridization, the anti-sense probe of mouse C6ST reacted with stromal cells in the bone marrow, and stromal cells in the marginal zone and red pulp of the spleen, but the sense probe did not (Figure 3).
Figure 2. Northern and Southern blot analyses of mouse C6ST. (A) Total RNA samples (20 µg) from various tissues of the adult mouse was analyzed. (B) Total RNA samples (10 µg) from the whole embryos on 11.5, 13.5, 15.5, and 17.5 embryonic days were analyzed. (C) Total RNA samples (10 µg) from the mouse cerebrum (cereb.) and cerebellum (cerebe.) on the postnatal 1, 2, 3 and 4 weeks were analyzed. The GAPDH cDNA probe was used to test for the equal RNA loading. The exposure time for the C6ST mRNA was 16 h in (A), and 60 h in (B) and (C), and that for GAPDH mRNA was 1 h. (D) Genomic Southern blot analysis of the mouse C6ST. Hybridization gave single bands of expected size on digestion with EcoRI, HindIII, HincII, KpnI, SacI, and EcoRV, respectively.
Figure 3. In situ localization of mouse C6ST transcripts in adult mouse spleen and bone marrow. Organs from mice (C57 BL/6J) were fixed in 4% paraformaldehyde in PBS and processed as described in Materials and methods. (A) Hematoxylin and eosin staining of the spleen section. (B) An adjacent section hybridized with the mouse C6ST antisense cRNA probe exhibits the prominent signal in the marginal zone (m) and the red pulp (r), with no signal in the white pulp (w). (C) The boxed area in B is enlarged. Positive cells are stromal cells (arrowheads). (D) Hematoxylin and eosin staining of the bone marrow section. (E) Expression of C6ST mRNA in the bone marrow. (F) Hybridization with the sense probe yields no signal. (G) The boxed area in (E) is enlarged. Stromal cells express C6ST mRNA (arrowheads). Scale bars, 50 µm. Genomic Southern blot analysis of mouse C6ST
On Southern blot analysis after digestion with restriction endonucleases, which do not cut the probe, a single band was detected (Figure 2D) with the SmaI-HincII fragment of mL1 cDNA (nucleotide numbers 1125-2103, in Figure 1A) as a probe. This finding indicates that the C6ST gene is not present in multicopies.
Table I
Plasmid
C6ST activity
(pmol/min/mg protein)KSST activitya
(pmol/min/mg protein)
None
7.1 ± 1.7
0.9 ± 0.1
pcDNA3-mC6STA
7.8 ± 1.3
0.8 ± 0.2
pcDNA3-mC6ST
231 ± 28
7.1 ± 0.7
Chromosomal localization of the mouse C6ST gene
Fluorescence in situ hybridization analysis revealed the mouse C6ST gene is located in the C region of chromosome 9 (Figure 4).
Figure 4. Fluorescence in situ hybridization with mouse C6ST cDNA as a probe. Two sections of normal mouse metaphase spreads are shown. (a) and (b); arrows indicate the fluorescent signals on chromosome 9C.
Discussion
Since sulfotransferases forming glycosaminoglycans have become the subject of molecular biology analysis quite recently, C6ST is the first mammalian sulfotransferase whose primary structure has been compared with that of the chick counterpart, and is among the two examples in which the chromosome localization has been determined: the other one is the human heparan sulfate-N-deacetylase/N-sulfotransferase (Dixon et al., 1995).
Generally, the homology among different species is limited to functionally critical domains of the molecules. The amino acid sequence of the catalytic domain of mouse C6ST shows 81% sequence identity with that of the chick enzyme in spite of the wide evolutionary gap. This reveals the significant evolutionary pressure for maintaining the primary structure of the enzyme. The expression of mouse C6ST cDNA in COS cells revealed the remarkable acceptor specificity for position 6 of the N-acetylgalactosamine residue of chondroitin, while very low enzyme activity toward keratan sulfate was observed. The availability of the conserved and non-conserved amino acid information opens the way for investigation of the structure and function relationships of this enzyme by means of recombinant DNA techniques, and also opens a way for isolating the C6ST gene family through homology cloning.
During the evolution of the glycosyltransferase genes, exon-shuffling and/or gene duplication events played roles in the generation of multiple gene families (Schachter, 1994). Thus, some segments of amino acid sequence similarity are found within a family which shares similar acceptor and/or donor substrates. Common amino acid sequences would be expected within sulfotransferases which share PAPS as a donor substrate. However, the three cloned sulfotransferases do not show any sequence homology. This suggests that each sulfotransferase has evolved from a different gene. The human heparan sulfate-N-deacetylase/N-sulfotransferase gene is in chromosome 5q32-q33.1 (Dixon et al., 1995), whose mouse equivalent is in chromosome 18. Thus, the two sulfotransferase genes so far mapped, i.e., these for C6ST and heparan sulfate-N-deacetylase/N-sulfotransferase, are in different chromosomes. Further genomic analysis of other sulfotransferases may provide additional information on the evolution of this gene family.
Murine cartilages, whose major proteoglycan is aggrecan, have chondroitin sulfate chains mostly 4-sulfated and have few keratan sulfate (Venn and Mason, 1985; Rostand et al., 1986). There is a good consistency of the properties and distribution of murine C6ST with the structural characteristics of murine cartilage glycosaminoglycans. Thus, murine C6ST had less KSST activity as compared to human or chicken C6ST. Furthermore, murine chondrocytes expressed very low levels of C6ST (K.Uchimura and T.Muramatsu, unpublished observations).
Generally speaking, murine C6ST showed spatially restricted expression, and the strongest expression occurring in the spleen, as revealed on Northern blot analysis. This finding is interesting from the viewpoint of its possible role in hematopoiesis. On in situ hybridization, a strong positive signals was also observed in bone marrow cells. These cells were not abundant, and were localized in the marginal zone and red pulp of the spleen. In terms of morphology, they are distinct from megakaryocytes or other hematopoietic cells. From the characteristic distribution and morphology, these cells may belong to a subpopulation of stromal cells, e.g., blanket cells that are implicated in hematopoiesis by providing hematopoietic stem cells with a suitable microenvironment, rather than in the construction of the tissue skeleton (Tavassoli, 1989; Roecklein and Torok-Storb, 1995). The present data are consistent with a previous report showing that chondroitin 6-sulfate comprises 80% of the glycosaminoglycan expressed in bone marrow stromal cells in the rabbit (Okayama et al., 1988). There are increasing lines of evidence that the combination of hematopoietic factors and stem cells is not sufficient for in vivo hematopoiesis. For example, stromal cells are required, in addition to erythropoietin, for erythrocytic hematopoiesis (Dexter et al., 1977). Chondroitin 6-sulfate, the product of C6ST, plays a critical role in hematopoiesis in the mouse bone marrow and spleen by providing a suitable microenvironment. Further studies on the C6ST positive cells in the spleen and bone marrow, and gene manipulation of C6ST, e.g., gene targeting, may provide new insights into hematopoiesis and the physiological role of C6ST.
Materials and methods
Assaying of chondroitin 6-sulfotransferase
The standard reaction mixture comprised 2.5 µmol of imidazole-HCl, pH 6.8, 1.25 µg (for chondroitin) or 3.75 µg (for keratan sulfate) of protamine chloride, 0.1 µmol of dithiothreitol, 25 nmol (as glucuronic acid) of chondroitin or 25 nmol (as glucosamine) of keratan sulfate, 25 pmol of [35S]PAPS (2.5 × 105 c.p.m.), and the enzyme in a final volume of 50 µl. The reaction mixtures were incubated at 37°C for 20 min, and the reaction was stopped by boiling for 1 min. Subsequently, 35S-labeled glycosaminoglycans were isolated by precipitation with ethanol and then gel chromatography as described previously (Habuchi et al., 1993). The radioactivities of fractions were determined with a scintillation counter (Beckman LS-5000TD).
Construction of a mouse ATDC5 cell cDNA library
Total RNA was prepared from ATDC5 cells, which is a cell line derived from a differentiating culture of mouse AT805 teratocarcinoma cells (Atsumi et al., 1990) (a generous gift from Dr. Seki, University of Osaka). Poly(A)+ RNA was purified by oligo(dT)-cellulose column chromatography. The synthesis of oligo(dT)- or random-primed cDNA, and ligation of the cDNA to EcoRI-digested [lambda]gt11 vector arms (Pharmacia) were carried out with a TimeSaver cDNA synthesis kit (Pharmacia). The ligated cDNA was used for the construction of a cDNA library as described previously (Fukuta et al., 1995).
Screening of the mouse ATDC5 cDNA library
Approximately 2 × 106 plaques of the mouse ATDC5 cDNA library were screened. Nylon membrane (Hybond-N+ Amersham) replicas of the plaques were prehybridized in a solution comprised of 50% formamide, 5× SSPE, 5× Denhardt's solution, 0.5% SDS, 0.04 mg/ml of denatured salmon sperm DNA, and 0.004 mg/ml of E.coli DNA for 3 h at 42°C, after the alkali fixation method. Hybridization was carried out in the same buffer overnight at 42°C. The chicken C6ST cDNA 2354 bp fragment (Fukuta et al., 1995) was used as a probe for the cDNA library. 32P-Labeling of the probe was performed with a Random labeling kit (TAKARA). The membranes were washed at 55°C in 1× SSPE, 0.1% SDS, and subsequently in 0.1× SSPE, 0.1% SDS. Positive clones were detected by autoradiography.
Construction of pcDNA3-mC6ST and pcDNA3-mC6STA
A cDNA fragment which encodes the open reading frame of the mouse C6ST, 472 amino acid residues, was amplified by the RT-PCR method following reverse-transcription (SUPERSCRIPT II RT, GIBCO BRL) with an oligo(dT)-primer, using mouse spleen total RNA as a template. The sense primer, 5[prime]-CAGAATTCATGGAGAAAGGACTCGCTTTGC-3[prime], and the antisense primer, 5[prime]-CGGAATTCCTACGTGACCCAGAAGGTGCC-3[prime], were designed based on the sequence of mouse C6ST genomic DNA (K.Uchimura and T.Muramatsu, unpublished observations) and mL1 cDNA (Figure 1C), respectively. Then, both primers were used for PCR amplification, which was carried out at 94°C for 3 min, with 35 cycles of 94°C for 0.5 min, 64°C for 1 min and 72°C for 1.5 min. The PCR product, mRT1 (Figure 1C) was digested with EcoRI and then subcloned into a site of pBluescript II SK- (STRATAGENE) followed by determination of the DNA sequence (ABI) (Sanger et al., 1977). Then, the EcoRI fragment was subcloned into the pcDNA3 expression vector (Invitrogen). Recombinant plasmids were analyzed by restriction mapping to confirm the correct orientation of pcDNA3-mC6ST. The plasmid that contained the EcoRI fragment in the reverse orientation, termed pcDNA3-mC6STA, was used in control experiments.
Transient expression of mouse chondroitin 6-sulfotransferase cDNA in COS-7 cells
COS-7 cells (3 × 106 cells per 10 cm-dish) were transfected with 15 µg of pcDNA3-mC6ST or pcDNA3-mC6STA by the DEAE-dextran method (Aruffo, 1991). After 65 h incubation, the cells were washed with PBS, scraped off, and then homogenized with a Dounce homogenizer in 1.5 ml/dish of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.2, and 0.5% Triton X-100. The homogenates were centrifuged at 10,000×g for 15 min, and then the activities of C6ST and KSST in the supernatant fractions were measured as described above.
Northern blot analysis
Total RNA was prepared from mouse (C57 BL/6J) tissues as described (Chomczynski and Sacchi, 1987), and then electrophoresed on a 1.0% agarose gel containing 5% formaldehyde (v/v). The radioactive probe was the same as that used for the screening of the mouse ATDC5 cell cDNA library described above. The blots were washed at 55°C in 2× SSPE, 0.1% SDS, and finally in 0.1× SSPE, 0.1% SDS at 55°C. The membrane was exposed to a BAS-imaging plate and then the radioactivity on the membrane was determined with a BAS2000 radioimage analyzer (Fuji Film).
Genomic Southern analysis
Genomic DNA (10 µg) prepared from the mouse D3 cell line, which was derived from the mouse 129 line, was digested for 4 h with appropriate restriction enzymes. Hybridization was performed as described above for Northern blot analysis, except for the probe. The SmaI-HincII fragment of mL1 cDNA (nucleotide numbers 1125-2103 in Figure 1A) was used as the 32P-labeled probe.
In situ hybridization
Mice (C57 BL/6J) were anesthetized with Nembutal, and then perfused with 10 ml of saline followed by 50 ml of 4% paraformaldehyde in PBS. Tissues were removed and fixed in 4% paraformaldehyde in PBS at 4°C overnight. After embedding in paraffin, sections of 5 µm thickness were cut, placed on saline-coated slide glasses, and then subjected to hematoxylin-eosin staining or in situ hybridization. As the C6ST probe, a SmaI-ApaI fragment of mL1 cDNA (nucleotide numbers 1125-1806 in Figure 1A) was subcloned into a site of pBluescript II KS+ (STRATAGENE). Sense and antisense probes were prepared a DIG RNA labeling kit (Boehringer Mannheim, Germany). In situ hybridization was performed essentially as described previously (Kurosawa et al., 1997).
Chromosomal mapping of the mouse C6ST gene
Mouse lymphocyte cultures and fluorescence in situ hybridization (FISH) were established following methods for human lymphocytes with slight modifications (Kaname et al., 1993). In brief, lymphocytes were isolated from the spleen of adult C57BL/6J male mice, and then cultured with 30 ml RPMI1640, 20% fetal bovine serum, 3 µg/ml concanavalin-A (type IV-S, Sigma), 10 µg/ml lipopolysaccharide (Sigma), and 5 × 10-5 M mercaptoethanol. Thymidine (300 µg/ml) was added to the cultures 44 h later and continued for 14 h. The cells were washed with RPMI 1640 medium, and then were cultured with the medium containing 20 µg/ml BrdU for 4.5 h. At 30 min before harvesting, colcemid was added. Chromosome preparation was as described. A 1.4 kbp C6ST cDNA fragment, mRT1 (Figure 1C) was used as a probe. Both probe labeling procedure by biotin and in situ hybridization were as described (Kaname et al., 1993). Biotin was detected by binding with fluorescein isothiocyanate-labeled avidin with an Olympus AX70 and Olympus filter combination U-MWIB (excitation at 460-490 nm; Kaname et al., 1993). The chromosomes were photographed with Ektachrome Dyna400 film and were identified by banding patterns.
5[prime]-Rapid amplification of cDNA ends (RACE) analysis
Amplification of the 5[prime]end of mouse C6ST cDNA was performed essentially according to the procedure of Frohman et al. cDNA was synthesized by reverse transcription (SUPERSCRIPT II RT, GIBCO BRL) of 2.5 µg of mouse spleen poly(A)+RNA using a primer C6-SP0, 5[prime]-CTGCTTCAGCTTGTCGGAGACC-3[prime] (complementary to the mouse C6ST coding strand, nucleotide numbers 376-397 in Figure 1A). The excess primers and deoxynucleotides were removed by passage of the cDNA through a MicroSpin S-400 column (Pharmacia). The cDNA was A-tailed with 0.6 U of terminal deoxynucleotidyltransferase (Boehringer Mannheim) using 0.05 mM dATP. Two consecutive PCRs were performed with two nested sets of primers. For pair 1, the forward primer was NotI-(dT)18 (Pharmacia), and the reverse primer was C6-SP1, 5[prime]-AGGAACAAGACGTATCTGCCTCGA-3[prime] (complementary to the coding strand, nucleotide numbers 295-318 in Figure 1A). For pair 2, the forward primer was as above but without the T-tail, 5[prime]-AACTGGAAGAATTCGCGGCCGCAGGAA-3[prime], and the reverse primer was C6-SP2 (5[prime]-ATCTTTAGGCTGTGTACAAGGTCC-3[prime]; complementary to nucleotides 271-294 of mouse C6ST cDNA in Figure 1A). The cDNA was amplified for 35 cycles of a step program (94°C, 40 s; 55°C, 40 s; 72 °C 60 s). The amplification products were digested with NotI and NcoI and then subcloned into pGEM-5Zf (Promega) followed by determination of the DNA sequence (ABI) (Sanger et al., 1977).
Acknowledgments
We thank Dr. Masafumi Ito for histological evaluation, and Ms. Asako Horisawa and Ms. Akemi Miyata for their secretarial assistance. This work was supported by the Grants-in-Aid for Scientific Research No. 05274103 and 90001535 from the Ministry of Education, Science, and Culture, Japan, and a grant from the Japanese Science and Technology Agency. K.U. is a Research Fellow of the Japan Society for the Promotion of Science.
Abbreviations
C6ST, chondroitin 6-sulfotransferase; KSST, keratan sulfate sulfotransferase; PAPS, 3[prime]-phosphoadenosine 5[prime]-phosphosulfate; CSPG, chondroitin sulfate proteoglycan; RT-PCR, reverse transcription-polymerase chain reaction; 5[prime]-RACE, 5[prime]-rapid amplification of cDNA ends; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
References
4To whom correspondence should be addressed
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S. Bhakta, A. Bartes, K. G. Bowman, W.-M. Kao, I. Polsky, J. K. Lee, B. N. Cook, R. E. Bruehl, S. D. Rosen, C. R. Bertozzi, et al. Sulfation of N-Acetylglucosamine by Chondroitin 6-Sulfotransferase 2 (GST-5) J. Biol. Chem., December 15, 2000; 275(51): 40226 - 40234. [Abstract] [Full Text] [PDF]
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T. O. Akama, J. Nakayama, K. Nishida, N. Hiraoka, M. Suzuki, J. McAuliffe, O. Hindsgaul, M. Fukuda, and M. N. Fukuda Human Corneal GlcNAc 6-O-Sulfotransferase and Mouse Intestinal GlcNAc 6-O-Sulfotransferase Both Produce Keratan Sulfate J. Biol. Chem., May 4, 2001; 276(19): 16271 - 16278. [Abstract] [Full Text] [PDF]
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K. Uchimura, K. Kadomatsu, H. Nishimura, H. Muramatsu, E. Nakamura, N. Kurosawa, O. Habuchi, F. M. El-Fasakhany, Y. Yoshikai, and T. Muramatsu Functional Analysis of the Chondroitin 6-Sulfotransferase Gene in Relation to Lymphocyte Subpopulations, Brain Development, and Oversulfated Chondroitin Sulfates J. Biol. Chem., January 4, 2002; 277(2): 1443 - 1450. [Abstract] [Full Text] [PDF]
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