Department of Life Science, Aichi University of Education, Igaya-cho, Kariya, Aichi 4488542, Japan
Received on June 3, 1999; revised on July 19, 1999; accepted on July 21, 1999.
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Abstract |
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Key words: sialyl N-acetyllactosamine/keratan sulfate/sulfotransferase/fetuin/sialyl Lewis x
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Introduction |
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Although the sulfotransferases involved in the sulfation of oligosaccharides were found to share some molecular features such as being type II transmembrane protein and having putative PAPS-binding sites (Kakuta et al., 1998) with glycosaminoglycan sulfotransferases so far cloned, our knowledge about the relation of the specificity between the two sulfotransferase groups has been limited. We previously showed that chondroitin 6-sulfotransferase (C6ST), which transfers sulfate to position 6 of GalNAc residues of chondroitin (Habuchi et al., 1993
), transferred sulfate to position 6 of Gal residue of keratan sulfate (Fukuta et al., 1995
; Habuchi et al., 1996
) and position 6 of sialyl N-acetyllactosamine oligosaccharides (Habuchi et al., 1997
). A sulfotransferase which transferred sulfate to position 6 of nonreducing terminal GlcNAc residue and promoted the formation of 6-sulfo sialyl Lewis x in the transfected COS-7 cells was cloned and was found to have a significant sequence homology with C6ST (Uchimura et al., 1998
). A putative sulfotransferase, NSIST, showing relatively high sequence homology to C6ST was cloned by expression cloning using mAb 3B3, which recognized a carbohydrate-containing epitope expressed on dystroglycan and other constituent of the postsynaptic membranes of Torpedo electric organ (Nastuk et al., 1998
). The epitope recognized by mAb 3B3 was sensitive to the digestion with neuraminidase but not to the digestion with chondroitinase, keratanase or heparitinase (Bowe et al., 1994
).
Keratan sulfate Gal-6-sulfotransferase (KSGal6ST), which catalyzes transfer of sulfate to position 6 of Gal residue of keratan sulfate, was cloned from the fetal human brain library (Fukuta et al., 1997). When the cloned cDNA was transfected in COS-7 cells, the expressed sulfotransferase transferred sulfate to position 6 of Gal residue of keratan sulfate, but not to chondroitin. Recently KSGal6ST was shown to be involved in the formation of L-selectin ligand (Bistrup et al., 1999
). In this paper we investigated whether KSGal6ST could transfer sulfate to sialyl N-acetyllactosamine oligosaccharides in vitro as observed in C6ST. We also investigated whether oligosaccharides attached to intact fetuin could be sulfated by KSGal6ST. As a result we found that sialyl N-acetyllactosamine oligosaccharides were found to serve as acceptors for KSGal6ST as efficiently as keratan sulfate.
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Results |
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Discussion |
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A microsomal sulfotransferase preparation obtained from the rat spleen was reported to have the ability to sulfate position 6 of Gal residues of glycoprotein oligosaccharides (Spiro and Bhoyroo, 1998). The substrate specificities of the rat spleen sulfotransferase was essentially the same as those of KSGal6ST; the rat spleen sulfotransferase catalyzed the transfer of sulfate to 3'SLN and fetuin oligosaccharides but not to SLex tetrasaccharide. In the human tissues, KSGal6ST mRNA was expressed in the brain (Fukuta et al., 1997
) and in the various immunologically relevant tissues including spleen (Figure 7). On the other hand, the activity of 6-O-sulfation of the Gal residue was also found in various tissues including the brain (Spiro and Bhoyroo, 1998
). The similarity in the specificity and distribution between KSGal6ST and the rat Gal-6-O-sulfotransferase suggests that the rat Gal-6-O-sulfotransferase activity may be carried by a rat counterpart of KSGal6ST or by a hypothetical isoform of KSGal6ST with the specificity similar to that of KSGal6ST. However, the possibility that the Gal-6-O-sulfotransferase activity in the spleen may be partly due to C6ST, since C6ST is also expressed in the spleen (Fukuta et al., 1998
).
GlyCAM-1 is one of high endothelial venule-associated ligands (Imai et al., 1991; Lasky et al., 1992
) and was reported to contain O-linked sugar chains containing sulfated sialyl Lewis x structure (Hemmerich and Rosen, 1994
; Hemmerich et al., 1995
). Structural analysis of GlyCAM-1 has identified Gal(6SO4) and GlcNAc(6SO4) as the major sulfated sugars (Hemmerich et al., 1994
). Recently, KSGal6ST has been shown to be able to sulfate L-selectin ligand when COS cells with a cDNA encoding a GlyCAM-1/IgG chimera were transfected with a cDNA encoding KSGal6ST (Bistrup et al., 1999
). When CHO/fucosyltransferase VII/core 2 ß1-6 N-acetylglucosaminyl transferase were transfected with KSGal6ST and HEC-GlcNAc6ST cDNAs (singly or in combination), the resulting cells showed positive binding to L-selectin/IgM. The combination of the two sulfotransferase cDNAs synergistically enhanced the binding of L-selectin/IgM. These interesting observations may be correlated with our findings that sulfation of Gal residues with KSGal6ST was strongly stimulated by the presence of sulfate group on the adjacent GlcNAc residue. Since KSGal6ST showed no activity toward SLex in vitro, introduction of sulfate to Gal residue catalyzed by KSGal6ST should precede the introduction of fucose.
Chiba et al. reported the structure of major oligosaccharides bound to -dystroglycan, Neu
2-3Galß1-4GlcNAcß1-2Man (Chiba et al., 1997
). They showed that, even after the neuraminidase digestion, a significant portion of the oligosaccharide fractions released by ß-elimination from
-dystroglycan was still absorbed to the anion exchange column. The structure of the neuraminidase-resistant negatively charged oligosaccharide is not known. It remains to be investigated whether sulfate group is present on the dystroglycan oligosaccharides. A mAb 3B3, which recognized agrin-binding proteins, was raised using the synaptic membrane proteins as the antigen (Bowe et al., 1994
). One of the epitope-bearing proteins was found to be dystroglycan. The reactivity of the mAb was decreased by the digestion with neuraminidase but not affected by the digestion with chondroitinase or keratanase, suggesting that the epitope for the mAb may not be glycosaminoglycans but oligosaccharides with sialic acid. NSIST cDNA was cloned from Torpedo electric organ by detecting the expression of the epitope recognized by mAb 3B3 on the surface of COS cells (Nastuk et al., 1998
). NSIST shows significant sequence homology to both C6ST and KSGal6ST; identity of amino acid sequence between NSIST and chick C6ST is 56% and identity of amino acid sequence between NSIST and human KSGal6ST is 38%. Such similarities in the amino acid sequence suggest that NSIST may be a novel sulfotransferase and that, in addition to sialic acid, sulfate group may be involved in the formation of the structure of the epitope for mAb 3B3. Although the substrate specificity of NSIST has not been revealed yet, it may possibly be similar to those of KSGal6ST.
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Materials and methods |
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Construction of pCXNKSGal6ST and preparation of keratan sulfate Gal 6-sulfotransferase from COS-7 cells transfected with pCXNKSGal6ST
The human KSGal6ST cDNA was inserted in an expression vector pCXN2 (pCXN2 was generously donated from Dr. Jun-ichi Miyazaki, Department of Disease-related Gene Regulation, Faculty of Medicine, University of Tokyo) and pCXNKSGal6ST was constructed as described previously (Fukuta et al., 1997). Transient expression of KSGal6ST cDNA in COS-7 cells and the preparation of the partially purified KSGal6ST with DEAE-Sephadex A-50 and heparin-Sepharose CL 6B were described previously (Fukuta et al., 1997
).
Preparation of a FLAG-KSGal6ST fusion protein
Recombinant KSGal6ST was also expressed as a fusion protein with FLAG peptide. A DNA fragment which codes for full open reading frame was amplified by PCR using human KSGal6ST cDNA as a template. The 5' and 3' primers were CGCAAGCTTATGCAATGTTCCTGGAAGGCC and CAGGAATTCTCACGAGAAGGGGCGGAAGTC, respectively. At the 5'-end of the oligonucleotide primers, restriction enzyme recognition sites were introduced; HindIII site for the sense primer and EcoRI site for the antisense primer. The PCR product was digested with EcoRI and HindIII, and subcloned into these sites of pFLAG-CMV-2 plasmid (Kodak, New Haven, CT). The resulting plasmid was transfected in COS-7 cells as described previously (Fukuta et al., 1997) and the fusion protein produced was extracted from the cells with buffer B containing 10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 10 mM MgCl2, 2 mM CaCl2, 0.5% Triton X-100, 20% glycerol by gentle shaking on a rotatory shaker for 30 min at 4°C. The extracts were centrifuged at 10,000 x g for 10 min. The supernatant fraction (crude extract) was applied to an anti-FLAG mAb-conjugated affinity column (Kodak) equilibrated with the buffer B. The absorbed materials were eluted with FLAG peptide under the conditions recommended by the manufacturer.
Incorporation of 35SO4 into oligosaccharides, keratan sulfate, and fetuin
The reaction mixture contained 2.5 µmol of imidazole-HCl, pH 6.4, 0.5 µmol of CaCl2, 0.1 µmol dithiothreitol, 0.025 µmol of oligosaccharides or 0.025 µmol (as glucosamine) of keratan sulfate or 0.025 µmol (as sialic acid) of fetuin, 50 pmol [35S]PAPS (about 5 x 105 cpm), and the partially purified KSGal6ST or FLAG-KSGal6ST in a final volume of 50 µl. The reaction mixtures were incubated at 37°C for 60 min for the partially purified KSGal6ST or 20 min for FLAG-KSGal6ST and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. After the reaction was stopped, 35S-labeled products were separated from 35SO4 and [35S]PAPS by Superdex 30 gel chromatography, and the radioactivity was determined. As a control, reaction mixture without acceptors was applied to the Superdex 30 column, and the radioactivity observed in the control was subtracted. When fetuin was used as an acceptor, the reaction was stopped by placing on ice and the reaction mixture was immediately injected into a Superdex 30 column. When keratan sulfate was used as acceptor, sulfotransferase reaction proceeded linearly up to 1.5 µg of the partially purified KSGal6ST and up to 60 min under the conditions described above.
Neuraminidase digestion and NaBH4 reduction of 35S-labeled 3'SLN
35S-Labeled 3'SLN was prepared using the partially purified KSGal6ST (2 µg as protein) as described above except that concentration of [35S]PAPS was increased to 6.8-fold and incubation was carried out for 20 h. The 35S-labeled 3'SLN eluted from the Superdex 30 column was lyophilized, purified by paper electrophoresis, and digested with neuraminidase as described below. Aliquot of the sample after neuraminidase digestion was dried, dissolved in 10 µl of 0.5 M NaBH4/0.2 M Na2CO3, pH 10.2. After 2 h at 0°C, 10 µl of the same solution was added and the reduction was continued for further 2 h at 0°C. After the reduction, excess NaBH4 was destroyed by addition of 10 µl of 3 M acetic acid. The reaction mixtures were dried under N2 stream, dissolved in a small volume of water, and applied to a Dowex 50 H+ column (bed volume 0.2 ml). The flow through fraction was dried and suspended in methanol. Boric acid was removed as methyl ester by drying in vacuo.
N-Deacetylation, deamination, and NaBH4 reduction of 35S-labeled oligosaccharides released from the sulfated fetuin
35S-Labeled oligosaccharides were released from the sulfated fetuin by the digestion with Actinase E and neuraminidase, and separated with Superdex 30 chromatography. The oligosaccharides were then subjected to N-deacetylation, deaminative cleavage and NaBH4 reduction as described (Shaklee and Conrad, 1986; Habuchi et al., 1996
) using nonradioactive NaBH4. The final sample was dissolved in 60 µl of water and spotted on a strip of Whatman 3 paper and developed with the solvent described below.
Digestion with neuraminidase, ß-galactosidase, keratanase I, and Actinase E
Digestion with neuraminidase was carried out for 60 min at 37°C in the reaction mixture containing, in a final volume of 50 µl, 35S-labeled oligosaccharide, 5 µmol of potassium acetate buffer, pH 6.5, 0.5 µmol of CaCl2 and 20 mU of neuraminidase (Kiyohara et al., 1974). Reaction mixture for ß-galactosidase digestion contained 35S-labeled L1L1, 25 nmol of L1L1, 2.5 µmol of sodium acetate buffer, pH 5.5, and 5 mU of the enzyme in a final volume of 50 µl (Kiyohara et al., 1976
). The reaction mixtures were incubated at 37°C for 20 h. Keratanase I digestion was carried out for 15 h at 37°C in the reaction mixture containing, in a final volume of 25 µl, 35S-labeled keratan sulfate or 35S-labeled fetuin, 1.25 µmol of Tris-HCl, pH 7.4, 100 mU of keratanase I, and protease inhibitors (50 µM Na
-p-tosyl-L-lysine chloromethyl ketone, 30 µM N-tosyl-L-phenylalanine chloromethyl ketone, 300 µM phenylmethyl sulfonyl fluoride). Reaction mixture for Actinase E contained 35S-labeled fetuin, 2 µmol of Tris-HCl, pH 8.0 and 250 µg of Actinase E in a final volume of 40 ml. The reaction mixtures were incubated at 37°C for 20 h.
Superdex 30 chromatography, paper electrophoresis, paper chromatography, and HPLC
Hiload Superdex 30 16/60 column was equilibrated with 0.2 M NH4HCO3. The flow rate was 1 ml/min; 1 ml or 0.5 ml fractions were collected, mixed with 4 ml Clearsol (Nakarai Tesque, Kyoto), and the radioactivity was determined. Oligosaccharides were monitored by absorption at 210 nm. Paper electrophoresis was carried out on Whatman No. 3 paper (2.5 cm x 57 cm) in pyridine/acetic acid/water (1:10:400, by volume, pH 4) at 30 V/cm for 40 min. Samples for paper chromatography was spotted on a Whatman No. 3 paper (2.5 cm x 57 cm) and developed with 1-butanol/acetic acid/1 M NH3 (3:2:1, by volume). The dried paper strips after paper electrophoresis or paper chromatography were cut into 1.25 cm segments and radioactivity was determined by liquid scintillation counting. HPLC separation of 35S-labeled disaccharide alditols was carried out on a Whatman Partisil 10-SAX column (4.5 x 25 cm) equilibrated with 5 mM KH2PO4. The column was developed with 5 mM KH2PO4. The flow rate was 1 ml/min and the column temperature was 40°C; 0.5 ml fractions were collected, mixed with 4 ml Clearsol, and the radioactivity was determined.
Determination of glucosamine and sialic acid
The glucosamine contents of oligosaccharides were determined by the Elson-Morgan method as modified by Strominger et al. (Strominger et al., 1959) after hydrolysis of the glycosaminoglycans with 6 M HCl at 100°C for 4 h. Sialic acid was determined by thiobarbituric acid method (Aminoff, 1961
) after hydrolysis with 0.1 M H2SO4 at 80°C for 60 min.
Northern blot hybridization
Human Multiple Tissue Northern Blot Filters (Clontech), on which 2 µg poly (A)+ RNAs from various adult human tissues were blotted, were prehybridized in a solution containing 50% formamide, 5x SSPE, 5x Denhardts solution, 0.5% SDS, and 0.1 mg/ml of denatured salmon sperm DNA for 3 h at 42°C. Hybridization was carried out in the same buffer containing 32P-labeled probe for 16 h at 42°C. The radioactive probe was prepared from the EcoRI fragment containing 2415 bp cDNA (Fukuta et al., 1997) by the random oligonucleotide-primed labeling method using [
-32P]dCTP (Amersham) and a DNA random labeling kit (Takara Shuzo). The filters were washed at 65°C in 2x SSPE, 0.1% SDS, and subsequently in 1x SSPE, 0.1% SDS. The membrane was exposed to x-ray film for 26 h with an intensifying screen at 80°C.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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