Glycobiology Program, Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 USA
Received on December 22, 2000; revised on January 19, 2001; accepted on January 23, 2001.
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Abstract |
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Key words: GalNAc 4-O-sulfotransferase/pituitary hormone glycoproteins/mucin-type O-glycans/HNK-1 sulfotransferase gene family
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Introduction |
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Sulfation of carbohydrates is carried out by a sulfotransferase that catalyzes a specific reaction on specific acceptors. These sulfotransferases have type II membrane topology and almost exclusively function in the Golgi apparatus. Molecular cloning of these sulfotransferases was initially successful for chondroitin 6-O-sulfotransferase and keratan sulfate 6-O-sulfotransferase based on the amino acid sequences of purified enzymes (Fukuta et al., 1995, 1997). Although it was not apparent that these sulfotransferases share homologous sequences, molecular cloning of HNK-1 sulfotransferase (HNK-1ST) revealed that there is a homologous sequence motif among the sulfotransferases cloned to date (Ong et al., 1998
). This motif, ZZRDPXXXZ, where X and Z denote any amino acid and a hydrophobic amino acid, respectively, was then found to be a part of the 3'-phosphate binding site for the donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) (Kakuta et al., 1997
, 1998; Ong et al., 1999
). Crystallographic studies also demonstrated that the amino acid sequences responsible for binding to 5'-phosphosulfate are conserved among different sulfotransferases (Kakuta et al., 1998
).
Based on the presence of the weak but discernible similarity among different sulfotransferases, additional sulfotransferases have been identified by their similarity to previously cloned sulfotransferases. One such example is the molecular cloning of an L-selectin ligand sulfotransferase (LSST or HEC-GlcNAc6ST) that adds a sulfate to the 6-position of N-acetylglucosamine, leading to the formation of 6-sulfo sialyl Lewis x, NeuNAc2
3Galß1
4[Fuc
1
3(sulfo
6)]GlcNAcß1
R (Hiraoka et al., 1999
; Bistrup et al., 1999
), which functions as an L-selectin ligand. This cloning was based on its similarity to chondroitin 6-O-sulfotransferase (Fukuta et al., 1995
) and keratan sulfate 6-O-sulfotransferase (Fukuta et al., 1997
).
Recent studies demonstrated that chondroitin 4-O-sulfotransferases (C4ST) could be cloned based on their similarity to HNK-1ST (Hiraoka et al., 2000; Yamauchi et al., 2000
). HNK-1ST adds a sulfate to the 3-position of glucuronic acid (GlcA), which is in turn linked to the 3-position of galactose in the context of N-acetyllactosamine. On the other hand, C4ST adds a sulfate to the 4-position of N-acetylgalactosamine, which is linked to the 4-position of GlcA. Moreover, HNK-1ST adds a sulfate to GlcA at the nonreducing end, while C4ST apparently adds sulfate groups to a chondroitin polymer. These results indicate that sulfotransferases, utilizing entirely different acceptors, can be related to each other. Similarly, a novel chondroitin 6-O-sulfotransferase, chondroitin 6-O-sulfotransferase-II (Kitagawa et al., 2000
), was found to be related more to GlcNAc-6-O-sulfotransferase, which adds a sulfate at C-6 of the nonreducing terminal N-acetylglucosamine (Uchimura et al., 1998
), than the previously cloned chondroitin 6-O-sulfotransferase (Fukuta et al., 1995
).
During the molecular cloning of C4ST-1 and C4ST-2, we noticed that there are two additional DNA sequences in the expression sequence tag (EST) and genomic DNA database that are related to HNK-1ST, C4ST-1, and C4ST-2. Expression of full-length cDNAs revealed that they do not encode HNK-1ST or C4ST. Instead, we herein demonstrate that these cDNAs encode novel N-acetylgalactosamine 4-O-sulfotransferases that add a sulfate to the 4-position of N-acetylgalactosamine in GalNAcß14GlcNAcß1
R attached to pituitary hormones (Green et al., 1985
; Skelton et al., 1991
; Smith et al., 1993
). Moreover, we found that these two GalNAc-4-O-sulfotransferases (GalNAc4ST-1 and GalNAc4ST-2) can add sulfate to GalNAcß1
4GlcNAcß1
R in both N-glycans and core 2 branched O-glycans. The expression of these two enzymes exhibits reciprocal tissue distribution, indicating that GalNAc4ST-1 and -2 play complementary roles in different tissues.
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Results |
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Comparison of the amino acid sequences
The comparison of the amino acid sequences of GalNAc4ST-1, GalNAc4ST-2, C4ST-1, C4ST-2, and HNK-1ST is shown in Figure 2. The amino acid sequence of GalNAc4ST-1 is more homologous to GalNAc4ST-2 (63.7% identity in 278 amino acids) than that between C4ST-1 and C4ST-2 (41.8% identity). Both GalNAc4ST-1 and -2 is related slightly more to C4ST-1 (4042% identity) than HNK-1ST (3237% identity) or C4ST-2 (3336% identity). Figure 2 also illustrates that all members of the HNK-1ST gene family are highly homologous to each other in the catalytic domains. As described previously (Hiraoka et al., 2000), the 5'-phosphosulfate binding and 3'-phosphate binding sites are well conserved. Moreover, three additional regions (A, B, and C) are conserved among all these sulfotransferases, whereas the fourth, fifth, and sixth homologous regions (D, E, and F) are only conserved between GalNAc4ST-1 and -2 (Figure 2). These conserved regions (AF) likely correspond to domain structures that are important for the proper sterical structure maintained in all of the HNK-1ST gene family (A, B, and C) and both GalNAc4ST-1 and -2 (D, E, and F).
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We then tested GalNAcß14GlcNAcß1
2Man
1
6Manß1
octyl as an acceptor, because GalNAc4-O-sulfotransferase and chondroitin 4-O-sulfotransferase catalyze similar reactions on similar acceptors. As shown in Figure 3, both GalNAc4ST-1 and GalNAc4ST-2 exhibited significant activity on this acceptor. Similarly, GalNAc4ST-1 and -2 added sulfate to GalNAcß1
4GlcNAcß1
6Man
1
6Manß1
octyl. Moreover, both enzymes exhibited strong activity toward a core 2based O-glycan, GalNAcß1
4GlcNAcß1
6(Galß1
3) GalNAc
1
octyl (Figure 3). In contrast, none of the oligosaccharides containing galactose or N-acetylglucosamine as terminal sugars served as an acceptor. These results indicate that GalNAc4ST-1 and -2 act on GalNAcß1
4GlcNAcß1
R in both N- and O-glycans.
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Identification of reaction products
To formally demonstrate that GalNAc4ST-1 and GalNAc4ST-2 add a sulfate to the 4-position of GalNAc residues, GalNAcß14GlcNAcß1
2Man
1
6Manß1
octyl was incubated with [35S]-PAPS and the enzyme preparation. The reaction product was purified by a Sep-Pak C18 cartridge column and then subjected to Bio-Gel P-4 gel filtration or QAE-Sephadex column chromatography. As shown in Figure 4A, the product eluted as a monosulfated compound on QAE-Sephadex column chromatography. The same product eluted at an elution position close to that of GalNAcß1
4GlcNAcß1
2Man
1
6Manß1
octyl in Bio-Gel P-4 gel filtration (Figure 4B). Previous experiments demonstrated that a tetrasaccharide elutes at almost the same position regardless of whether it is monosulfated or not (Hiraoka et al., 1999
). The purified product was also subjected to mild acid hydrolysis to release sulfated monosaccharide. On high-performance liquid chromatography (HPLC) analysis, the majority of 35S-sulfated monosaccharide eluted at the same elution position as that of 4-O-sulfated GalNAc, separating from 6-O-sulfated GalNAc, 3-O-sulfated GalNAc, or 4,6-di-O-sulfated GalNAc (Figure 4C). These results demonstrate that GalNAc4ST-1 and -2 transfer a sulfate group to C-4 of GalNAc in the context of GalNAcß1
4GlcNAcß1
R.
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Discussion |
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Comparison of the amino acid sequences of GalNAc4ST-1, GalNAc4ST-2, and the other three enzymes that belong to the HNK-1ST gene family showed that these five enzymes significantly share conserved sequences in the 5'-phosphosulfate and 3'-phosphate binding sites (Figure 3). Two additional homologous regions (A and B in Figure 3) were also noticed in the previous studies when C4ST-1, C4ST-2, and HNK-1ST were compared (Hiraoka et al., 2000). Moreover, GalNAc4ST-1 and 2 share conserved sequences in the vicinity of the 3'-phosphate binding site and the COOH-terminal (D, E, and F in Figure 3). These results indicate that a subfamily of sulfotransferases may be recognized by the presence of conserved sequences that are not apparent in other members of the same gene superfamily. It is important to note, however, that GalNAc4ST-1 and -2 clearly belong to the HNK-1ST gene family based on a phylogenetic tree analysis carried out previously (Hiraoka et al., 2000
).
Sulfation at C-4 of N-acetylgalactosamine in glycoproteins has been extensively studied (Green et al., 1985; Fiete et al., 1991
; Skelton et al., 1991
; Stockell Hartree and Renwick, 1992
; Siciliano et al., 1993
, 1994; Bergwerff et al., 1995
; Manzella et al., 1996
). These studies indicate that sulfo
4GalNAcß1
4GlcNAcß1
R is present in the N-glycans of pituitary hormones, such as luteinizing hormone, proopiomelanocortin, and thyroid-stimulating hormone. This sulfated terminal structure in these hormones is recognized by a receptor present in hepatic endothelial cells. It has been suggested that the rise and fall in circulating luteinizing hormone levels is regulated by its uptake through the recognition of sulfo
4GalNAcß1
4GlcNAcß1
R by a mannose-binding protein on endothelial cells (Fiete et al., 1991
, 1997). Because GalNAc4ST-1 is highly expressed in the pituitary gland, it is likely that this enzyme is responsible for the 4-O-sulfation of pituitary hormones.
While we were preparing this manuscript, the cloning of GalNAc4ST-1 was reported by others (Okuda et al., 2000; Xia et al., 2000
). Although the results obtained in our study are very similar to those reported, there is a slight difference in the expression of GalNAc4ST-1 transcripts in different tissues. We and Okuda et al. (2000)
showed that GalNAc4ST-1 is expressed widely in different tissues including the pituitary gland (Figures 6 and 7), while the expression of the same transcripts were more restricted to the pituitary gland in the studies by Xia et al. (2000)
. This difference may be due to a difference in the blots used. Nevertheless, all of these studies agree that GalNAc4ST-1 is highly expressed in the pituitary gland, indicating its role in the sulfation of pituitary gland glycoproteins.
GalNAc4ST-2, on the other hand, is highly expressed in the trachea, where a large amount of mucin-type glycoproteins are synthesized. This expression profile is the same as that recently reported for GalNAc4ST-2 (Kang et al., 2001). It has been shown that proopimelanocortin contains sulfo
4GalNAcß1
4GlcNAcß1
6(Galß1
3)GalNAc as a major O-glycan (Siciliano et al., 1994
), in addition to the same sulfated terminal in N-glycans (Siciliano et al., 1993
; Skelton et al., 1992
). This sulfated core2 branched O-glycan may play a role in determining the turnover of proopiomelanocortin following its secretion (Siciliano et al., 1994
). Because proopimelanocortin is synthesized in the pituitary gland, GalNAc4ST-1 is mainly responsible for sulfation in O-glycans, although GalNAc4ST-2 must contribute to sulfation of N- and O-glycans in the same tissue, to some extent. Other reports demonstrate that human urokinase and Tamm-Hosfall glycoprotein, mainly synthesized in kidney cells, also contain sulfo
4GalNAcß1
4GlcNAcß1
R (Hard et al., 1992
; Bergwerff et al., 1995
). Our results show that GalNAc4ST-1 transcripts are apparently expressed more in human adult kidney than are GalNAc4ST-2 transcripts, suggesting that GalNAc4ST-1 may be mainly responsible for the sulfation of these glycoproteins in adult kidney cells. On the other hand, GalNAc4ST-2 is probably involved in the sulfation of tissue factor pathway inhibitor synthesized in the human embryonic kidney 293 cell line (Smith et al., 1992
), because the transcripts of GalNAc4ST-2 are expressed in fetal kidney.
The present study also demonstrated that a side chain derived from the 6-position of 2,6-linked mannose was as good an acceptor as a side chain from the 2-position of
2,6-linked mannose for both GalNAc4ST-1 and GalNAc4ST-2 (Figure 5). In nature, a side chain derived from the 6-position of
2,6-linked mannnose is present in tetra-antennary N-glycans. The structural analyses thus far reported, however, show that sulfo
4GalNAcß1
4GlcNAcß1
R in pituitary gland glycoproteins are present in biantennary N-glycans and core 2 branched O-glycans (Green et al., 1985
; Skelton et al., 1991
, 1992; Stockell Hartree and Renwick, 1992
; Siciliano et al., 1993
, 1994; Smith et al., 1993
; Bergwerff et al., 1995
; Manzella et al., 1996
). These results suggest that the N-acetylgalactosaminyltransferase that adds ß1,4-linked GalNAc to GlcNAc may have a strict acceptor requirement for carbohydrate moieties as well as proteins that attach those carbohydrates (Mengeling et al., 1995
). This may also be the reason why luteinizing hormone but not chorionic gonadotropin contains a sulfo
4GalNAcß1
4GlcNAc terminal structure (Endo et al., 1979
; Smith et al., 1993
). In contrast, human urinary kallidinogenase synthesized in the kidney contain GalNAcß1
4GlcNAcß1
R in side chains derived from the 6- or 2-position of
-mannose (Tomiya et al., 1993
). These findings are consistent with the studies showing that there are at least two different N-acetylgalactosaminyltransferases: one in the pituitary gland that recognizes a specific amino acid motif, and another in other tissues such as the kidney that does not (Dharmesh et al., 1993
). On the other hand, mouse LSST preferentially adds a sulfate to core 2 branched O-glycans and less efficiently to N-glycans (Hiraoka et al., 1999
). These combined results indicate that sulfation of oligosaccharides can be regulated by acceptor substrate specificity of glycosyltransferases which form a precursor or that of sulfotransferase itself.
It has been demonstrated that sulfo4GalNAcß1
4GlcNAcß1
2Man can be bound to the mannose/GalNAc-4-SO4-receptor (Fiete et al., 1997
). It has not been determined, however, if the mannose/GalNAc-4-SO4 receptor also binds to sulfo
4GalNAcß1
4GlcNAcß1
R in mucin-type O-glycans. Future studies will need to determine whether the same acceptor recognizes sulfo
4GalNAcß1
4GlcNAcß1
R in both N- and O-glycans.
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Materials and methods |
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To obtain 5'-sequences, 5'-RACE was carried out using poly(A)+RNA isolated from human lymph node (Clontech). For reverse transcription, an anti-sense primer corresponding to nucleotides 565546 (nucleotides 13 encode the initiation methionine) was used under the conditions described previously (Hiraoka et al., 1999). PCR was then carried out using a nested 3'-primer corresponding to nucleotides 524503. PCR products were cloned into pBluescript II SK(+) that had been digested with EcoRV and reacted with Taq polymerase, and colony hybridization was performed to identify the 5'-RACE products using a 32P-labeled DNA fragment encompassing nucleotides 182524. The latter probe DNA was obtained by PCR. The nucleotide sequence of the PCR product revealed a mismatch during PCR, resulting in a frame shift. To obtain the correct sequence in this region, RT-PCR was carried out to amplify nucleotides 227948 using poly(A)+RNA from human lymph node. The PCR product was excised with HindIII and EcoRI, replacing the corresponding sequence in the first product of 5'-RACE. The resultant 5'-RACE product was digested with NotI and EcoRI, and this fragment was appended to the 5'-end EcoRI site of AA417127 (encompassing nucleotides 6711966), producing a full length cDNA encoding GalNAc4ST-2. The ligated cDNA cloned into the NotI sites of pcDNA3.1/Hygro (Invitrogen) resulted in pcDNA3.1GalNAc4ST-2 (the name of GalNAc4ST-2 was given after the determination of its acceptor specificity).
The second gene was identified by searching the human genomic DNA database using the amino acid sequence for GalNAc4ST-2 as a probe. Comparison of the identified genomic sequence, registered as ACO67911, with that of GalNAc4ST-2 revealed that the upstream sequence from the 5'-phosphosulfate binding site to the stop codon appeared to be encoded in one exon. Accordingly, RT-PCR was carried out using a 5'-primer (nucleotides 540559), a 3'-primer (nucleotides 10521032), and Marathon cDNA prepared from human pituitary gland (Clontech) as a template. Because the product of this reaction showed an expected sign of 512 base pairs, 5'-RACE and PCR of the 3'-region of the cDNA were carried out using the above Marathon pituitary cDNA as a template. Finally, PCR was carried out using the above Marathon cDNA from human pituitary gland as a template, a 5'-primer (nucleotides 3 to 16), and a 3'-primer (nucleotides 12801261), which were synthesized based on the sequences obtained by the above 5'-RACE and PCR experiments. These primers also contained EcoRI and XbaI sites, and the PCR product was cloned into the same sites of pcDNA 3.1/Hyg, resulting in pcDNA3.1GalNAc4ST-1.
Synthesis of acceptor oligosaccharides
Galß14GlcNAcß1
2Man
1
6Manß1
octyl, GlcNAcß1
2Man
1
6Manß1
octyl, Galß1
4GlcNAcß1
6Man
1
6Manß1
octyl, GlcNAcß1
6Man
1
6Manß1
octyl, GlcAß1
3Galß1
4GlcNAcß1
octyl, and Galß1
4GlcNAcß1
O(CH2)8COOCH3 were synthesized as described previously (Tsuboi et al., 1996
; Ding et al., 1998
; Ujita et al., 1998
; McAuliffe et al., 1999
). Galß1
4GlcNAcß1
6(Gal-ß1
3)GalNAc
1
octyl and GlcNAcß1
6(Galß1
3)GalNAc
1
octyl were synthesized as described below. The suitably protected Galß1
3GalNAc
1
octyl (compound 1) was synthesized by the coupling of methyl 2,3,4,6-tetra-O-benzoyl-1-thio-ß-D-galactopyranoside with octyl 2-acetamido-4,6-O-benzylidene-2-deoxy-
-D-galactopyranoside in a dimethyl(methylthio)sulfonium triflate promoted reaction (68% yield) (Fügedi and Garegg, 1986
). Benzylidene cleavage under acidic conditions then yielded the disaccharide diol having two free hydroxyl groups at C-4 and C-6 of the GalNAc residue (compound 1). In parallel, ethyl 3,6-di-O-benzyl-2-deoxy-2-phthlimido-1-thio-ß-D-glucopyranoside was coupled with 2,6-di-O-acetyl-3,4-di-O-chloroacetyl-
-D-galactopyranosyl chloride in the presence of AgOTf to give the required bifunctional N-acetyllactosamine donor (compound 2) in 73% yield. Reaction of the disaccharide donor (compound 2) with the core 1 disaccharide acceptor (compound 1) in the presence of dimethyl(methylthio)sulfonium triflate gave the tetrasaccharide (compound 3) in 61% yield. Dechloroacetylation of compound 3 using hydrazinedithiocarbonate in 2,6-lutidine-HOAc (3:1) then furnished the tetrasaccharide triol in 74% yield. Conventional deprotection of the tetrasaccharide triol produced Galß1
4GlcNAcß1
6(Galß1
3)GalNAc
1
octyl (compound 4) with a yield of 56% after purification on Sephadex LH-20. GlcNAcß1
6(Galß1
3)GalNAc
1
octyl (compound 5) was obtained on a 5 mg scale from compound 4 by digestion with ß-galactosidase from jack beans as described elsewhere (McAuliffe et al., 1999
).
Partial NMR (500 MHz; D2O) data are as follows: 4: 4.83 (d, J1,2 = 3.6 Hz, 1H, H-1), 4.52 (d, J1¢¢,2¢¢ = 7.8 Hz, 1H, H-1''), 4.42 (2d, J1¢,2¢¢ = 7.8 Hz and J1¢¢¢,2¢¢¢ = 7.8 Hz, 2H, H-1' and H-1'''), 4.26 (dd, 1H, H-3), 4.17 (bs, 1H, H-4), 2.0 and 2.02 (2s, each 3H, 2 NHAc); 13C NMR:
105.4, 103.7, 102.2 and 97.5. 5:
4.86 (d, J1,2 = 3.0 Hz, 1H, H-1), 4.52 (d, J1¢,2¢ = 8.5 Hz, 1H, H-1¢), 4.46 (d, J1¢¢,2¢¢ = 8.0 Hz, 1H, H-1''), 4.30 (dd, 1H, H-3), 4.21 (bs, 1H, H-4), 2.02 and 2.01 (2s, each 3H, 2 NHAc); 13C NMR:
105.3, 102.1, 97.2. m/z (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF]) are: 4, Calculated for C36H64O21N2 (M+Na+) 883.3894, found 883.3910; 5, calculated for C30H54O16N2 (M+Na+) 721.3366, found 721.3365. Detailed procedures of the synthesis will be published elsewhere (Misra et al., 2001
).
Addition of ß1,4-linked GalNAc to GlcNAc-terminated compounds was carried out as a secondary reaction of milk ß1,4-galactosyltransferase (ß4Gal-TI) as described previously (Palcic and Hindsgaul, 1991; Do et al., 1995
).
-Lactalbumin (10 mg/ml) was added to facilitate N-acetylgalactosaminylation (Do et al., 1995
). The reaction products were separated from UDP-GalNAc using Sep-Pak C-18 cartridge column chromatography and then fractionated by HPLC using an AX-5 NH2-bonded column (4.0 mm x 30 cm) (Bierhuizen and Fukuda, 1992
). The column was initially equilibrated with solution A (15% H2O/85% acetonitrile) and then eluted by a linear gradient to 95% solution A and 5% solution B (60% 15 mM KH2PO4/40% acetonitrile) in 10 min. The column was then eluted by a linear gradient to 50% solution A and 50% solution B in 55 min. Under these conditions, GlcNAcß1
2Man
1
6Manß1
octyl and GalNAcß1
4GlcNAcß1
2Man
1
6Manß1
octyl eluted at 31 min and 40 min, respectively. Similarly, GalNAcß1
4GlcNAcß1
6Man
1
6Manß1
octyl and GalNAcß1
4GlcNAcß1
6(Galß1
3)GalNAc
1
octyl were separated from GlcNAcß1
6Man
6Manß1
octyl and GlcNAcß1
6(Galß1
3)GalNAc
1
octyl, respectively.
Sulfotransferase assay
CHO cells were transfected with pcDNA3.1-GalNAc4ST-1 or pcDNA3.1-GalNAc4ST-2 as described previously (Hiraoka et al., 2000). Sixty-two hours after the transfection, the cells attached to plates were washed with phosphate-buffered saline, scraped, and homogenized; the supernatant after brief centrifugation was used as an enzyme preparation, as described previously (Hiraoka et al., 2000
).
GalNAc-4-O-sulfotransferase activity was measured in the reaction mixture (50 µl) containing 50 mM imidazole-HCl, pH 6.8, 0.015% protamine chloride, 40 mM 2-mercaptoethanol, 0.1% Triton X-100, 10 mM NaF, 2 mM ATP, an acceptor oligosaccharide (0.5 mM), 0.2 nmole [35S]-PAPS (1.97 Ci/mmol), and 25 µl of the enzyme solution. The composition of this reaction mixture was determined based on the results shown by Skelton et al. (1991). After incubation for 2 h at 28°C, the reaction mixture was boiled for 2 min, adjusted to 0.25 M ammonium formate, pH 4.0 (Ong et al., 1998
), and then applied to a Sep-Pak C-18 cartilage column. The column was washed with the same solution, then the product was eluted with 30% acetonitrile in water and the radioactivity was measured by scintillation counting.
To characterize the product sulfated by GalNAc4ST, 35S-sulfate labeled product was prepared using GalNAcß14GlcNAcß1
2Man
1
6Manß1
octyl as an acceptor. The product, purified by a Sep-Pak C-18 cartilage column, was then applied to a column (1.0 x 120 cm) of Bio-Gel P-4 equilibrated with 0.1 M ammonium acetate buffer (pH 6.7) as described elsewhere (Hiraoka et al., 1999
). Separation of oligosaccharides with different numbers of sulfate groups was achieved by QAE-Sephadex A-25 column chromatography. The column (0.7 x 0.8 cm) was equilibrated with 10 mM pyridine-acetate buffer (pH 5.5) and stepwisely eluted with 70 mM, 120 mM, 140 mM, 500 mM, and 1 M NaCl in the the same buffer. Mono- and disulfated oligosaccharides elute with 70 mM and 120 mM NaCl, respectively (Hiraoka et al., 1999
).
To determine the position of a sulfate attached to GalNAc, the purified 35S-sulfated product was hydrolyzed in 40 mM HCl at 100°C for 120 min (Habuchi and Conrad, 1985). The hydrolysate was directly subjected to HPLC using a Whatman Partisil SAX-10 column (4.6 mm x 25 cm) equilibrated with 10 mM KH2PO4 at room temperature. In the first 20 min, the column was isocratically eluted with 10 mM KH2PO4 and then eluted with a linear gradient from 10 mM KH2PO4 to 400 mM KH2PO4 over the next 20 min. Standard sulfated monosaccharides were purchased from Sigma.
Chondroitin 4-O-sulfotransferase and HNK-1ST activities were assayed as described previously (Ong et al., 1998; Hiraoka et al., 2000
).
Incorporation of 35S-sulfate into glycoprotein acceptors
Thirty micrograms of human lutropin (Sigma) was incubated with the enzyme preparation for GalNAc4ST-1 or GalNAc4ST-2. After incubation for 18 h at 28°C under the same conditions described above, a portion of the product was digested with N-glycanase. The digested and undigested products were subjected to SDSPAGE and fluorography.
Northern blot analysis
Northern blots of multiple human tissues (Clontech) or human RNA Master BlotTM (Clontech) were hybridized with cDNA fragments isolated from pcDNA3.1GalNAc4ST-1 and pcDNA3.1GalNAc4ST-2 after 32P-labeling using a nick translation kit (PrimIt-RmT) from Stratagene.
RT-PCR
One hundred nanograms of poly(A)+RNA were reverse transcribed by using MMLV reverse transcriptase (Stratagene) with 300 ng of oligo dT in 50 µl of reaction mixture. One microliter of RT mixture was used as a template for PCR. To detect GalNAc4ST-2 transcripts, samples were denatured for 2 min at 94°C, followed by 35 cycles of 1 min at 94°C, 30 s at 56°C, and 30 s at 72°C. For GalNAc4ST-1, the annealing temperature was raised to 60°C. Oligonucleotide primers were used for specific amplification of human GalNAc4ST-2 (nucleotides 710729 for 5'-primer and nucleotides 962940 for 3'-primer) and human GalNAc4ST-1 (nucleotides 540559 for 5'-primer and nucleotides 838819 for 3'-primer).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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1-3)-fucosylated N-acetylgalactosamine(ß1-4)-N-acetylglucosamine elements. Eur. J. Biochem., 228, 10091019.[Abstract]
Bierhuizen, M.F., and Fukuda, M. (1992) Expression cloning of a cDNA encoding UDP-GlcNAc:Gal ß13-GalNAc-R (GlcNAc to GalNAc) ß1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. Proc. Natl. Acad. Sci. USA, 89, 93269330.[Abstract]
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