Ectopic expression of a GlcNAc 6-O-sulfotransferase, GlcNAc6ST-2, in colonic mucinous adenocarcinoma

Akira Seko2, Koji Nagata3, Suguru Yonezawa3 and Katsuko Yamashita1,2

2 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, 2-3, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; and 3 Second Department of Pathology, Kagoshima University School of Medicine, 8-35–1, Sakuragaoka, Kagoshima 890-8520, Japan

Received on January 21, 2002; revised on February 25, 2002; accepted on February 26, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The content of sulfated glycans having 6-O-sulfated GlcNAc residues alters in the course of colonic carcinogenesis. We previously characterized two GlcNAc 6-O-sulfotransferases (SulTs), SulT-a and -b, expressed in colonic normal tissues and adenocarcinomas [Seko et al. (2000)Go Glycobiology, 10, 919–929]. Levels of the enzymatic activities of SulT-a in normal colonic mucosa are higher than those in colonic adenocarcinomas, and the enzymatic activities of SulT-b are detected only in mucinous adenocarcinomas. To determine which GlcNAc 6-O-SulTs cloned so far correspond to SulT-a and -b, we expressed seven enzymes of a Gal/GalNAc/GlcNAc 6-O-SulT family in COS-7 cells and examined their substrate specificities in comparison with those of SulT-a and -b. GlcNAc6ST-2 (HEC-GlcNAc6ST, LSST, or GST-3) can recognize GlcNAcß1->3GalNAc{alpha}1-O-pNP as a good acceptor as well as other O-linked- and N-linked-type oligosaccharides, and its substrate specificity was similar to that of SulT-b. GlcNAc6ST-3(I-GlcNAc6ST or GST-4{alpha}) preferred Galß1->3(GlcNAcß1->6)GalNAc{alpha}1-O-pNP as an acceptor to the other oligosaccharides examined, and its specificity was similar to that of SulT-a. To confirm these correspondences, we further performed quantitative analyses of transcripts for GlcNAc6ST-2 and -3 genes by competitive RT-PCR. As a result, GlcNAc6ST-2 gene was expressed in almost all the mucinous adenocarcinomas examined and hardly expressed in normal colonic mucosa and nonmucinous adenocarcinoma. Expression levels of transcript for GlcNAc6ST-3 in normal mucosa were significantly higher than those in adenocarcinomas. From these results, it was indicated that GlcNAc6ST-2 corresponds to mucinous adenocarcinoma-specific SulT-b and that expression of GlcNAc6ST-3 is down-regulated in colonic adenocarcinomas.

Key words: colon cancer/mucinous adenocarcinoma/substrate specificity/sulfated oligosaccharide/sulfotransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It has been elucidated that expression levels of various sulfated glycans decrease in the course of human colonic carcinogenesis (Felipe, 1969Go; Yamori et al., 1987Go; Yamashita et al., 1987Go; Fukushima et al., 1995Go). We previously performed comparative structural studies of N-linked glycans in carcinoembryonic antigen (CEA) and its counterpart in normal tissues, normal fecal antigen-2 (NFA-2) and found that the relative contents of 6-O-sulfated GlcNAc residues in NFA-2 are higher than those in CEA (Yamashita et al., 1987Go; Fukushima et al., 1995Go). The results suggested that expression levels of a GlcNAc 6-O-sulfotransferase (SulT) are down-regulated in colonic adenocarcinomas. Our following studies about characterization of GlcNAc 6-O-SulTs present in colonic tissues and cancers showed the occurrence of two GlcNAc 6-O-SulT activities, SulT-a and -b (Seko et al., 2000Go).

SulT-a is present in normal colonic mucosa and adenocarcinomas, and levels of the enzymatic activities in normal mucosa are significantly higher than those in adenocarcinomas, suggesting that SulT-a is responsible for sulfation of N-linked glycans in CEA and NFA-2. On the other hand, SulT-b is not detected in normal mucosa and nonmucinous adenocarcinoma, but the strong enzymatic activities are found in mucinous adenocarcinoma or adenocarcinoma with a mucinous component (Seko et al., 2000Go). Mucinous adenocarcinomas are defined by the World Health Organization as those in which over 50% of the microscopic area is occupied with mucus (Morson and Sobin, 1990Go). The remarkable difference in biochemical properties between SulT-a and -b is in their substrate specificities; SulT-a acts efficiently on Galß1->3(GlcNAcß1->6)GalNAc{alpha}1-O-p-nitrophenyl (core 2 O-pNP), weakly on GlcNAcß1->2Man and GlcNAcß1->3Galß1->4Glc (GL), but not on GlcNAcß1->3GalNAc{alpha}1-O-pNP (core 3 O-pNP), whereas SulT-b acts efficiently on these acceptor substrates (Seko et al., 2000Go). From the broad substrate specificity of SulT-b, it could be speculated that some sulfated glycans are specifically synthesized in mucinous adenocarcinomas and that the sulfated moieties may serve as a good clinical marker against colonic mucinous adenocarcinomas. Therefore, it is important to determine which GlcNAc 6-O-SulTs so far cloned correspond to SulT-a and -b.

A Gal/GalNAc/GlcNAc 6-O-SulT family comprises four GlcNAc 6-O-SulTs (GlcNAc6ST-1, -2, -3, and -5), one GlcNAc/GalNAc 6-O-SulT (GlcNAc6ST-4/Ch6ST-2), one Gal/GalNAc 6-O-SulT(Ch6ST-1), and one Gal 6-O-SulT (KS6ST) (Fukuta et al., 1995Go, 1997, 1998; Uchimura et al., 1998aGo,b,c, 2000; Tsutsumi et al., 1998Go; Mazany et al., 1998Go; Li and Tedder, 1999Go; Bistrup et al., 1999Go; Hiraoka et al., 1999Go; Lee et al., 1999Go; Kitagawa et al., 2000Go; Akama et al., 2000Go; Sakaguchi et al., 2000Go; Bhakta et al., 2000Go; Hemmerich et al., 2001aGo). They share approximately 30–90% identity of amino acid sequences and three well-conserved domains, two of which are involved in the binding to a donor substrate, adenosine 3'-phosphate 5'-phosphosulfate (PAPS) (Negishi et al., 2001Go; Fukuda et al., 2001Go). However, expression profiles of these genes in human tissues and their putative biological roles are diverse. Habuchi et al. (1993)Go purified chondroitin 6-O-SulT (C6ST or Ch6ST-1) from chick embryo chondrocytes, and Fukuta et al. (1995)Go isolated a cDNA for the SulT. KS6ST (keratan sulfate 6-O-SulT [KSGal6ST]) has been cloned by Fukuta et al. (1997)Go through cross-hybridization with the cDNA for Ch6ST-1. GlcNAc6ST-1 was identified by Uchimura et al. (1998b,c) as the first GlcNAc 6-O-SulT (GlcNAc6ST) with rather broad expression profile in human and mouse, and GlcNAc6ST-2 (HEC-GlcNAc6ST or LSST), the second GlcNAc 6-O-SulT cloned by Bistrup et al. (1999)Go and Hiraoka et al. (1999)Go, exhibits a limited expression in high endothelial venules among human and mouse normal tissues.

KS6ST and GlcNAc6ST-1 and -2 (Uchimura et al., 1998bGo,c; Bistrup et al., 1999Go; Hiraoka et al., 1999Go) were shown to be involved in the biosynthesis of L-selectin ligand moieties, Gal-6-O-sulfated and/or GlcNAc-6-O-sulfated sialyl Lewis X (Hemmerich et al., 1995Go; Mitsuoka et al., 1998Go; Yeh et al., 2001Go). L-selectin on lymphocytes binds to specific sulfated glycoproteins on the surface of the endothelial cells of high endothelial venules in lymph nodes and mediates recruitment of lymphocytes (Yeh et al., 2001Go; Hemmerich et al., 2001bGo). GlcNAc6ST-3 (I-GlcNAc6ST), which is expressed predominantly in small intestine and colon, is the third GlcNAc 6-O-SulT (Lee et al., 1999Go). GlcNAc6ST-5 (CGn6ST) (Akama et al., 2000Go; Hemmerich et al., 2001aGo), which shares over 85% identity of the amino acid sequence with GlcNAc6ST-3, was found to be the gene responsible for macular corneal dystrophy, a hereditary eye disorder (Akama et al., 2000Go).

There exists a discrepancy about the substrate specificity of GlcNAc6ST-4/Ch6ST-2. Kitagawa et al. (2000)Go isolated the cDNA for GlcNAc6ST-4/Ch6ST-2 and characterized the enzyme as a chondroitin 6-O-SulT (C6ST-2); in contrast, Uchimura et al. (2000)Go independently found and characterized the enzyme as an N-linked glycan-preferable GlcNAc 6-O-SulT (GlcNAc6ST-4). Bhakta et al. (2000)Go also showed GlcNAc6ST-4/Ch6ST-2 as a GlcNAc 6-O-SulT. The involvement of these SulTs in several biological phenomena has been elucidated as described, but their precise substrate specificities for comparison to those of SulT-a and -b have not yet been clarified.

In this article, we comparatively studied the substrate specificities of all members of Gal/GalNAc/GlcNAc 6-O-SulT family. We show that GlcNAc6ST-2 and -3 have similar substrate specificities to those of SulT-b and -a, respectively. We further revealed by the competitive reverse-transcription polymerase chain reaction (RT-PCR) that transcript for GlcNAc6ST-2 selectively occurs in colonic mucinous adenocarcinomas and that expression levels of transcript for GlcNAc6ST-3 in normal colonic mucosa are significantly higher than those in colonic adenocarcinomas. From these results, we conclude that GlcNAc6ST-2 and -3 are dominant components of the enzymatic activities of SulT-b and -a, respectively.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Expression of seven enzymes of Gal/GalNAc/GlcNAc 6-O-SulT family in COS-7 cells
We isolated cDNAs encoding for the seven SulTs from the genome or human testis cDNA library and inserted them into pcDNA3 expression vector. The plasmids were expressed in COS-7 cells, and the crude membrane fractions were isolated as enzyme sources. The crude membrane fraction derived from pcDNA3-transfected COS-7 cells had no detectable Gal/GalNAc/GlcNAc 6-O-SulT activity for the acceptor substrates described, whereas the crude membranes from COS-7 cells transfected with pcDNA3-GlcNAc6ST-1, -2, -3, -5, -GlcNAc6ST-4/Ch6ST-2, -KS6ST, and -Ch6ST-1 had SulT activities; the specific activities were calculated as 169 pmol/min/mg of protein for Ch6ST-1 (using chondroitin, sodium salt [CS] as a substrate), 127 for KS6ST (KS), 42.4 for GlcNAc6ST-1 (core 2 O-pNP), 2.6 for GlcNAc6ST-2 (core 2 O-pNP), 38.7 for GlcNAc6ST-3 (core 2 O-pNP), 32.4 for GlcNAc6ST-5 (core 2 O-pNP), and 56.3 for GlcNAc6ST-4/Ch6ST-2 (core 2 O-pNP). The results indicate that these SulT activities in the membrane fractions are derived from the transfected cDNAs for respective SulTs. We examined effects of pH, divalent cations, and detergents on each SulT activity and determined the optimum conditions as described in Materials and methods (data not shown). In the enzymatic reactions of these SulTs, sulfated products increased linearly for at least 1 h.

Structural analysis of 35S-labeled oligosaccharides produced by the seven SulTs
To determine the substrate specificities of these SulTs, six oligosaccharides, CS, and KS were used as substrates (Table I). The structure of 35S-labeled products was analyzed in combination with Psathyrella velutina lectin (PVL)- or Trichosanthes japonica agglutinin-I (TJA-I)-immobilized column chromatography and glycosidase digestion.


View this table:
[in this window]
[in a new window]
 
Table I. Summary of substrate specificities of seven Gal/GalNAc/GlcNAc 6-O-SulTs and human colon GlcNAc 6-O-SulTs
 
[35S]sulfated core 2 O-pNP
After enzyme reaction, the mixture containing the GlcNAc6ST-1 membrane fraction and core 2 O-pNP as a substrate, was applied on paper electrophoresis at pH 5.4 (Figure 1). [35S]sulfated product was clearly separated from [35S]PAPS and [35S]sulfate, and the product indicated by bar was recovered. To determine the linkage position of [35S]sulfate, the [35S]sulfated core 2 O-pNP was treated with jackbean ß-N-acetylhexosaminidase and then applied on a PVL-Sepharose column. The enzyme cleaves nonsubstituted ß-GlcNAc but not 6-O-sulfated ß-GlcNAc (Seko et al., 2000Go). PVL binds to nonsubstituted ß-GlcNAc and 6-O-substituted ß-GlcNAc at nonreducing termini but not to 3-O- or 4-O-substituted ß-GlcNAc (Kochibe and Matta, 1989Go). When applied to a PVL-Sepharose column, the 35S-labeled digest bound to the column and was eluted with 0.3 M GlcNAc (Figure 2A), indicating that [35S]sulfate is incorporated at the C-6 position of GlcNAc residue as shown in Figure 3 (product 1). As for 35S-labeled core 2 O-pNP synthesized by GlcNAc6ST-2, -3, -5, and GlcNAc6ST-4/Ch6ST-2, the same results were also obtained.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Paper electrophoresis (at pH 5.4) of the 35S-labeled reaction mixture provided by GlcNAc6ST-1 using core 2 O-pNP as an acceptor substrate. Arrows indicate the positions of standard compounds and enzymatic reaction products; a, [35S]SO42–; b, [35S]PAPS; c, [35S]sulfated GlcNAcß1->2Man; d, [35S]sulfated core 3 O-pNP; e, [35S]sulfated GL; f, [35S]sulfated monoGP; g, [35S]sulfated biGP. [35S]sulfated core2 O-pNP indicated by bar was extracted with water and analyzed for the linkage position of [35S]sulfate.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. PVL-Sepharose (A) and TJA-I-Sepharose (B) chromatographies of [35S]sulfated compounds. (A) Jackbean ß-N-acetylhexosaminidase digest of [35S]sulfated core 2 O-pNP provided by GlcNAc6ST-1. The same elution patterns were obtained in the cases of GlcNAc6ST-1, -2, -3, and -5, or GlcNAc6ST-4/Ch6ST-2, using core 2 O-pNP, core 3 O-pNP, GlcNAcß1->2Man, GL, biGP, or monoGP as acceptor sustrates. (B) Jack bean ß-N-acetylhexosaminidase digest of [35S]sulfated GL provided by KS6ST. The same elution pattern was obtained in the case of Ch6ST-1. Vo, void volume. Elution was started with 4 ml TBS containing 1 mM CaCl2 and 1 mM MgCl2 (A) or TBS (B), followed by the respective buffer containing 0.3 M GlcNAc (A) or 0.1 M lactose (B) (indicated by arrows).

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Structure of sulfated oligosaccharides produced by Gal/GalNAc/GlcNAc 6-O-SulTs.

 
In the cases of core 3 O-pNP, GlcNAcß1->2Man, GlcNAcß1->2Man{alpha}1->3/6Manß1->4GlcNAc (monoGP), and GlcNAcß1->2Man{alpha}1->3(GlcNAcß1->2Man{alpha}1->6)Manß1->4GlcNAcß1->4GlcNAc (biGP) as acceptor substrates, jackbean ß-N-acetylhexosaminidase digests also completely bound to a PVL-Sepharose column and were eluted with 0.3 M GlcNAc, indicating that [35S]sulfate is incorporated at the C-6 position of GlcNAc or branching GlcNAc residues of these substrates as shown in Figure 3 (products 2, 3, 4, and 5, respectively).

[35S]sulfated GlcNAcß1->3Galß1->4Glc
All of the seven SulTs were capable of acting on GlcNAcß1->3Galß1->4Glc (GL) (Table I), but the linkage positions of [35S]sulfate were different as follows. When jack bean ß-N-acetylhexosaminidase digest of [35S]sulfated GL synthesized by GlcNAc6ST-1, -2, -3, -5, or GlcNAc6ST-4/Ch6ST-2 was applied to PVL-Sepharose chromatography, each radioactive product bound to the column and was eluted with 0.3 M GlcNAc (data not shown), indicating the incorporation of [35S]sulfate at the C-6 of GlcNAc (Figure 3, product 6). On the other hand, when jack bean ß-N-acetylhexosaminidase digest derived from KS6ST or Ch6ST-1 was applied on a PVL-Sepharose column, the 35S-labeled compounds flowed through the column (data not shown). Instead, the digests bound to a TJA-I-Sepharose column and were eluted with 0.1 M lactose (Figure 2B, shown in the case of KS6ST). TJA-I specifically binds to Neu5Ac{alpha}2->6Galß1->4GlcNAc/Glc and SO3->6Galß1->4GlcNAc/Glc structure, but not to Neu5Ac{alpha}2->3(±SO3->6)Galß1->4GlcNAcß1-> and Galß1-> 4GlcNAcß1-> (Yamashita et al., 1992Go; Seko and Yamashita, unpublished data). These results indicate that the digests are SO3->6Galß1->4Glc and that Ch6ST-1 and KS6ST transfer [35S]sulfate to the C-6 position of Gal residue of GL (Table I and Figure 3, product 7). This result is consistent with those reported by Torii et al. (2000)Go, who showed that KS6ST can sulfate the proximal Gal residue in Galß1->4GlcNAcß1-> 3Galß1->4GlcNAc structure.

Substrate specificities of the seven SulTs
From the results described, substrate specificities of the SulTs are summarized in Table I. The substrate specificities of SulT-a and -b, which are GlcNAc 6-O-SulTs detected in human colonic mucosa and mucinous adenocarcinoma, respectively (Seko et al., 2000Go), are also shown. Several unique characters of the SulTs were found with regard to those substrate specificities: (1) only GlcNAc6ST-2 and GlcNAc6ST-5 can act on core 3 O-pNP, but the relative activity of GlcNAc6ST-2 for core 3 O-pNP is greater than that of GlcNAc6ST-5, suggesting that GlcNAc6ST-2 corresponds to SulT-b. (2) The specificity of SulT-a is similar to that of GlcNAc6ST-3, suggesting that GlcNAc6ST-3 corresponds to SulT-a. (3) Core 2 O-pNP is the best substrate for GlcNAc6ST-1, -2, -3, -5, and GlcNAc6ST-4/Ch6ST-2 among the oligosaccharides examined. (4) The main difference in the substrate specificities between GlcNAc6ST-3 and GlcNAc6ST-5 is that GlcNAc6ST-5 acts on GlcNAcß1->2Man and GL more efficiently than GlcNAc6ST-3. (5) BiGP and monoGP are poor substrates for GlcNAc6ST-1, -2, -3, -5 and GlcNAc6ST-4/Ch6ST-2.

Determination of expression levels of GlcNAc6ST-2 and -3 in human normal colonic mucosa and adenocarcinomas
From the results in Table I, SulT-a and -b are likely to correspond to GlcNAc6ST-3 and -2, respectively. To further confirm this, we examined expression levels of transcripts for GlcNAc6ST-2 and -3 in human normal colonic mucosa and adenocarcinomas by the competitive RT-PCR method. We previously found that the enzymatic activity of SulT-b is not present in normal mucosa and nonmucinous adenocarcinoma but in mucus-pool depositing adenocarcinomas and that levels of the enzymatic activities of SulT-a in normal mucosa are higher than those in adenocarcinomas (Seko et al., 2000Go). The competitive RT-PCR products were applied on agarose electrophoresis and detected by staining with ethidium bromide. Typical results for GlcNAc6ST-2, -3, and ß-actin are shown in Figure 4A, and relative amounts of transcripts for GlcNAc6ST-2 and -3 to that for ß-actin are shown in Table II.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4. Quantitative analysis of transcripts for GlcNAc6ST-2 and -3 genes in normal colonic mucosa and adenocarcinomas by the competitive RT-PCR method. (A) Typical patterns of electrophoresis in a 1.0% agarose gel visualized by staining with ethidium bromide. The electrograms of GlcNAc6ST-2, -3, and ß-actin were in the cases of no. 16 (tumor), no. 2 (normal), and no. 12 (normal), respectively. (B and C) Comparison of relative values of transcripts for GlcNAc6ST-3 (B) and GlcNAc6ST-2 (C) in normal mucosa (Normal) with those in nonmucinous adenocarcinoma (Non-muc) and mucinous adenocarcinoma or adenocarcinoma with a mucinous component (Muc). Columns, mean; bars, SE.

 

View this table:
[in this window]
[in a new window]
 
Table II. Major clinicopathological features of the 18 patients with colonic adenocarcinomas and the relative values of transcripts for GlcNAc6ST-2 and GlcNAc6ST-3 obtained by the competitive RT-PCR method
 
The transcript for GlcNAc6ST-2 was not detected in 18 specimens of normal mucosa and were present only in one specimen of nonmucinous adenocarcinoma, whereas it was expressed in five of mucinous adenocarcinoma or adenocarcinoma with a mucinous component among six specimens examined here. The result suggests that expression of GlcNAc6ST-2 gene is closely associated with mucinous adenocarcinomas (Figure 4C), supporting that GlcNAc6ST-2 corresponds to SulT-b. On the other hand, relative expression levels of GlcNAc6ST-3 gene in normal mucosa (32 ± 5.6 [x 103], N = 18] were significantly higher than those in nonmucinous (6.4 ± 2.6 [x 103], N = 12, P < 0.001) and mucinous adenocarcinomas (5.6 ± 1.7 [x 103], N = 6, P < 0.01) (Figure 4B), as the enzymatic activities previously shown (Seko et al., 2000Go). From this result and the substrate specificities in Table I, it is indicated that GlcNAc6ST-3 corresponds to SulT-a.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this article, we constructed expression vectors containing cDNAs encoding for seven members of Gal/GalNAc/GlcNAc 6-O-SulT family, expressed them in COS-7 cells, and examined their substrate specificities. We determined linkage positions of [35S]sulfate in various acceptor substrates with use of a combination of two lectin affinity chromatographies and jack bean ß-N-acetylhexosaminidase.

Our first aim in this study is to identify a GlcNAc 6-O-SulT (SulT-b) that is specifically expressed in human colonic mucinous adenocarcinoma or adenocarcinoma with a mucinous component (Seko et al., 2000Go). SulT-b activity is not detected in human normal colonic mucosa and nonmucinous adenocarcinoma. The substrate specificity of SulT-b is broader than that of SulT-a, which occurs in normal colonic mucosa and adenocarcinomas (Table I). The conspicuous difference between both enzymes is that SulT-b can act on core 3 O-pNP, whereas SulT-a cannot. Considering these facts, we examined the substrate specificities of seven enzymes of Gal/GalNAc/GlcNAc 6-O-SulT family and found that GlcNAc6ST-2 and -5 can act on core 3 O-pNP (Table I). However, GlcNAc6ST-5 utilizes core 3 O-pNP with a quite low efficiency compared with SulT-b, and Bartes et al. (2001)Go showed that mRNA for GlcNAc6ST-5 is expressed in human normal colon by the northern blot analysis. These facts exclude the possibility that GlcNAc6ST-5 is a major component of SulT-b. On the other hand, the expression of GlcNAc6ST-2 gene was shown to be limited in high endothelial venules among human and mouse normal tissues (Bistrup et al., 1999Go; Hiraoka et al., 1999Go). Our results obtained by the competitive RT-PCR suggest that GlcNAc6ST-2 is hardly expressed in colonic normal mucosa and nonmucinous adenocarcinoma, but found in mucinous adenocarcinomas. From these facts, we conclude that GlcNAc6ST-2 is dominant component of SulT-b activity and that GlcNAc6ST-2 is ectopically expressed in human mucinous adenocarcinoma cells. Relative values of the enzymatic activities of SulT-b for GlcNAcß1->2Man, core 3 O-pNP, and GL are slightly lower than those of GlcNAc6ST-2 (Table I). The result suggests that a SulT specific for core 2 O-pNP is present as a minor component in mucinous adenocarcinoma. One candidate is GlcNAc6ST-3, which has strong activity for core 2 O-pNP (Table I) and is weakly expressed even in mucinous adenocarcinomas (Table II).

On the other hand, SulT-a is present in human normal mucosa and adenocarcinomas (Seko et al., 2000Go). The substrate specificity of SulT-a is similar to that of GlcNAc6ST-3, which is expressed in small intestine and colon (Lee et al., 1999Go). We previously showed that levels of SulT-a activities in nonmucinous adenocarcinomas are lower than those in normal mucosa (Seko et al., 2000Go) and, in this study, that expression levels of transcript for GlcNAc6ST-3 in colonic adenocarcinomas are lower than those in normal mucosa (Table II and Figure 4B). From these results, we conclude that SulT-a corresponds to GlcNAc6ST-3.

The contents of a sulfated glycan moiety, Galß1->4(SO3 ->6)GlcNAcß1->, in CEA derived from colon cancer patients are lower than those in NFA-2 (Yamashita et al., 1987Go; Fukushima et al., 1995Go). GlcNAc6ST-3 can be responsible for the synthesis of the sulfated glycans in CEA and NFA-2, and the down-regulation in colon cancer may cause decrease of the content of the sulfated glycans in CEA. It has been shown that mRNAs for GlcNAc6ST-1 and -5 are also expressed in normal colon (Uchimura et al., 1998cGo; Bartes et al., 2001Go, respectively). However, as shown in Table I, the relative activity of GlcNAc6ST-1 for GlcNAcß1->2Man and that of GlcNAc6ST-5 for GL are greater than those of SulT-a and GlcNAc6ST-3, suggesting that the contribution of GlcNAc6ST-1 and -5 activities to SulT-a seems to be quite low.

GlcNAc6ST-3 and –5 share over 85 % identity of the amino acid sequence (Akama et al., 2000Go; Hemmerich et al., 2001aGo), but their substrate specificities are different; GL is relatively good substrate for GlcNAc6ST-5, but it is poor one for GlcNAc6ST-3 (Table I). Akama et al. (2000, 2001) showed that GlcNAc6ST-5 is the gene responsible for macular corneal dystrophy and is involved in biosynthesis of keratan sulfate in corneas, whereas GlcNAc6ST-3 is unable to contribute to the biosynthesis. This fact seems to be in agreement with our result that GlcNAc6ST-5, not GlcNAc6ST-3, can efficiently act on GL, an analog of poly-N-acetyllactosamine that is the backbone structure of KS.

It has been reported that Galß1->3[(6SO3)Glc-NAcß1->6]GalNAc{alpha}1-> structure is present in hen ovomucin (Strecker et al., 1987Go), human tracheobronchial mucous glycoproteins (Mawhinney et al., 1992aGo), mucous glycoproteins from patients suffering from cystic fibrosis (Mawhinney et al., 1992bGo; Lo-Guidice et al., 1994Go) and chronic bronchitis (Lo-Guidice et al., 1997Go), GlyCAM-1 (Hemmerich et al., 1995Go), oviducal mucins (Maes et al., 1997Go), and human colonic carcinoma mucin (Capon et al., 1997Go). These structures should be synthesized by GlcNAc6ST-1, -2, -3, -5, or GlcNAc6ST-4/Ch6ST-2, because core 2 O-pNP is a good substrate for the five GlcNAc 6-O-SulTs. In spite of various distribution of expression of the five SulTs, information on the occurrence of GlcNAc-6-O-sulfated core 2 structure is rather limited. Because ß1,6GlcNAc-transferases responsible for core 2 formation are mainly expressed in mucus-producing tissues and immunity-related tissues (Bierhuizen and Fukuda, 1992Go; Yeh et al., 1999Go; Schwientek et al., 1999Go, 2000), the core 2–synthesizing enzymes may be limiting factors for biosynthesis of GlcNAc-6-O-sulfated core 2.

BiGP and monoGP are relatively poor substrates for GlcNAc6ST-1 to -5 in comparison with GlcNAcß1->2Man. It has been shown that 6-O-sulfated GlcNAc residues linked to trimannosyl core moiety are present in porcine thyroglobulin (De Waard et al., 1991Go) and Tribolodon hakonensis hyosophorin (Taguchi et al., 1996Go). In this study, we could not reveal N-linked biantennary glycan-preferable GlcNAc 6-O-SulT among the seven SulTs. Uchimura et al. (2000)Go showed that mouse GlcNAc6ST-4/Ch6ST-2 recognizes GlcNAcß1->2Man and GlcNAcß1->6Man-O-Me as good acceptors and acts on core2 O-pNP and GL with a lower efficiency. Human GlcNAc6ST-4/Ch6ST-2 recognizes core2 O-pNP as a good acceptor as well as GlcNAcß1->2Man (Table I). It still remains unclear whether or not the difference in their substrate specificities is due to the difference in their sources.

On the other hand, Spiro et al. (1996)Go showed that a GlcNAc 6-O-SulT in rat liver can act on both GlcNAcß1->2Man-{alpha}1->3(GlcNAcß1->2Man{alpha}1->6)Man, an analog of biGP, and GlcNAcß1->2Man with a similar good efficiency, but can not act on GL. It is interesting whether or not the rat liver SulT corresponds to rat GlcNAc6ST-4/Ch6ST-2.

In this study, we revealed two GlcNAc 6-O-SulTs, expression levels of which are dependent on pathological type of colon cancer. One of those is GlcNAc6ST-2, which is selectively expressed in mucinous adenocarcinomas. Biological role and substrate glycoproteins of GlcNAc6ST-2 in the mucus-depositing cancer cells are unclear now, but considering the broad substrate specificity of GlcNAc6ST-2, mucinous adenocarcinomas may express unique sulfated glycan(s), which is(are) absent in normal mucosa and nonmucinous adenocarcinoma. Such a glycan might serve as a good clinical marker for detecting mucinous adenocarcinomas.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
[35S]PAPS (62.5 GBq/mmol) was purchased from NEN Life Science Products (Boston, MA). GlcNAcß1->2Man, core 2 O-pNP, and core 3 O-pNP were obtained from Funakoshi (Tokyo). BiGP was prepared from egg yolk sialoglycopeptide (Seko et al., 1997Go) by mild acid hydrolysis and Streptococcus 6646K ß-galactosidase digestion (Kiyohara et al., 1976Go) (Seikagaku, Tokyo). Jackbean ß-N-acetylhexosaminidase was prepared by the method of Li and Li (1972)Go. MonoGP was obtained from the urine of GM1-gangliosidosis patients (Yamashita et al., 1981Go) followed by Streptococcus 6646K ß-galactosidase digestion. GL was prepared from lacto-N-tetraose (Kobata, 1972Go) by digestion with Streptococcus 6646K ß-galactosidase. PVL was prepared according to the methods of Kochibe and Matta (1989)Go and conjugated to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. TJA-I-Sepharose (Yamashita et al., 1992Go), KS (sodium salt, bovine cornea) and CS (prepared from chondroitin sulfate A by desulfation) were purchased from Seikagaku.

Cloning of cDNAs encoding for Gal/GalNAc/GlcNAc 6-O-SulTs
cDNAs encoding for Ch6ST-1 and GlcNAc6ST-1 were amplified from Super ScriptTM human testis cDNA library (Life Technologies) by PCR, and cDNAs encoding for KS6ST, GlcNAc6ST-2, -3, and -5, and GlcNAc6ST-4/Ch6ST-2 were amplified by PCR from the genome prepared from lymphocytes of the A. Seko. The open reading frames for KS6ST and GlcNAc6ST-1 to -5 are encoded within single exons (Hemmerich et al., 2001aGo). Oligonucleotide primers used for the PCR were:

• 5'-tttaagcttATGGAGAAAGGACTCACT-3' (forward primer for Ch6ST-1)

• 5'-ttttctagaCTACGTGACCCAGAAGGT-3' (reverse primer for Ch6ST-1)

• 5'-tttaagcttGGAGCAGTCCCT-3' (forward primer for KS6ST)

• 5'-ttttctagaTCACGAGAAGGGGCGGA-3' (reverse primer for KS6ST)

• 5'-tttgaattcTCTCGGAATGAAGGTG-3' (forward primer for GlcNAc6ST-1)

• 5'-tttctcgagATCAGGTCTCCTGGGAA-3' (reverse primer for GlcNAc6ST-1)

• 5'-tttaagcttAGCACAATGCTACTGCCT-3' (forward primer for GlcNAc6ST-2)

• 5'-ttttctagaTTAGTGGATTTGCTCAGG-3' (reverse primer for GlcNAc6ST-2)

• 5'-tttaagcttATGTGGCTGCCACGGTT-3' (forward primer for GlcNAc6ST-3)

• 5'-ttttctagaTCAGTCAGGCGATGCCCA-3' (reverse primer for GlcNAc6ST-3)

• 5'-tttaagcttAGCAGTCAGCATGTGGCT-3' (forward primer for GlcNAc6ST-5)

• 5'-ttttctagaGCGCCTGCTACAACTGT-3' (reverse primer for GlcNAc6ST-5)

• 5'-tttaagcttCGGTGAACGATGAAGGGC-3' (forward primer for GlcNAc6ST-4/Ch6ST-2)

• 5'-ttttctagaAGGGATGGGAGGCTACGT-3' (reverse primer for GlcNAc6ST-4/Ch6ST-2)

Sequences in lowercase letters contain appropriate restriction sites.

Amplified cDNAs for Ch6ST-1, KS6ST, GlcNAc6ST-2, -3, -5, and GlcNAc6ST-4/Ch6ST-2 were digested with HindIII and XbaI (as for GlcNAc6ST-1, with EcoRI and XhoI) and cloned between the respective sites of pcDNA3 (Invitrogen). The constructed plasmids were named pcDNA3-Ch6ST-1; -KS6ST; -GlcNAc6ST-1, -2, -3, and -5; and -GlcNAc6ST-4/Ch6ST and were sequenced using Applied Biosystems PRISM 310 Genetic Analyzer (PE Biosystems). The nucleic acid sequences of the seven SulTs cloned were identical to the sequences with accession number AB017915 (Tsutsumi et al., 1998Go) for Ch6ST-1; U65637 (Mazany et al., 1998Go), AB003791 (Fukuta et al., 1997Go), and AF090137 (Li and Tedder, 1999Go) for KS6ST; AB021124 (Sakaguchi et al., 2000Go) and AF083066 (Li and Tedder, 1999Go) for GlcNAc6ST-1; AF149783 (Yeh et al., 2001Go) and AF280088 (Hemmerich et al., 2001aGo) for GlcNAc6ST-2; AF176838 (Lee et al., 1999Go) and AF246718 (Akama et al., 2000Go) for GlcNAc6ST-3; AF219990 (Akama et al., 2000Go) and AF280086 (Hemmerich et al., 2001aGo) for GlcNAc6ST-5; AB037187 (Kitagawa et al., 2000Go) and AB040711 (Uchimura et al., 2000Go) for GlcNAc6ST-4/Ch6ST-2.

Expression of SulTs in COS-7 cells
The plasmids (1 µg) were transfected into COS-7 cells on 35-mm dishes using Lipofectin Reagent (Life Technologies) according to the manufacturer’s instructions. After 48 h, the cells were washed twice with phosphate-buffered saline, scraped off from the dishes in 10 mM HEPES–NaOH (pH 7.2) and 0.25 M sucrose, and homogenized. The homogenates were ultracentrifuged at 100,000 x g for 1 h. The precipitated crude membranes were suspended in 20 mM HEPES–NaOH (pH 7.2) and kept at –80°C until use.

Assay of SulT activity
In the cases of GlcNAc6ST-1, -2, -3, and -5, 20 µl of reaction mixture consisting of 0.1 M sodium cacodylate (pH 6.8), 10 mM MnCl2, 0.1% (w/v) digitonin, 50 µg/ml protamine chloride, 2 mM dithiothreitol, 0.1 M NaF, 2 mM ATP-Na2, 6.5 µM [35S]PAPS (4.9 x 105 dpm), 1 mM acceptor substrate, and the membrane fraction approximately diluted were incubated at 37°C for 1 h. In the cases of Ch6ST-1, KS6ST, and GlcNAc6ST-4/Ch6ST-2, 0.1 M sodium cacodylate (pH 6.4) and 0.1% (v/v) Triton X-100 were added to the reaction mixture in place of sodium cacodylate (pH 6.8) and digitonin, respectively. The 35S-labeled products were purified by paper electrophoresis (pyridine:acetic acid:water, 3:1:387, pH 5.4). After drying, the paper was monitored for radioactivity with a radiochromatogram scanner, and the 35S-labeled products were extracted with water and analyzed for the linkage position of [35S]sulfate. Enzymatic activity values were calculated as the means of three independent experiments.

Lectin affinity chromatography
35S-labeled compounds were applied to PVL-Sepharose column (3 mg/ml gel; 0.7 x 2.5 cm). Elution was started with 4 ml of 10 mM Tris-HCl (pH 8.0)–0.15 M NaCl (TBS) containing 1 mM CaCl2 and 1 mM MgCl2, followed by the same buffer containing 0.3 M GlcNAc. As for TJA-I-Sepharose column (3 mg/ml gel; 0.7 x 2.5 cm), elution was started with 4 ml of TBS, followed by the same buffer containing 0.1 M lactose.

ß-N-acetylhexosaminidase digestion
[35S]sulfated oligosaccharides were digested with jackbean ß-N-acetylhexosaminidase (0.1 U) in 50 µl of 0.1 M citrate-phosphate buffer (pH 6.0) at 37°C for 20 h. After incubation, reaction mixtures were heated at 100°C for 2 min to stop the reaction and applied to lectin affinity chromatography.

Determination of protein concentrations
The protein concentrations in the preparations of crude membranes were estimated using the Bio-Rad Protein Assay dye reagent with bovine serum albumin as a standard.

Preparation of normal human colonic mucosa and adenocarcinoma tissues
Fresh samples of normal colonic mucosa and carcinomas from 18 patients were stored frozen at –80 °C before use. Table II shows the Dukes’ staging system, the histological character of the adenocarcinomas, and the serum CEA levels of the patients before surgery. Eight of the 18 patients were assessed as "B," 6 patients were "C," 4 patients were "C(D)," according to Dukes’ staging system. Twelve cases (cases 1–12) were classified as nonmucinous adenocarcinoma, two (cases 15 and 17) were classified as mucinous adenocarcinoma, and four (cases 13, 14, 16, and 18) were classified as adenocarcinoma with a mucinous component. Four patients had a focus of distant metastasis in the liver (cases 12, 16–18). Serum CEA levels were within the normal range (less than 2.5 ng/ml) in these eight patients (cases 2, 4, 5, 7, 12, 13, 15, and 17), whereas elevation of serum CEA levels was seen in other nine patients.

Normal mucosal tissue was obtained from an area 10 cm distant from the margin of the carcinoma, and it was separated completely from the muscle layer. The carcinoma tissue was obtained from the surface portion of the carcinoma, which was rich in carcinoma cells and had little interstitial tissue.

The study was approved by the Kagoshima University Faculty of Medicine Human Investigation Committee (No. H13–4).

Quantitation of GlcNAc6ST-2 and -3 transcripts by the competitive RT-PCR method
Levels of GlcNAc6ST-2 and -3 transcripts were measured by competitive PCR (Gilliland et al., 1990Go) based on the amounts of their respective cDNAs, which were reverse-transcribed from RNA samples. For distinction of a target cDNA from its competitor DNA, pcDNA3-GlcNAc6ST-2 and -3 were digested with SmaI and ligated. The treatment generated the deletion of 177- and 147-bp fragments, respectively, within the region corresponding to their cDNAs. The competitor cDNA for ß-actin was generated by deleting 218-bp fragment from the single-stranded cDNAs of normal colon tissue by PCR using oligonucleotides having the sequence 5'-ctcaagcttGATATCGCCGCGCTCGTCGTCGAC-3' as a forward primer and 5'-ctcggatccCAGGAAGGAAGGCTGGAAGAGTGCCAGTCAGGTCCCGGCCAGG-3' as a reverse primer. Amplified HindIII–BamHI fragment was inserted into the HindIII–BamHI site of pBluescript II SK+/–.

From 5 µg of total RNA isolated by lysis of normal colon tissues or adenocarcinomas in ISOGEN (Nippon Gene, Japan), cDNAs were synthesized using oligo(dT) primers and SuperScript II (Gibco BRL) in a total volume of 21 µl. To check for contamination by genomic DNA in RNA samples, cDNA synthesis was performed in the absence of SuperScript II using a small aliquot of isolated total RNA. One microliter of appropriately diluted cDNAs was subjected to the quantitative PCR analysis according to the methods of Sasaki et al. (1994)Go. After incubation at 95°C for 5 min, PCR was performed in a volume of 20 µl for 30 cycles of 95°C for 30 s, 65°C for 1 min, and 72°C for 2 min. The primers used were:

• 5'-ATGGAACCTGGTCCCCGGAGACA-3' (forward primer for GlcNAc6ST-2)

• 5'-AAGCGTGGTCACCCATGCCCTTG-3' (reverse primer for GlcNAc6ST-2)

• 5'-CCGCCTTTTTCAACTGGGCAACGA-3' (forward primer for GlcNAc6ST-3)

• 5'-AGGCCTCGATTGGCTTGCCGATC-3' (reverse primer for GlcNAc6ST-3)

• 5'-GATATCGCCGCGCTCGTCGTCGAC-3' (forward primer for ß-actin)

• 5'-CAGGAAGGAAGGCTGGAAGAGTGCC-3' (reverse primer for ß-actin)

After amplification, 10 µl aliquots were subjected to electrophoresis in 1.0% agarose gels, followed by staining with ethidium bromide. Amplified DNA fragments were quantified by scanning with a fluoro-image analyzer FLA-2000 (Fuji Photo Film, Japan). The amounts of target cDNAs were normalized by those of ß-actin.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported in part by grants-in-aid 12470046 and 13220016 to S.Y. from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. During processing of this manuscript, Uchimura et al. (2002)Go reported the substrate specificities of GlcNAc6ST-1, -2, and -3 and semiquantitative analysis of expression levels of their transcripts in two specimens of colon cancer tissues. Their results were consistent with ours in this paper, and they also suggested that GlcNAc6ST-2 corresponds to SulT-b.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
biGP, GlcNAcß1->2Man{alpha}1->3(GlcNAcß1->2Man{alpha}1->6)Man-ß1->4GlcNAcß1->4GlcNAc; CEA, carcinoembryonic antigen; CS, chondroitin, sodium salt; GL, GlcNAcß1->3Galß1->4Glc; GlcNAc6ST, GlcNAc 6-O-sulfotransferase; KS, keratan sulfate; monoGP, GlcNAcß1->2Man{alpha}1->3/6Manß1->4GlcNAc; NFA-2, normal fecal antigen-2; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; PCR, polymerase chain reaction; pNP, p-nitrophenyl; PVL, Psathyrella velutina lectin; RT, reverse transcription; SulT, sulfotransferase; TBS, 10 mM Tris-HCl (pH 8.0)–0.15 M NaCl; TJA-I, Trichosanthes japonica agglutinin-I.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: yamashita{at}sasaki.or.jp Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Akama, T.O., Nishida, K., Nakayama, J., Watanabe, H., Ozaki, K., Nakamura, T., Dota, A., Kawasaki, S., Inoue, Y., Maeda, N., and others. (2000) Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene. Nature Genet., 26, 237–241.[CrossRef][ISI][Medline]

Akama, T.O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M.N. (2001) Human corneal GlcNAc 6-O-sulfotransferase and mouse intestinal GlcNAc 6-O-sulfotransferase both produce keratan sulfate. J. Biol. Chem., 276, 16271–16278.[Abstract/Free Full Text]

Bartes, A., Bhakta, S., and Hemmerich, S. (2001) Sulfation of endothelial mucin by corneal keratan N-acetylglucosamine 6-O-sulfotransferase(GST-4ß). Biochem. Biophys. Res. Commun., 282, 928–933.[CrossRef][ISI][Medline]

Bhakta, S., Bartes, A., Bowman, K.G., Kao, W.-M., Polsky, I., Lee, J.K., Cook, B.N., Bruehl, R.E., Rosen, S.D., Bertozzi, C.R., and Hemmerich, S. (2000) Sulfation of N-acetylglucosamine by chondroitin 6-O-sulfotransferase-2(GST-5). J. Biol. Chem., 275, 40226–40234.[Abstract/Free Full Text]

Bierhuizen, M.F. and Fukuda, M. (1992) Expression cloning of a cDNA encoding UDP-GlcNAc:Galß1-3GalNAc-R(GlcNAc to GalNAc)ß1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. Proc. Natl Acad. Sci. USA, 89, 9326–9330.[Abstract]

Bistrup, A., Bhakta, S., Lee, J.K., Belov, Y.Y., Gunn, M.D., Zuo, F.-R., Huang, C.-C., Kannagi, R., Rosen, S.D., and Hemmerich, S. (1999) Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin. J. Cell Biol., 145, 899–910.[Abstract/Free Full Text]

Capon, C., Wieruszeski, J.-M., Lemoine, J., Byrd, J.C., Leffler, H., and Kim, Y.S. (1997) Sulfated Lewis x determinants as a major structural motif in glycans from LS174T-HM7 human colon carcinoma mucin. J. Biol. Chem., 272, 31957–31968.[Abstract/Free Full Text]

De Waard, P., Koorevaar, A., Kamerling, J.P., and Vliegenthart, J.F.G. (1991) Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase-F. J. Biol. Chem., 266, 4237–4243.[Abstract/Free Full Text]

Felipe, M.S. (1969) Value of histochemical reactions for mucosubstances in the diagnosis of certain pathological conditions of the colon and rectum. Gut, 10, 577–586.

Fukuda, M., Hiraoka, N., Akama, T.O., and Fukuda, M.N. (2001) Carbohydrate-modifying sulfotransferases: structure, function, and pathophysiology. J. Biol. Chem., 276, 47747–47750.[Free Full Text]

Fukushima, K., Ohkura, T., Kanai, M., Kuroki, M., Matsuoka, Y., Kobata, A., and Yamashita, K. (1995) Carbohydrate structures of a normal counterpart of the carcinoembryonic antigen produced by colon epithelial cells of normal adults. Glycobiology, 5, 105–115.[Abstract]

Fukuta, M., Uchimura, K., Nakashima, K., Kato, M., Kimata, K., Shinomura, T., and Habuchi, O. (1995) Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase. J. Biol. Chem., 270, 18575–18580.[Abstract/Free Full Text]

Fukuta, M., Inazawa, J., Torii, T., Tsuzuki, K., Shimada, E., and Habuchi, O. (1997) Molecular cloning and characterization of human keratan sulfate Gal 6-O-sulfotransferase. J. Biol. Chem., 272, 32321–32328.[Abstract/Free Full Text]

Fukuta, M., Kobayashi, Y., Uchimura, K., Kimata, K., and Habuchi, O. (1998) Molecular cloning and expression of human chondroitin 6-sulfotransferase. Biochim. Biophys. Acta, 1399, 57–61.[ISI][Medline]

Gilliland, G., Perrin, S., Blanchard, K., and Bunn, F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl Acad. Sci. USA, 87, 2725–2729.[Abstract]

Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and Noda, M. (1993) Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes. J. Biol. Chem., 268, 21968–21974.[Abstract/Free Full Text]

Hemmerich, S., Leffler, H., and Rosen, S.D. (1995) Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin. J. Biol. Chem., 270, 12035–12047.[Abstract/Free Full Text]

Hemmerich, S., Lee, J.K., Bhakta, S., Bistrup, A., Ruddle, N.R., and Rosen, S.D. (2001a) Chromosomal localization and genomic organization for the galactose/N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferase gene family. Glycobiology, 11, 75–87.[Abstract/Free Full Text]

Hemmerich, S., Bistrup, A., Singer, M.S., van Zante, A., Lee, L.K., Tsay, D., Peters, M., Carminati, J.L., Brennan, T.J., Carver-Moore, K., and others. (2001b) Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes. Immunity, 15, 237–247.[ISI][Medline]

Hiraoka, N., Petryniak, B., Nakayama, J., Tsuboi, S., Suzuki, M., Yeh, J.-C., Izawa, D., Tanaka, T., Miyasaka, M., Lowe, J.B., and Fukuda, M. (1999) A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis x, an L-selectin ligand displayed by CD34. Immunity, 11, 79–89.[ISI][Medline]

Kitagawa, H., Fujita, M., Ito, N., and Sugahara, K. (2000) Molecular cloning and expression of a novel chondroitin 6-O-sulfotransferase. J. Biol. Chem., 275, 21075–21080.[Abstract/Free Full Text]

Kiyohara, T., Terao, T., Shioiri-Nakano, K., and Osawa, T. (1976) Purification and characterization of ß-N-acetylhexosaminidase and ß-galactosidase from Streptococcus 6646K. J. Biochem. (Tokyo), 80, 9–17.[Abstract]

Kobata, A. (1972) Isolation of oligosaccharides from human milk. Methods Enzymol., 28, 262–271.

Kochibe, N. and Matta, K.L. (1989) Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom. J. Biol. Chem., 264, 173–177.[Abstract/Free Full Text]

Lee, J.K., Bhakta, S., Rosen, S.D., and Hemmerich, S. (1999) Cloning and characterization of a mammalian N-acetylglucosamine-6-sulfotransferase that is highly restricted to intestinal tissue. Biochem. Biophys. Res. Commun., 263, 543–549.[CrossRef][ISI][Medline]

Li, Y.T. and Li, S.C. (1972) {alpha}-Mannosidase, ß-N-acetylhexosaminidase, and ß-galactosidase from jack bean meal. Methods Enzymol., 28, 702–713.

Li, X. and Tedder, T.F. (1999) CHST1 and CHST2 sulfotransferases expressed by human vascular endothelial cells: cDNA cloning, expression and chromosomal localization. Genomics, 55, 345–347.[CrossRef][ISI][Medline]

Lo-Guidice, J.-M., Wieruszeski, J.-M., Lemoine, J., Verbert, A., Roussel, P., and Lamblin, G. (1994) Sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis. J. Biol. Chem., 269, 18794–18813.[Abstract/Free Full Text]

Lo-Guidice, J.-M., Herz, H., Lamblin, G., Plancke, Y., Roussel, P., and Lhermitte, M. (1997) Structures of sulfated oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b-) patient suffering from chronic bronchitis. Glycoconj. J., 14, 113–125.[CrossRef][ISI][Medline]

Maes, E., Florea, D., Delplace, F., Lemoine, J., Plancke, Y., and Strecker, G. (1997) Structural analysis of the oligosaccharide-alditols released by reductive ß-elimination from oviducal mucins of Rana temporaria. Glycoconj. J., 14, 127–146.[CrossRef][ISI][Medline]

Mawhinney, T.P., Adelstein, E., Gayer, D.A., Landrum, D.C., and Barbero, G.J. (1992a) Structural analysis of monosulfated side-chain oligosaccharides isolated from human tracheobronchial mucous glycoproteins. Carbohydr. Res., 223, 187–207.[CrossRef][ISI][Medline]

Mawhinney, T.P., Landrum, D.C., Gayer, D.A., and Barbero, G.J. (1992b) Sulfated sialyl-oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis. Carbohydr. Res., 235, 179–197.[CrossRef][ISI][Medline]

Mazany, K.D., Peng, T., Watson, C.E., Tabas, I., and Williams, K.J. (1998) Human chondroitin 6-sulfotransferase: cloning, gene structure and chromosomal localization. Biochim. Biophys. Acta, 1407, 92–97.[ISI][Medline]

Mitsuoka, C., Sawada-Kasugai, M., Ando-Furui, K., Izawa, M., Nakanishi, H., Nakamura, S., Ishida, H., Kiso, M., and Kannagi, R. (1998) Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X. J. Biol. Chem., 273, 11225–11233.[Abstract/Free Full Text]

Morson, B.C. and Sobin, L.H. (1990) Histological typing of intestinal tumors, 2d ed. Springer-Verlag, Berlin.

Negishi, M., Pedersen, L.G., Petrotchenko, E., Shevtsov, S., Gorokhov, A., Kakuta, Y., and Pedersen, L.C. (2001) Structure and function of sulfotransferases. Arch. Biochem. Biophys., 390, 149–157.[CrossRef][ISI][Medline]

Sakaguchi, H., Kitagawa, H., and Sugahara, K. (2000) Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to ß-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence. Biochim. Biophys. Acta, 1523, 269–276.[ISI][Medline]

Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) Expression cloning of a novel {alpha}1, 3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes. J. Biol. Chem., 269, 14730–14737.[Abstract/Free Full Text]

Schwientek, T., Nomoto, M., Levery, S.B., Merkx, G., van Kessel, A.G., Bennett, E.P., Hollingsworth, M.A., and Clausen, H. (1999) Control of O-glycan branch formation: molecular cloning of human cDNA encoding a novel ß1, 6-N-acetylglucosaminyltransferase forming core 2 and core 4. J. Biol. Chem., 274, 4504–4512.[Abstract/Free Full Text]

Schwientek, T., Yeh, J.-C., Levery, S.B., Keck, B., Merkx, G., van Kessel, A.G., Fukuda, M., and Clausen, H. (2000) Control of O-glycan branch formation: molecular cloning and characterization of a novel thymus-associated core 2 ß1, 6-N-acetylglucosaminyltransferase. J. Biol. Chem., 275, 11106–11113.[Abstract/Free Full Text]

Seko, A., Koketsu, M., Nishizono, M., Enoki, Y., Ibrahim, H.R., Juneja, L.R., Kim, M., and Yamamoto, T. (1997) Occurrence of a sialylglycopeptide and free sialylglycans in hen’s egg yolk. Biochim. Biophys. Acta, 1335, 23–32.[ISI][Medline]

Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Biochemical differences between two types of N-acetylglucosamine:->6sulfotransferases in human colonic adenocarcinomas and the adjacent normal mucosa: specific expression of a GlcNAc:->6sulfotransferase in mucinous adenocarcinoma. Glycobiology, 10, 919–929.[Abstract/Free Full Text]

Spiro, R.G., Yasumoto, Y., and Bhoyroo, V. (1996) Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in ß-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides. Biochem. J., 319, 209–216.[ISI][Medline]

Strecker, G., Wieruszeski, J.-M., Martel, C., and Montreuil, J. (1987) Determination of the structure of sulfated tetra- and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. A fast atom bombardment-mass spectrometric and 1H-NMR spectroscopic study. Glycoconj. J., 4, 329–337.[ISI]

Taguchi, T., Iwasaki, M., Muto, Y., Kitajima, K., Inoue, S., Khoo, K.-H., Morris, H.R., Dell, A., and Inoue, Y. (1996) Occurrence and structural analysis of highly sulfated multiantennary N-linked glycan chains derived from a fertilization-associated carbohydrate-rich glycoprotein in unfertilized eggs of Tribolodon hakonensis. Eur. J. Biochem., 238, 357–367.[Abstract]

Torii, T., Fukuta, M., and Habuchi, O. (2000) Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase. Glycobiology, 10, 203–211.[Abstract/Free Full Text]

Tsutsumi, K., Shimakawa, H., Kitagawa, H., and Sugahara, K. (1998) Functional expression and genomic structure of human chondroitin 6-O-sulfotransferase. FEBS Lett., 441, 235–241.[CrossRef][ISI][Medline]

Uchimura, K., Kadomatsu, K., Fan, Q.W., Muramatsu, H., Kurosawa, N., Kaname, T., Yamamura, K., Fukuta, M., Habuchi, O., and Muramatsu, T. (1998a) Mouse chondroitin 6-sulfotransferase: molecular cloning, characterization and chromosomal mapping. Glycobiology, 8, 489–496.[Abstract/Free Full Text]

Uchimura, K., Muramatsu, H., Kadomatsu, K., Fan, Q.-W., Kurosawa, N., Mitsuoka, C., Kannagi, R., Habuchi, O., and Muramatsu, T. (1998b) Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase. J. Biol. Chem., 273, 22577–22583.[Abstract/Free Full Text]

Uchimura, K., Muramatsu, H., Kaname, T., Ogawa, H., Yamakawa, T., Fan, Q.-W., Mitsuoka, C., Kannagi, R., Habuchi, O., Yokoyama, I., and others. (1998c) Human N-acetylglucosamine-6-O-sulfotransferase involved in the biosynthesis of 6-sulfo sialyl Lewis x: molecular cloning, chromosomal mapping and expression in various organs and tumor cells. J. Biochem. (Tokyo), 124, 670–678.[Abstract]

Uchimura, K., Fasakhany, F., Kadomatsu, K., Matsukawa, T., Yamakawa, T., Kurosawa, N., and Muramatsu, T. (2000) Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities. Biochem. Biophys. Res. Commun., 274, 291–296.[CrossRef][ISI][Medline]

Uchimura, K., El-Fasakhany, F.M., Hori, M., Hemmerich, S., Blink, S.E., Kansas, G.S., Kanamori, A., Kumamoto, K., Kannagi, R., and Muramatsu, T. (2002) Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression. J. Biol. Chem., 277, 3979–3984.[Abstract/Free Full Text]

Yamashita, K., Ohkura, T., Okada, S., Yabuuchi, H., and Kobata, A. (1981) Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups. J. Biol. Chem., 256, 4789–4798.[Abstract]

Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Structural studies of the carbohydrate moieties of carcinoembryonic antigens. Cancer Res., 47, 3451–3459.[Abstract]

Yamashita, K., Umetsu, K., Suzuki, T., and Ohkura, T. (1992) Purification and characterization of a Neu5Ac{alpha}2->6Galß1->4GlcNAc and HSO3->6Galb1->4GlcNAc specific lectin in tuberous roots of Trichosanthes japonica. Biochemistry, 31, 11647–11650.[ISI][Medline]

Yamori, T., Kimura, H., Stewart, K., Ota, D.M., Cleary, K.R., and Irimura, T. (1987) Differential production of high molecular weight sulfated glycoproteins in normal colonic mucosa, primary colon carcinoma and metastases. Cancer Res., 47, 2741–2747.[Abstract]

Yeh, J.-C., Ong, E., and Fukuda, M. (1999) Molecular cloning and expression of a novel ß-1, 6-N-acetylglucosaminyltransferase that forms core 2, core 4, and I branches. J. Biol. Chem., 274, 3215–3221.[CrossRef]

Yeh, J.-C., Hiraoka, N., Petryniak, B., Nakayama, J., Ellies, L.G., Rabuka, D., Hindsgaul, O., Marth, J.D., Lowe, J.B., and Fukuda, M. (2001) Novel sulfated lymphocyte homing receptors and their control by a core 1 extention ß1, 3-N-acetylglucosaminyltransferase. Cell, 105, 957–969.[CrossRef][ISI][Medline]