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

Akira Seko, Jun-ichi Sumiya, Suguru Yonezawa2, Koji Nagata2 and Katsuko Yamashita1

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

Received on February 9, 1999; revised on March 5, 2000; accepted on March 13, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
6-O-Sulfation of ß-GlcNAc is an initial step in the biosynthesis of N-linked and O-linked sulfated glycans, which are widely distributed in colonic tissues. However, the biochemical mechanism of this sulfation in human colonic carcinogenesis was still unclear. In this study, we found two types of GlcNAc:->6sulfotransferases (SulT) in human colonic adenocarcinomas and the adjacent normal mucosa, and we determined their enzymatic characteristics. One SulT, named SulT-a, was present in the adjacent normal mucosa and in non-mucinous adenocarcinomas, whereas the other SulT, named SulT-b, was present only in mucinous adenocarcinomas and adenocarcinomas with a mucinous component. SulT-a preferentially acted on Galß1->3(GlcNAcß1->6)GalNAc{alpha}1-p-nitrophenyl (pNP) and GlcNAcß1->2Man, whereas SulT-b could act not only on these two glycans, but also on GlcNAcß1->3GalNAc{alpha}1-pNP and GlcNAcß1->3Galß1->4Glc. The levels of SulT-a activity were significantly lower in non-mucinous adenocarcinomas than in the adjacent mucosa. In contrast, SulT-b was expressed in mucinous adenocarcinomas and in adenocarcinomas with a mucinous component. These results indicate that there are at least two types of GlcNAc:->6SulT, SulT-a and -b, in colonic mucosa and adenocarcinomas, and that the occurrence of these enzymes is closely correlated with colonic cancer and the presence of areas of mucin accumulation.

Key words: sulfotransferase/adenocarcinoma/mucinous carcinoma/ carcinoembryonic antigen/sulfomucin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sulfation at the C-6 position of ß-GlcNAc is catalyzed by GlcNAc:->6sulfotransferase (SulT). The GlcNAc:->6SulT enzymatic activities in rat stomach (Carter et al., 1988Go) rat corpus (Goso and Hotta, 1993Go), rat liver (Spiro et al., 1996Go), human respiratory mucosa (Degroote et al., 1997Go), and porcine lymph nodes (Bowman et al., 1998Go) have been characterized, and recently the genes for three types of GlcNAc:->6SulT have been cloned (Uchimura et al., 1998aGo,b; Bistrup et al., 1999Go; Hiraoka et al., 1999Go; Lee et al., 1999Go). Although N-linked and O-linked glycoproteins containing GlcNAc-6-O-sulfate residues in human colonic tissues and cell lines have been reported (Yamashita et al., 1987Go, 1989; Fukushima et al., 1995Go; Capon et al., 1997Go), the biochemical properties of GlcNAc:->6SulT in human colonic mucosa and adenocarcinomas have scarcely been investigated.

We previously determined the structure of N-linked glycans in carcinoembryonic antigens (CEA) from foci of liver metastases and the counterpart glycoprotein in normal colonic mucosa, normal fecal antigen-2 (NFA-2) (Yamashita et al., 1987Go; Fukushima et al., 1995Go). The content of GlcNAc-6-O-sulfate residues was consistently found to be ~40% in the sugar chains released from five NFA-2 samples. On the other hand, the content of sulfated glycans in two CEA samples was much lower (10%) than that in the case of NFA-2, and the content of sulfated glycans in one CEA sample, in contrast, was much higher (50%) (Fukushima et al., 1995Go). These results led us to speculate that human colon tissues have at least two GlcNAc:->6sulfotransferases. Because CEA is a tumor-associated glycoprotein containing 50–60%(w/w) of N-linked sugar chains (Yamashita and Kobata, 1996Go) and because it has been suggested that CEA may be involved in cell-to-cell interaction as an adhesion molecule (Benchimol et al., 1989Go), it seemed important to study the GlcNAc:->6SulT expressed in the course of human colonic carcinogenesis in order to clarify the role of the glycan chains in the biological functions of CEA.

In this study, we examined the biochemical characteristics of GlcNAc:->6SulT in human colonic adenocarcinomas and the adjacent normal mucosa. As a result, we found that one type of SulT activity is expressed at a lower level in non-mucinous adenocarcinomas than in the adjacent normal mucosa, and, moreover, that another type of GlcNAc:->6SulT occurs in mucinous adenocarcinomas and adenocarcinomas with a mucinous component.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We prepared crude membrane fractions from human colonic adenocarcinomas from 37 patients and the adjacent, pathologically normal mucosa, and assayed comparatively the GlcNAc:->6SulT activities in these fractions, using GlcNAcß1->2Man as the acceptor substrate. The adenocarcinoma specimens were divided into two groups on the basis of the changes in levels of SulT activity observed in the course of carcinogenesis. In one group of adenocarcinomas, the specific activity of SulT was found to be higher than that in the adjacent mucosa, whereas the other group showed the converse (described below). Moreover, in the former group of adenocarcinomas, small-scale deposition of mucin was often found (Figure 1). These findings led us to speculate that the SulT in the adenocarcinomas of the former group is associated with the presence of the mucinous component, and that it differs from the SulT in the adjacent mucosa. On the basis of these preliminary observations, we comparatively investigated the enzymatic characteristics of the SulT in the adenocarcinomas containing a mucinous component, which we named SulT-b, and those of the SulT in the normal adjacent mucosa, which we named SulT-a.



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Fig. 1. Histological findings of the normal mucosa (A) and histological types of the carcinoma examined (BF) stained by hematoxylin-eosin. (A) Normal mucosa distant from carcinoma (case No. 1); 60x. (B) Tubular adenocarcinoma (case No. 8); 120x. (C) Mucinous adenocarcinoma (case No. 36); 60x. (D) Mixed type of mucinous adenocarcinoma (upper area) and tubular adenocarcinoma (lower area) (case No. 37); 60x. (E) Mixed type of mucinous adenocarcinoma (left area) and tubular adenocarcinoma (right area) (case No. 32); 60x. (F) Area of tubulo-villous adenoma producing plentiful mucin in adenocarcinoma (case No.25); 60x.

 
Characterization of the SulT products
To determine the linkage position of [35S]sulfate, the [35S]-labeled products generated by SulT-a were first digested with jack bean ß-N-acetylhexosaminidase, which cleaves nonsubstituted ß-GlcNAc residues, but not 6-O-sulfated ß-GlcNAc residues (A.Seko and K.Yamashita, unpublished observations).

After digestion, the [35S]-labeled digestion products were applied to a PVL-Sepharose column. PVL is a lectin that recognizes nonsubstituted ß-GlcNAc or ß-GlcNAc substituted at the C-6 position, but it does not bind to ß-GlcNAc substituted at the C-3 or C-4 position (Kochibe and Matta, 1989Go). A fraction of the [35S]-labeled digestion products bound to the PVL-Sepharose column and was eluted with 0.3 M GlcNAc (Figure 2A), indicating the incorporation of [35S]sulfate at the C-6 position of GlcNAc residues. Rechromatography of the pass-through fraction did not show any radioactivity in the bound and sequential eluent with 0.3M GlcNAc. The [35S]-labeled product in the pass-through fraction in Figure 2 was 3'-O-[35S]sulfo-Galß1->3GalNAc{alpha}-pNP, because the [35S]-labeled product was retarded on a MAL-Sepharose column (data not shown), which recognizes SO3->3Galß1-> 3GalNAc as well as Neu5Ac{alpha}2->3Galß1->4GlcNAc3. Gal:->3SulT activities were reported to be present in human normal colon and colon cancer (Kuhns et al., 1995Go; Chandrasekaran et al., 1997Go).



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Fig. 2. Analysis of the sulfate linkages of [35S]SO3-core 2 synthesized by human colonic GlcNAc:->6SulT-a. (A) PVL-Sepharose column chromatography of [35S]SO3-core 2 digested by jack bean ß-N-acetylhexosaminidase. Elution was started with 6 ml of 10 mM Tris-HCl (pH 8), 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, followed by the same buffer containing 0.3 M GlcNAc (indicated by an arrow). Vo; void volume. (B) and (C), cellulose TLC of the [35S]-labeled derivative using the solvent system ethyl acetate/pyridine/acetic acid/water = 5:5:1:3 (B) or 1-butanol/pyridine/water = 6:4:3 (C). The [35S]-labeled derivative was obtained from the PVL-Sepharose-bound [35S]SO3-core 2 component by hydrazinolysis-nitrous acid deamination and following NaBH4 reduction. The thin layer plates were assayed for radioactivity using a radiochromatogram scanner. a, b, and c show the positions of authentic 2,5-anhydro-6-sulfo-[3H]mannitol, 2,5-anhydro-[3H]mannitol, and the front of the developing solvent, respectively. The same results were obtained in the case of SulT-b.

 
Furthermore, when the PVL-Sepharose-bound product was sequentially treated by hydrazinolysis, nitrous acid-deamination, and reduction with NaBH4, the final reaction product was found at the same position as authentic 2,5-anhydro-6-sulfo-[3H]mannitol by TLC using either of the solvents employed, pyridine/ethyl acetate/acetic acid/water = 5:5:1:3 (Figure 2B) or 1-butanol/pyridine/water = 6:4:3 (Figure 2C). This result also supports the view that the [35S]sulfate in the PVL-bound product was linked to the C-6 position of GlcNAc. The same results were obtained in the case of SulT-b (data not shown).

Biochemical properties of SulT-a and SulT-b
In the enzymatic reactions of SulT-a and SulT-b, sulfated products increased linearly for at least 1.5 hr, or up to a protein concentration of 2.0 mg/ml (data not shown). We examined the effects of pH (Figure 3A,B), divalent cations (Figure 3C,D), and detergent (Figure 3E,F) on SulT-a and SulT-b. The optimum pH for activity was 6.8–8.2 in each instance (Figure 3A,B). The profiles of pH dependency were similar, but SulT-a showed higher activity in cacodylate buffer (pH 5.53–6.81) than SulT-b. In the presence of 10–20 mM MnCl2, the levels of SulT-a and SulT-b activity increased 2.8- and 3.5~4-fold, respectively (Figure 3C, D, solid circles). MgCl2 and CaCl2 had no effect on the activity of these enzymes (Figure 3C, D, open circles and solid triangles, respectively). Chelation of divalent cations by addition of EDTA did not decrease the activity of these enzymes. These results indicate that MnCl2 has a positive effect on the activity of both types of SulT, but is not essential for their activity.



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Fig. 3. The effects of pH (A and B), divalent cations (C and D), detergents (E and F) and substrate concentrations (G and H) on SulT-a (A, C, E, G) and SulT-b (B, D, F, H) activities. One millimolar core 2 as the acceptor substrate was used in A–F. (A) and (B), the enzymatic activity was assayed using 50 mM sodium cacodylate buffer (pH 5.53–6.81, solid circles), 50 mM HEPES-NaOH buffer (pH 6.78–8.13, open circles), or 50 mM TAPS-NaOH buffer (pH 8.19–8.84, open triangles). The ionic strength was adjusted to 0.1 with NaCl. (C) and (D), the enzymatic activity was assayed in the presence of the indicated concentrations of MnCl2 (solid circles), MgCl2 (open circles), or CaCl2 (solid triangles). The enzymatic activity in the presence of 5 mM EDTA (open triangles) is also indicated. (E) and (F), the enzymatic activity was assayed in the presence of the indicated concentrations of digitonin (solid circles), Triton X-100 (solid triangles), n-octyl-ß-D-glucopyranoside (open triangles), CHAPS (open circles), or taurodeoxycholate (solid squares). (G) and (H), the enzymatic activity at various concentrations of core 2 (solid circles), GlcNAcß1->2Man (open circles), GlcNAcß1->3Galß1->4Glc (solid triangles), core 3 (open triangles), or GlcNAc2·Man3·GlcNAc2 (solid squares). The concentration of PAPS was 12 µM.

 
In the case of both SulT-a and SulT-b, the activity increased in the presence of 0.1–0.5% CHAPS or 0.1–1% digitonin, whereas, conversely, n-octyl-ß-D-glucopyranoside and taurodeoxycholate inhibited both types of SulT (Figure 3E,F). Especially, even 0.1% taurodeoxycholate completely inhibited the activity of these enzymes.

Substrate specificities of SulT-a and SulT-b
Next, we examined the acceptor substrate specificities of SulT-a and SulT-b. As shown in Figure 3, G and H, SulT-a recognized core 2 as a good substrate and, to a lesser extent, GlcNAcß1->2Man. In contrast, SulT-b recognized core 2, core 3, GlcNAcß1->2Man, and GlcNAcß1->3Galß1->4Glc as good substrates. Interestingly, in the case of both types of SulT, the activity decreased at higher concentrations of these acceptor substrates. Table II shows the relative levels of activity of the SulTs derived from non-mucinous adenocarcinomas (cases 3, 10, 13), mucinous adenocarcinomas (cases 29, 34), adenocarcinomas with a mucinous component (case 33), and their adjacent normal mucosa. The relative level of SulT activity in the adjacent mucosa, that of SulT-a, was similar in various specimens, regardless of the histological character of the colon cancer; these GlcNAc:->6SulTs preferentially act on core 2 oligosaccharide and, to a lesser extent, GlcNAcß1->2Man. Core 3 oligosaccharide was not recognized at all by the GlcNAc:->6SulT in normal mucosa. The relative level of SulT activity in non-mucinous adenocarcinomas was similar to that in the adjacent mucosa. In contrast, the GlcNAc:->6SulT derived from mucinous adenocarcinomas or adenocarcinomas with a mucinous component, SulT-b, showed different behavior. SulT-b efficiently acted on GlcNAcß1-> 3Galß1->4Glc and core 3, as well as core 2 and GlcNAcß1->2Man. We estimated the Michaelis constants (Km) of SulT-a and SulT-b for these substrates based on data obtained at low concentrations (Table III). The Km values of SulT-b for GlcNAcß1->2Man, core 3, GlcNAcß1-> 3Galß1->4Glc, or core 2 were 1.5, 0.27, 0.42, and 0.28 mM, respectively, and those of SulT-a for GlcNAcß1->2Man or core 2 were 7.7 and 0.30 mM, respectively. The Km values of SulT-a and –b for PAPS using core 2 as the acceptor substrate were the same (4 µM).



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Table II. Substrate specificities of GlcNAc:->6SulTs derived from human normal colonic mucosa, non-mucinous adenocarcinomas, and mucinous adenocarcinomas

aThe concentrations were 1 mM.

bAdjacent normal mucosa.

cCarcinoma.

 


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Table III. Michaelis constants of GlcNAc:->6SulT-a and -b derived from human colonic mucosa (case 3) and mucinous carcinoma (case 29)

aNot determined.

bThe acceptor substrate was Galß1->3(GlcNAcß1->6)GalNAc{alpha}-pNP(core 2, 1 mM).

 
To assess whether the SulT-b activity observed with GlcNAcß1->2Man, core 3, GlcNAcß1->3Galß1->4Glc, and core 2 is a single enzyme, substrate competition experiments were performed. When two acceptor substrates compete for one enzyme, the total velocity (v) is estimated as: v = (v1c1/K1+v2c2/K2)/(1+c1/K1+c2/K2), where c1 and c2 are the concentrations of the acceptor substrates, v1 and v2 are the respective maximum velocities, and K1 and K2 are the respective Michaelis constants. Alternatively, when two acceptor substrates are utilized by two different enzymes, the total velocity is the simple sum of reaction velocities of the individual enzymes (Dixon and Webb, 1979Go). As summarized in Table IV, the observed velocities for every combination of the four acceptor substrates were similar to the theoretical values for a single enzyme.


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Table IV. Substrate competition experiments with GlcNAc:->6SulT activity derived from mucinous carcinoma
 
Furthermore, there is a possibility that the SulT-a and SulT-b activities are due to a common enzyme protein and that in crude membrane fractions, a certain modulator exists which can change the substrate specificity of SulT, such as {alpha}-lactalbumin against ß1->4GalT-I (Brodbeck et al., 1967Go; Brew et al., 1968Go). To exclude this possibility, we mixed the crude membrane fraction from the adjacent mucosa in case 3 (SulT-a) with that from the adenocarcinoma in case 29 (SulT-b), and assayed the enzymatic activities with core 2, core 3, GlcNAcß1->2Man, and GlcNAcß1->3Galß1->4Glc. As the result, the levels of SulT activity observed were simply the sum of the values obtained when singly assayed (data not shown). Accordingly, it is suggested that no modulator is present in the crude membrane fractions. These results suggest that the SulT-b activity is due to a single enzyme capable of acting on all four of these substrates and its broad substrate specificity is clearly different from the substrate specificity of SulT-a.

Difference in GlcNAc:->6SulT activities between adenocarcinomas and the adjacent mucosa
As described above, the difference in substrate specificity between SulT-a and SulT-b is especially clear in the behavior with core 3 as the acceptor substrate; SulT-b recognizes core 3 well, whereas SulT-a does not recognize it at all. In view of these characteristics, we assayed the GlcNAc:->6SulT activity in all of the membrane fractions listed in Table I using GlcNAcß1->2Man and core 3 as acceptor substrates. As summarized in Table V and Figure 4, (1) in the case of the non-mucinous adenocarcinomas, the levels of GlcNAc:->6SulT activity with GlcNAcß1->2Man as the acceptor substrate were significantly lower in adenocarcinomas than in the adjacent mucosa (1.5 ± 0.27 pmol/min/mg protein in adenocarcinomas on average and 4.0 ± 0.52 pmol/min/mg protein in the adjacent mucosa, p < 0.0002) (Figure 4A). Except for a few specimens, GlcNAc:->6SulT activity was not detected with core 3 as the acceptor substrate (Figure 4C, Table V). (2) In the case of mucinous adenocarcinomas and adenocarcinomas with a mucinous component (Table I and Figure 1), conversely, the levels of activity with GlcNAcß1->2Man as the acceptor substrate were significantly higher in the adenocarcinomas than in the adjacent mucosa (6.1 ± 1.7 pmol/min/mg protein in adenocarcinomas and 2.1 ± 0.56 pmol/min/mg protein in the adjacent mucosa, p < 0.05) (Figure 4B). The occurrence of SulT-b in the adenocarcinomas might contribute to the rather high levels of GlcNAc:->6SulT activity with GlcNAcß1-> 2Man in the adenocarcinomas, because SulT-b can efficiently act on GlcNAcß1->2Man as well as core 3 (see Table II). SulT activity was not detected in any specimens of adjacent mucosa with core 3 as substrate, but this activity was detected in almost all of these adenocarcinomas (4.6 ± 1.3 pmol/min/mg protein in adenocarcinomas, p < 0.002) (Figure 4D). These results indicate that SulT-b occurs at a high frequency in mucinous adenocarcinomas and adenocarcinomas with a mucinous component, regardless of the proportion of the specimen occupied by the mucinous area as determined microscopically, and that SulT-b is scarcely detected in non-mucinous adenocarcinomas or the adjacent mucosa (Figure 4C).


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Table I. Major clinicopathological features of the 37 patients with colonic non-mucinous adenocarcinoma, mucinous adenocarcinoma, or adenocarcinoma with a mucinous component
 

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Table V. Summary of GlcNAc:->6SulT activities detected in adjacent normal mucosa (A) and colonic non-mucinous adenocarcinoma tissues, mucinous adenocarcinoma, or adenocarcinoma tissues with a mucinous component (C) using GlcNAcß1->2Man(GN-M) or GlcNAcß1->3GalNAc{alpha}-pNP (core 3) as acceptors
 


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Fig. 4. Comparison of the levels of GlcNAc:->6SulT activity in the adjacent normal mucosa (A, under panels) with those in adenocarcinomas (C, under panels). (A) and (C), the levels of GlcNAc:->6SulT activity in non-mucinous adenocarcinomas and the adjacent mucosa as determined using GlcNAcß1->2Man (A) and core 3 (C) as acceptor. (B) and (D), the levels of GlcNAc:->6SulT activity in mucinous adenocarcinomas or adenocarcinomas with a mucinous component, and the adjacent mucosa as determined using GlcNAcß1->2Man (B) or core 3 (D) as acceptors. Columns, mean; bars, bracketed range is SE.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We clearly demonstrated in this paper that (1) at least two types of GlcNAc:->6SulT activity exist in human colonic adenocarcinomas and the adjacent mucosa, (2) in the case of non-mucinous adenocarcinomas, the levels of GlcNAc:-> 6SulT activity (SulT-a) in the adjacent mucosa are significantly higher than those in adenocarcinomas, (3) on the other hand, in the case of mucinous carcinomas and adenocarcinomas with a mucinous component, another type of GlcNAc:->6SulT activity (SulT-b) prominently occurs, (4) SulT-b has a broader substrate specificity than SulT-a. To the best of our knowledge, this is the first report demonstrating that there is a difference in the specific activity of GlcNAc:->6SulT-a between the activity in non-mucinous colonic adenocarcinomas and that in the adjacent mucosa, and that the expression of SulT-b is correlated with the presence of a mucinous area in colonic carcinomas.

Colonic mucinous carcinomas are defined by WHO as those in which at least 50% of the area evaluated microscopically is covered with mucus (Morson and Sobin, 1990Go). We should point out that in this study SulT-b activity was detected not only in the mucinous adenocarcinomas (cases 30, 35, 36) but also in the adenocarcinomas with a mucinous component. These phenomena suggest that it is necessary to carefully examine the differences in biochemical characteristics when comparing mucinous adenocarcinomas and those with a mucinous component.

Moreover, the levels of SulT activity in the mucosa adjacent to non-mucinous adenocarcinomas (4.0 ± 0.52 pmol/min/mg protein on average) were found to be significantly (p < 0.03) higher than those in the mucosa adjacent to mucinous adenocarcinomas (2.1 ± 0.56 pmol/min/mg protein on average), using GlcNAcß1->2Man as the acceptor substrate (Figure 4). It seems that two possibilities may explain the result. (1) The mucosa adjacent to non-mucinous carcinomas may differ biochemically from that adjacent to mucinous carcinomas, although there appears to be no difference in the pathological features of the mucosa adjacent to these two types of adenocarcinomas. (2) Among the specimens examined, about half of the mucinous adenocarcinomas and about half of those with a mucinous component were present in the ascending colon. The levels of SulT-a activity in the ascending colon might be lower than those in the sigmoid colon and rectum. Further experiments are needed to clarify this matter. We are now searching for other biochemical and histochemical differences between the non-mucinous adenocarcinomas and those with a mucinous component.

This study suggests that the levels of SulT-a and SulT-b activity are correlated with the content of GlcNAc-6-O-sulfate residues in NFA-2 and CEA(Yamashita et al., 1987Go; Fukushima et al., 1995Go). The N-glycans containing GlcNAc-6-O-sulfate residues in CEA molecules may modify the biological properties of the CEA molecules including the cell-to-cell or cell-to-virus/bacteria recognition properties.

We showed that SulT-b has a broader substrate specificity than SulT-a; core 3 and GlcNAcß1->3Galß1->4Glc are poor acceptor substrates for SulT-a, but good ones for SulT-b. It has been unclear whether or not the core 3 structure exists substantially in O-linked glycans of mucins produced by mucinous adenocarcinomas. Yang et al. (1994)Go showed that the levels of enzymatic activity of UDP-GlcNAc:GalNAc-R ß3N-acetylglucosaminyltransferase which synthesizes O-glycan core 3, decrease in cases of colon cancer. Accordingly, it seems necessary to determine the structure of the O-linked, sulfated glycans of mucin-type glycoproteins derived from mucinous adenocarcinoma tissue in the near future.

It has been reported that the abundance of sulfomucins decreases in the course of colonic carcinogenesis (Felipe, 1969Go; Yamori et al., 1987Go). Kuhns et al. (1995)Go showed that the level of Gal:->3SulT activity is lower in the case of colon cancer than in normal colonic tissue, indicating that the level of Gal:->3SulT activity reflects the abundance of sulfomucins. Recently, it has been determined that 3'-sulfo-Lewisa is an antigenic determinant on sulfomucins, and expression of this determinant is also decreased in adenocarcinomas (Matsushita et al., 1995Go; Loveless et al., 1998Go). Although a quantitative difference in the content of 6-O-sulfated GlcNAc in O-linked glycans between normal mucosa and adenocarcinomas has not yet been reported, downregulation of SulT-a might also contribute to the decrease in abundance of sulfomucins in the course of colonic carcinogenesis.

GlcNAc:->6SulTs activities have been identified and characterized in rat stomach (Carter et al., 1988Go), rat corpus (Goso and Hotta, 1993Go), rat liver (Spiro et al., 1996Go), human respiratory mucosa (Degroote et al., 1997Go), and porcine lymph nodes (Bowman et al., 1998Go). Recently, cDNAs for three GlcNAc:->6SulTs have been cloned (Uchimura et al., 1998aGo,b; Bistrup et al., 1999Go; Hiraoka et al., 1999Go; Lee et al., 1999Go). The SulT derived from rat liver seems to be different from our colonic SulTs; for example, in terms of the dependencies on divalent cations and pH. The rat liver SulT activity is completely inhibited by EDTA, and the optimal pH region is narrower than that of the SulT-a and SulT-b characterized in this study (Spiro et al., 1996Go). In terms of substrate specificity, the rat liver SulT can act on GlcNAcß1->2Man and GlcNAcß1->6Man{alpha}1-O-Me, but not on GlcNAcß1-> 3Galß1->4Glc at all (Spiro et al., 1996Go), whereas the human respiratory SulT can act on sialylated core 2 oligosaccharide, but not GlcNAcß1->3GalNAcol (Degroote et al., 1997Go). A high content of 6-O-sulfated GlcNAc residues in core 2 oligosaccharides is observed in respiratory mucins derived from cystic fibrosis patients (Lo-Guidice et al., 1994Go), indicating that the SulT in the respiratory mucosa may recognize core 2 oligosaccharide as a good acceptor. Uchimura et al. (1998b)Go isolated cDNA for a GlcNAc:->6SulT and showed that there is no difference in the amount of the corresponding mRNA expressed in human normal colonic mucosa as compared to adenocarcinomas. Hiraoka et al. (1999)Go showed that another GN:SulT, LSST, preferentially acts on core 2 O-glycans, catalyzing their sulfation in vivo, although LSST is unable to act on these oligosaccharides in vitro. We found that the substrate specificity of the GlcNAc:->6SulT reported by Uchimura et al. is similar to that of SulT-a (K.Uchimura, A.Seko, K.Yamashita, and T.Muramatsu, unpublished observations).

More recently, Lee et al. (1999)Go isolated cDNA for a GlcNAc:->6SulT, expression of which is highly restricted to small intestine and colon. Considering that SulT-b activity can not be detected in normal colon and small intestine (data not shown), SulT-b is an enzyme different from the three GlcNAc:->6SulTs so far cloned, and probably different from the rat liver SulT. Accordingly, since several GlcNAc:-> 6SulTs showing different enzymatic properties exist in various tissues, it may be necessary to investigate the enzymological properties of these SulTs in detail.

Colonic SulTs have a unique dependency on detergents (Figure 3E,F); digitonin or a low concentration of CHAPS increases the enzymatic activity, whereas n-octyl-ß-D-glucopyranoside and taurodeoxycholate inhibit these SulTs. These results suggest that the condition of solubilization strongly affects the efficiency of catalysis of each of the two SulTs. Since several groups have reported that phospholipids can activate the enzymatic activity of glycosyltransferases, including oligosaccharyltransferase (Chalifour and Spiro, 1988Go), ß1->4galactosyltransferase (Mitranic et al., 1983Go), {alpha}2-> 3sialyltransferase (Westcott et al., 1985Go), and glucuronyltransferase (Zakim et al., 1988Go; Terayama et al., 1998Go), it seems that colonic GlcNAc:->6SulTs may also require a certain lipid cofactor.

It has been proposed that 6-sulfo-3'-sialyl Lewis x is a ligand for L-selectin on the endothelial cells of high endothelial venules (Hemmerich et al., 1995Go; Sanders et al., 1996Go; Mitsuoka et al., 1998Go). The initial step in biosynthesis of this glycan is sulfation of the C-6 position of ß-GlcNAc by GlcNAc:->6SulT, and the following galactosylation seems to be catalyzed by GlcNAc-6-O-sulfate:ß1->4GalT which we recently found (Seko et al., 1998Go). In cases of colon cancer, 6-sulfo-3'-sialyl Lewis x is expressed on the mucous epithelial cells surrounding differentiated adenocarcinoma tissues (Kannagi et al., 1999Go). Although the biological role of this glycan moiety in colon cancer still remains unclear, this glycan moiety may be involved in tumor metastasis or growth of cancer cells. Identification of the SulT-a and SulT-b genes may contribute to clarification of the biological roles of sulfated glycans in colon cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Preparation of normal human colonic mucosa and adenocarcinoma tissues
Fresh samples of normal colonic mucosa and carcinomas from 37 patients were stored frozen at –80°C before use. Table I shows the Dukes’s staging system, the histological character of the adenocarcinomas, and the serum CEA levels of the 37 patients before surgery. Two of the 37 patients were assessed as "A," 13 patients were "B," 11 patients were "C," and the other 11 patients were "C(D)," according to Dukes’s staging system. Twenty-two cases (cases 1–22) were classified as non-mucinous adenocarcinoma, 3 (cases 30, 35, 36) were classified as mucinous adenocarcinoma, and 12 (cases 23–29, 31–34, 37) were classified as adenocarcinoma with a mucinous component (Figure 1). Twelve patients had a focus of distant metastasis in the liver (cases 17–22, 32–37). Serum CEA levels were within the normal range (less than 2.5 ng/ml) in these 12 patients, whereas elevation of serum CEA levels was seen in other 19 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.

Chemicals and enzymes
GlcNAcß1->2Man, GlcNAcß1->3GalNAc{alpha}1-p-nitrophenyl (pNP) (core 3), and Galß1->3(GlcNAcß1->6)GalNAc{alpha}1-pNP (core 2) were obtained from Funakoshi Co., Ltd. (Tokyo, Japan). GlcNAcß1->3Galß1->4Glc was prepared from lacto-N-tetraose (Kobata, 1972Go) by digestion with Streptococcus 6646K ß-galactosidase (Seko et al., 1996Go). GlcNAcß1->2Man{alpha}1->3(GlcNAcß1->2Man{alpha}1->6)Manß1-> 4GlcNAcß1->4GlcNAc was prepared from egg yolk SGP (Seko et al., 1997Go) by mild acid hydrolysis and ß-galactosidase digestion. 3'-Phosphoadenosine-5'-phospho[35S]sulfate ([35S]PAPS, 91.8 GBq/mmol) was purchased from DuPont/NEN (Boston, MA). 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium taurodeoxycholate were purchased from Sigma (St. Louis, MO). Triton X-100, N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), and N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) were purchased from Nacalai Tesque (Kyoto, Japan). n-Octyl-ß-D-glucopyranoside was obtained from Dojindo Laboratories (Kumamoto, Japan). Digitonin was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Psathyrella velutina lectin (PVL) was prepared according to the method described previously (Kochibe and Matta, 1989Go) and the lectin was conjugated to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s recommendations. Bio-Rad Protein Assay dye reagent was obtained from Bio-Rad Laboratories (Richmond, CA).

Preparation of microsomes from human colonic tissues
The microsome preparation procedures were described previously (Seko et al., 1996Go). Briefly, tissues (0.1–0.3 g) were immersed in 9 volumes of 10 mM HEPES-NaOH buffer (pH 7.2), 0.25 M sucrose at 4°C, homogenized with a Potter-Elvehjem type homogenizer, and then centrifuged. The extraction was further performed twice and the three supernatant fractions were mixed and ultracentrifuged at 100,000 x g for 1 hr. The precipitated microsomes were suspended in 20 mM HEPES-NaOH buffer (pH 7.2) and kept at –80°C until use.

Assay of GlcNAc:->6SulT
A 20 µl reaction mixture consisting of 0.1 M HEPES-NaOH (pH 7.4), 0.1% (w/v) digitonin, 10 mM MnCl2, 0.1 M NaF, 2 mM ATP-Na2, 1 mM acceptor substrate, 6.5 µM [35S]PAPS (2.8 x 105 dpm), and microsomes appropriately diluted, was incubated at 37°C for 1.5 h. The [35S]-labeled products were purified by paper electrophoresis. The Rf values of [35S]-labeled GlcNAcß1->2Man, [35S]-labeled core 2, and PAPS are 0.90, 0.59, and 1.89, respectively, when the Rf value of bromophenol blue is taken as 1.0. After extraction with water, the [35S]-labeled products using core 2 as substrate were first digested with jack bean ß-N-acetylhexosaminidase (1 unit; 37°C for 16 h, 100 µl of 0.1 M citrate-phosphate buffer pH 6.0) and then applied to PVL-Sepharose column (2 mg/ml; 0.35 x 2.5 cm). Elution was started with 6 ml of 10 mM Tris-HCl (pH 8), 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, followed by the same buffer containing 0.3 M GlcNAc.

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

Hydrazinolysis-nitrous acid deamination of [35S]sulfated products
This was performed as reported by Edge and Spiro (1984)Go. [35S]Sulfated oligosaccharides were de-N-acetylated by hydrazinolysis at 100°C for 24 h, and then deaminated by treatment with 0.2 M NaNO2 in 0.5 N acetic acid at room temperature for 2 h. After the deaminated fragments were reduced with NaBH4, the sulfated fragments were analyzed by thin layer chromatography (TLC) in the solvent, ethyl acetate/pyridine/acetic acid/water = 5:5:1:3 or 1-butanol/pyridine/water = 6:4:3. Authentic 2,5-anhydro-6-sulfo-[3H]mannitol was prepared as described previously (Fukushima et al., 1995Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported in part by grants-in aid from the Uehara Memorial Foundation, and by Grants-in-aid 10780373 and 10178104 (for Scientific Research on Priority Areas) from the Ministry of Education, Science, Sports, and Culture of Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CEA, carcinoembryonic antigen; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; core 2, Galß1->3(GlcNAcß1->6)GalNAc{alpha}1-p-nitrophenyl; core 3, GlcNAcß1->3GalNAc{alpha}1-p-nitrophenyl; Gal, galactose; GalNAc, N-acetylgalactosamine; GalT, galactosyltransferase; Glc, glucose; GlcNAc, N-acetylglucosamine; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Man, mannose; NFA-2, normal fecal antigen-2; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; pNP, p-nitrophenyl; PVL, Psathyrella velutina lectin; SulT, sulfotransferase; TAPS, N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid; TLC, thin layer chromatography.


    Footnotes
 
1 To whom requests for reprints should be addressed Back


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 Abstract
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 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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