ARTICLE |
Correspondence to: Jun Nakayama, Central Clinical Laboratories, Shinshu University Hospital, Asahi 3-1-1, Matsumoto 390-8621, Japan. E-mail: jun@hsp.md.shinshu-u.ac.jp
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Summary |
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1,4-N-acetylglucosaminyltransferase (
4GnT) is a glycosyltransferase that mediates transfer of GlcNAc to ßGal residues with
1,4-linkage, forming GlcNAc
1
4Galß
R structures. In normal human tissues, glycoproteins having GlcNAc
1
4Galß
R structures at non-reducing terminals are exclusively limited to the mucins secreted from glandular mucous cells of gastric mucosa, Brunner's gland of duodenum, and accessory gland of pancreaticobiliary tract. Recently, we have isolated a cDNA encoding human
4GnT by expression cloning. Although
4GnT plays a key role in producing this unique glycan in vitro, the actual localization of
4GnT was not determined. In this study we examined the localization of
4GnT in various human tissues, including gastrointestinal mucosa, using a newly developed antibody against human
4GnT. The specificity of the antibody was confirmed by analyses of human gastric adenocarcinoma AGS cells transfected by
4GnT cDNA. Expression of
4GnT was largely associated with the Golgi region of mucous cells that produce the mucous glycoproteins having GlcNAc
1
4Galß
R, such as the glandular mucous cells of stomach and Brunner's gland. An immunoprecipitation experiment disclosed that two distinct mucin proteins, MUC5AC and MUC6 present in gastric mucin, carried the GlcNAc
1
4Galß
R structures. These results indicate that
4GnT is critical to form the mucous glycoproteins having GlcNAc
1
4Galß
R on MUC6 and MUC5AC in vivo.
(J Histochem Cytochem 49:587596, 2001)
Key Words: glycosyltransferase, mucin core protein, O-glycan
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Introduction |
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GASTROINTESTINAL MUCINS of heavily glycosylated mucous glycoproteins play important roles in protecting gastrointestinal mucosa from a variety of physical, chemical, and microbial damage (
Recently, it was demonstrated that, in the gastrointestinal tract, distribution of the mucins identified by HIK1083 antibody specific for GlcNAc1
4Galß
R and that of Class III mucin were identical (
1
4Galß
R and (b) that PCS identified this particular carbohydrate moiety. To test these hypotheses, we have isolated a cDNA encoding
1,4-N-acetylglucosaminyltransferase (
4GnT) from a human stomach cDNA library by expression cloning and have established that Class III mucin identified by PCS characteristically contained terminal GlcNAc
1
4Galß
R structures (
1,4-linkage, forming GlcNAc
1
4Galß
R (
Extensive studies have been carried out to determine the tissue localization of glycosyltransferases such as ß1,4-galactosyltransferase-I and 1,3-fucosyltransferase-VI using immunohistochemistry (
4GnT, it was demonstrated by Northern blotting analysis that
4GnT was transcribed only in stomach and pancreas (
1
4Galß
R in gastric mucin were not clarified.
In the present study we have developed monospecific polyclonal antibody I17K against human 4GnT by immunizing rabbits with a synthetic peptide corresponding to the COOH-terminal end of this enzyme. The specificity of I17K antibody was confirmed by analyzing human gastric adenocarcinoma AGS cells transfected by
4GnT cDNA. Using I17K antibody, we have examined the immunohistochemical localization of
4GnT in various human tissues, including gastrointestinal tract. Double immunostaining of gastroduodenal mucosa with I17K antibody together with HIK1083 antibody was also carried out. Finally, we found that MUC5AC and MUC6 immunoprecipitated from gastric mucin carried GlcNAc
1
4Galß
R structures detected by Western blotting analysis.
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Materials and Methods |
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Production of Polyclonal Antibody Against Human 4GnT
The monospecific polyclonal antibody I17K, directed to the COOH-terminal end of human 4GnT, was generated against a synthetic peptide, IKGPEGSVTGELGPGNK, corresponding to the amino acid residues of 324340 (
Establishment of Human Gastric Adenocarcinoma AGS Cells Stably Expressing GlcNAc1
4Galß
R
To establish a cell line stably expressing 4GnT, human gastric carcinoma AGS cells were co-transfected with a mammalian expression vector encoding human
4GnT (pcDNAI-
4GnT) and pSV2neo encoding G418 resistance gene (10:1 by molar ratio) using LipofectAmine (Life Technologies; Grand Island, NY) as described previously (
4GnT. After the selection with G418, AGS cells stably expressing GlcNAc
1
4Galß
R were established by immunofluorescent staining with HIK1083 antibody specific for GlcNAc
1
4Galß
R (
Laser Confocal Microscopy
The AGS-4GnT cells as well as mock-transfected AGS cells were grown on coverslips in Dulbecco's modified Eagle's medium to 60% confluence. Then they were fixed with 20% formaldehyde in PBS for 20 min at room temperature. After washing with PBS, these cells were permeabilized with 0.2% saponin as described previously (
Tissue specimens of normal gastric fundic mucosa, duodenal mucosa, and pancreas and gallbladder showing gastric metaplasia were selected from the pathology files of the Central Clinical Laboratories (Shinshu University Hospital; Matsumoto, Japan). These tissue specimens were doubly immunostained with HIK1083 antibody and anti-MUC5AC antibody, CLH2 (Novocastra; Newcastle, UK), or HIK1083 antibody and anti-MUC6 antibody, CLH5 (Novocastra) after microwave irradiation for 25 min in a 1.0 mM EDTANaOH solution, pH 8.0 as described before (-chain specific) (Southern Biotechnology Associates) for anti-MUC5AC and anti-MUC6 antibodies were used. The localization of these molecules in the gastric mucosa was analyzed using the LSM510 laser confocal microscope.
Western Blotting Analysis
To test whether the I17K antibody specifically detected human 4GnT, cell lysates prepared from 5 x 105 of AGS-
4GnT cells or mock-transfected AGS cells in the presence of a protease inhibitor cocktail Complete (Roche Molecular Biochemicals; Mannheim, Germany) were separated by SDS-PAGE (10% gel) in the presence of ß-mercaptoethanol and then transferred onto nitrocellulose membranes. The blots were incubated with I17K antibody, and then with anti-rabbit immunoglobulins conjugated with HRP using an ECL Western blotting analysis system (Amersham Pharmacia Biotech; Piscataway, NJ). The peroxidase reaction was visualized using the same kit.
Immunohistochemistry
Various normal and metaplastic human tissues were selected from the pathology files of the Central Clinical Laboratories (Shinshu University Hospital). These tissue specimens sliced into 3-µm thickness were immunostained with the I17K antibody using a Super Sensitive detection kit (BioGenex Laboratories; San Romon, CA) based on the biotinstreptavidinperoxidase method (
For double immunostaining with I17K and HIK1083 antibodies, tissue sections of normal stomach and duodenum were incubated with the I17K antibody, followed by application of alkaline phosphatase-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals). Visualization was performed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (Roche Molecular Biochemicals). After washing with water, sections were incubated with HIK1083 and then reacted with biotinylated secondary antibody, followed by the streptavidinHRP conjugate using a HistoStain SP kit (Zymed Laboratories; South San Francisco, CA). The peroxidase reaction was visualized with a 3-amino-9-ethyl carbazole/H2O2 solution by using the same kit. After washing with water, sections were mounted with Glycergel (DAKO).
Immunoelectron Microscopy
Ultrastructural immunohistochemistry of 4GnT in the mucous neck cells of gastric mucosa was performed by the pre-embedding method as described previously, with slight modifications (
Immunoprecipitation
Biopsy specimens of the normal fundic mucosa were obtained from a volunteer with a gastroscope after informed consent was obtained. These samples (200 mg) were washed with PBS and homogenized in 5 ml of lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate with protease inhibitors, using an Immunoprecipitation Kit (Roche Molecular Biochemicals). Two ml of the lysates were pre-cleared by incubation with 50 µl of a 50% slurry of protein GAgarose beads for 3 hr at 4C. The extracts were then incubated with anti-MUC5AC antibody (CLH2) or anti-MUC6 antibody (CLH5) for 1 hr at 4C. Immunocomplexes were precipitated with 50 µl protein Gagarose overnight at 4C. After washing with high-salt wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% NP-40, and 0.05% sodium deoxycholate) and low-salt wash buffer (50 mM Tris-HCl, pH 7.5, 0.1% NP-40, and 0.05% sodium deoxycholate), immunoprecipitates were dissociated from protein GAgarose beads with electrophoresis sample buffer and then subjected to a 515% gradient polyacrylamide gel, followed by transfer to nitrocellulose membrane. The blot was then incubated with HIK1083 antibody for 1 hr at room temperature, followed by visualization with HRP-conjugated goat anti-mouse immunoglobulins using the ECL Western blotting analysis system (Amersham Pharmacia Biotech).
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Results |
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I17K Antibody Specifically Recognizes Human 4GnT Expressed in Transfected AGS Cells
To evaluate the specificity of the I17K antibody, we established a clonal cell line of human gastric adenocarcinoma AGS-4GnT cells stably expressing GlcNAc
1
4Galß
R by transfecting with
4GnT cDNA, then doubly immunostained with I17K and HIK1083 antibodies. In parallel, the mock-transfected AGS cells were used as a control. The I17K antibody specifically reacted with dot-like structures dispersed in the cytoplasm of AGS-
4GnT cells (Fig 1A) that expressed GlcNAc
1
4Galß
R at their cell surface (Fig 1B), whereas no immunoreactivity was found in the mock-transfected AGS cells that did not express GlcNAc
1
4Galß
R (Fig 1D1F). It is well known that most of glycosyltransferases reside in the Golgi (
4GnT cells with I17K antibody and antisera against Vti1a that is enriched in the Golgi membrane (
4GnT is largely co-localized with Vit1a (Fig 1G1I), indicating that
4GnT resides in the Golgi. AGS-
4GnT cells may be unique in that the Golgi apparatus is not confined to the perinuclear region.
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In parallel, we analyzed cell lysates made from AGS-4GnT cells and mock-transfected AGS cells using Western blotting with the I17K antibody. As shown in Fig 2, immunoreactive bands were detected at 40, 42, and 45 kD in AGS-
4GnT cells (Fig 2, Lane 1), but not in the mock-transfected AGS cells (Fig 2, Lane 2). These results, taken together, establish that the I17K antibody specifically recognizes human
4GnT expressed in AGS-
4GnT cells.
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Distribution of 4GnT in Normal and Metaplastic Human Tissues
We then examined the tissue distribution of 4GnT using I17K antibody specific for this enzyme (Table 1). Among the normal human tissues examined, expression of
4GnT was exclusively limited to the mucous neck cells, cardiac gland, and pyloric gland of gastric mucosa (Fig 3A and Fig 3C3E), Brunner's gland of duodenum (Fig 3F), and accessory gland of pancreaticobiliary tract, exhibiting a characteristic perinuclear Golgi staining pattern. On the other hand,
4GnT was also detected in gastric metaplasia found in pancreatic duct (Fig 3G) and gallbladder (Fig 3H). However, intestinal metaplasia found in the gastric mucosa did not express
4GnT. Other cells, such as the surface epithelial cells of gastric mucosa (Fig 3B), salivary gland (Fig 3I), submucosal gland of esophagus (Fig 3J), small intestine, and large intestine (Fig 3K) did not express
4GnT. No
4GnT immunoreactivity was detected in non-gastrointestinal tissues, including adrenal gland, Fallopian tube, heart, kidney, lung, lymph node, ovary, placenta, spinal cord, thyroid, and uterus (cervix and body).
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We then determined the subcellular distribution of 4GnT in the mucous neck cells of gastric mucosa using immunoelectron microscopy with the I17K antibody. As shown in Fig 4,
4GnT was clearly detected in the medial Golgi of the mucous neck cells.
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Human 4GnT Directs the Synthesis of Terminal GlcNAc
1
4Galß
R Structures In Vivo
To determine if 4GnT expressed in the mucous cells actually produces mucous glycoproteins carrying GlcNAc
1
4Galß
R, we doubly immunostained gastroduodenal mucosa with the I17K and HIK1083 antibodies. As shown in Fig 5,
4GnT was detected in the mucous cells such as mucous neck cells, pyloric gland, and Brunner's gland that produced GlcNAc
1
4Galß
R. However, the expression level of
4GnT in these mucous cells was variable, and occasionally mucous cells expressing GlcNAc
1
4Galß
R, but not
4GnT, were found, particularly in the pyloric gland (Fig 5B). In contrast, mucous cells expressing
4GnT but not GlcNAc
1
4Galß
R were not detected. These results indicate that
4GnT plays a major role in synthesis of GlcNAc
1
4Galß
R in the gastroduodenal mucosa.
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Terminal GlcNAc1
4Galß
R Structures in Gastric Mucin Are Attached to Two Distinct Mucin Core Proteins, MUC6 and MUC5AC
To determine the mucin core proteins carrying the terminal GlcNAc1
4Galß
R structures formed by
4GnT, co-expression of MUC5AC and GlcNAc
1
4Galß
R, or MUC6 and GlcNAc
1
4Galß
R was analyzed using laser confocal microscopy in gastroduodenal mucosa and in pancreatic duct and gallbladder showing gastric metaplasia. In the fundic mucosa of stomach, MUC5AC was predominantly expressed in the cytoplasm of surface epithelial cells located in the gastric pit (Fig 6A) as described previously (
1
4Galß
R structures were detected in the mucous neck cells found in the isthmus and glandular neck of fundic mucosa (Fig 6B). We also detected small but definite numbers of mucous cells doubly expressing MUC5AC and GlcNAc
1
4Galß
R in the isthmus (Fig 6C). On the other hand, MUC6 was predominantly expressed in the cytoplasm of mucous neck cells located in the glandular base of fundic mucosa (Fig 6D), as shown previously (
1
4Galß
R (Fig 6E and Fig 6F). Similar results were also found in the pyloric mucosa of stomach and in gastric metaplasia in pancreatic duct and gallbladder, i.e., most of the mucous cells secreting GlcNAc
1
4Galß
R simultaneously produced MUC6 and, in addition, small numbers of mucous cells actually co-expressed GlcNAc
1
4Galß
R and MUC5AC (data not shown). Interestingly, MUC5AC was not expressed in the duodenal mucosa, including Brunner's gland (Fig 6G) and accessory gland of pancreaticobiliary tract, and MUC6 was largely co-expressed with GlcNAc
1
4Galß
R in these mucous cells (Fig 6J6L). These results, taken together, suggest that GlcNAc
1
4Galß
R structures could be attached not only to MUC6 but also to MUC5AC in the gastric mucosa, as well as gastric metaplasia found in pancreatic duct and gallbladder, whereas in the Brunner's gland and accessory gland of pancreaticobiliary tract this glycan could be linked to MUC6.
To confirm that the two distinct mucin core proteins, MUC5AC and MUC6, actually carry GlcNAc1
4Galß
R, gastric mucin isolated from normal fundic mucosa was immunoprecipitated with anti-MUC5AC or anti-MUC6 antibody and then subjected to SDS-PAGE followed by Western blotting analysis using the HIK1083 antibody. As shown in Fig 7, a broad and high molecular band larger than 220 kD was detected in the immunoprecipitated samples with anti-MUC5AC (Fig 7, Lane 1) as well as anti-MUC6 antibody (Fig 7, Lane 2). These results establish that GlcNAc
1
4Galß
R structures present in gastric mucin are linked to MUC5AC and MUC6.
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Discussion |
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In the present study, we have clearly demonstrated that 4GnT, a key enzyme responsible for the biosynthesis of GlcNAc
1
4Galß
R structures, is expressed in the Golgi of the mucous neck cells and pyloric gland of gastric mucosa, Brunner's gland of duodenum, and pancreatic duct and gallbladder showing gastric metaplasia, using a newly produced polyclonal antibody I17K specific for human
4GnT. In addition, we have also shown that GlcNAc
1
4Galß
R structures were attached to the two distinct mucin core proteins, MUC5AC and MUC6, present in gastric mucosa.
The antibody I17K was prepared by immunizing rabbits with a synthetic peptide corresponding to the COOH-terminal end of human 4GnT composed of 17 amino acid residues. By using gastric adenocarcinoma AGS-
4GnT cells stably transfected with
4GnT cDNA, we have demonstrated that the I17K antibody specifically reacts with the Golgi of AGS-
4GnT cells. In addition, this antibody recognized the proteins of 40, 42, and 45 kD isolated from these cells. Because the deduced molecular weight of
4GnT is 39.497 kD (
4GnT are apparently utilized. Recently we have isolated a cDNA encoding the mouse counterpart of
4GnT and we found that no significant similarity was found in the COOH-terminal end of 17 amino acid residues between human and mouse
4GnTs (unpublished results). This result suggests that the I17K antibody should not recognize mouse
4GnT. In fact, we have shown that this antibody did not react with mouse gastric mucosa (Zhang and Nakayama, personal communication). It is also noteworthy that among the glycosyltransferases cloned thus far, extensive homology at the amino acid level (35% similarity) was found with recently cloned human
1,4-galactosyltransferase (
4GT) responsible for globo-series glycosphingolipid, Gb3 (Gal
1
4Galß1
4Glcß1
Cer), which is also known as blood group Pk (
4GT, indicating that the I17K antibody did not crossreact with
4GT. These results, taken together, indicate that the I17K antibody specifically recognizes human
4GnT.
4GnT is responsible for the biosynthesis of GlcNAc
1
4Galß
R structures. In human, mucous glycoproteins having this unique glycan are limited to the mucous cells (cardiac gland, mucous neck cells, pyloric gland) of stomach, Brunner's gland of duodenum, accessory gland of pancreaticobiliary tract, and pancreatic duct and gallbladder showing gastric metaplasia (
4GnT was transcribed in stomach and pancreas (
4GnT in various human tissues, including the gastrointestinal tract. As expected,
4GnT was significantly expressed in the mucous cells, such as mucous neck cells that produce GlcNAc
1
4Galß
R, indicating that
4GnT plays a key role in the biosynthesis of GlcNAc
1
4Galß
R in vivo. Interestingly, double immunolabeling using the I17K and HIK1083 antibodies apparently showed that this enzyme was not always associated with the mucous cells secreting GlcNAc
1
4Galß
R. On the other hand, GlcNAc
1
4Galß
R structures can be always found when
4GnT is expressed. These results suggest that
4GnT is not continuously but is intermittently expressed in these mucous cells. However it is also possible that
4GnT-related enzyme(s) might be present in the gastroduodenal mucosa and play a role in the production of GlcNAc
1
4Galß
R, as exemplified by the fact that polysialic acid is synthesized by two distinct polysialyltransferases, PST and STX (
Using immunoelectron microscopy, the subcellular localization of 4GnT in the mucous neck cells of gastric mucosa was determined to be in the medial Golgi. Sugar chains such as O-glycans are synthesized by sequential actions of glycosyltransferases in the Golgi (
4GnT most efficiently acts on the core 2 branched O-glycan, GlcNAc
1
6(Galß1
3)GalNAc, which is synthesized by C2GnT as its acceptor donor (
Among the mucin core proteins, MUC5AC and MUC6 are abundantly expressed in the gastric mucosa (1
4Galß
R are quite similar in the gastric mucosa, we considered that MUC6 was a candidate for the carrier protein of this glycan (
1
4Galß
R. However, mucous cells co-expressing MUC5AC and GlcNAc
1
4Galß
R were also detectable in the isthmus of the fundic mucosa. These results suggest that not only MUC6 but also MUC5AC can be a carrier protein of GlcNAc
1
4Galß
R. Indeed, we demonstrate that GlcNAc
1
4Galß
R structures present in gastric mucin are actually attached to MUC5AC and MUC6 by immunoprecipitation followed by Western blotting analysis (see, Fig 7).
1
4Galß
R structures are unique in that they are attached to both MUC5AC and MUC6 in gastric mucin. Regarding the mucous cells found in gastric metaplasia of pancreatic duct and gallbladder, we suggested that GlcNAc
1
4Galß
R structures could be linked to MUC5AC or MUC6, like gastric mucous cells. In addition, it was also suggested that GlcNAc
1
4Galß
R structures in Brunner's gland of duodenum and accessory gland of pancreaticobiliary tract could be linked to MUC6 but not MUC5AC (see Fig 6). Future study will be of significance to determine the actual mucin cores carrying this particular glycan in these mucous cells.
Recently, it was shown that mucous glycoproteins having GlcNAc1
4Galß
R was produced in gastric and pancreatic cancer (
4GnT is not expressed in normal lung and uterus, as shown in the present study, it is likely that
4GnT is aberrantly expressed in the lung or uterine cervix, in association with the production of GlcNAc
1
4Galß
R in these tumor cells. Further study will be of significance to determine whether this enzyme is actually expressed in these cancer cells.
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Acknowledgments |
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Supported by Grant-in-Aids for Scientific Research on Priority Area (10178104) from the Ministry of Education, Science, Sports, and Culture of Japan, and for the Ribosome Engineering Project in Organized Research Combination System (to JN), and by National Institutes of Health Grant CA48737 (to MF).
We are grateful to Dr Tsutomu Katsuyama for encouragement and discussion during this study, to Drs Kazuhiko Ishihara and Kyoko Hotta for the generous gift of the HIK1083 antibody, and to Dr Edgar Ong for critical reading of the manuscript.
Received for publication October 27, 2000; accepted December 20, 2000.
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