Glycobiology Advance Access originally published online on May 25, 2006
Glycobiology 2006 16(9):777-785; doi:10.1093/glycob/cwl005
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Apical Golgi localization of N,N'-diacetyllactosediamine synthase, ß4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa
2 Division of Oncological Pathology, Aichi Cancer Center Research Institute; 1-1 Kanokoden, Chiksa-ku, Nagoya 464–8681, Japan; 3 Glycogene Function Team and 4 Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305–8568, Japan
1 To whom correspondence should be addressed; e-mail: h.narimatsu{at}aist.go.jp
Received on October 26, 2005; revised on May 15, 2006; accepted on May 22, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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ß1,4-N-acetylgalactosaminyltransferase III (ß4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcß residue with ß1,4-linkage, forming the N,N'-diacetyllactosediamine, GalNAcß1,4GlcNAc (LacdiNAc or LDN). Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of ß4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of ß4GalNAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the ß4GalNAc-T3 gene into MKN45 cells, and the terminal ßGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). ß4GalNAc-T3 was localized in the supra-nuclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, ß4GalNAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAc
-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither ß4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of ß4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and ß4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa. Key words: gastric mucosa / N,N'-diacetyllactosediamine / ß4GalNAc-T3 expression
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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N,N'-diacetyllactosediamine (GalNAcß1,4GlcNAc/LacdiNAc, LDN) is a unique terminal structure in the outer chain moieties of human N-glycans, whereas N-acetyllactosamine (Galß1,4GlcNAc, LacNAc) is commonly present as a type II blood core carbohydrate structure (Manzella et al., 1996
). LDN has been found on glycoprotein hormones secreted from the pituitary glands, leutropin (LH) and thyrotropin (TSH). LH and TSH have two components, a common subunit and a specific ß subunit for each; the subunit is also shared by a follicular stimulation hormone (FSH) secreted from the pituitary glands and human chorionic gonadotropin (hCG) from the placenta (Pierce and Parsons, 1981; Green and Baenziger, 1988; Manzella et al., 1996). Interestingly, though these four hormones utilize the common subunit, LDN was found on the and ß subunits of LH and TSH but not on either or ß subunit of FSH and hCG (Smith and Baenziger, 1988). LDN has been found on many molecules other than LH and TSH, such as glycodelins in amnion of fetal tissue (Dell et al., 1995; Morris et al., 1996; Seppala et al., 2001) and CD36 in fat globules in bovine milk (Nakata et al., 1993; Sato et al., 1993) and membrane proteins of bovine pituitary glands (Taka et al., 1996).
A wide distribution of LDN has been examined histochemically using lectins (Taka et al., 1996; Sakiyama et al., 1998). A plant lectin, Wisteria floribunda agglutinin (WFA), which binds preferentially to the terminal GalNAc on N-glycans (Mengeling et al., 1991; Sakiyama et al., 1998; Kitamura et al., 2003), was used to explore the existence and localization of LDN. One study on the mammary glands demonstrated that WFA could histochemically detect the terminal GalNAc on N-glycans, distinguishing it from that on O-glycans (Kitamura et al., 2003). Thus, WFA was demonstrated to be an available tool for detecting LDN in the previous histochemical and lectin column studies. In this study, we employed WFA for histochemical detection of LDN epitopes.
The recent progress of molecular cloning on glycosyltransferases has revealed the presence of nine members of human ß4GalNAc-Ts that may be classified into three subgroups. The first group consists of ß4GalNAc-T1 (GM2/GD2 synthase) and ß4GalNAc-T2 [human Sd(a) immune epitope synthase] (GalNAcß4 [NeuAc2,3] Galß1, 4GlcNAc), whose acceptor substrates are sialo–galactose (Yamashiro et al., 1995; Dohi et al., 1996; Montiel et al., 2003). The second group contains two chondroitin sulfate N-acetylgalactosaminyltransferases, I and II (CSGalNAc-T1 and -T2), and three chondroitin sulfate synthases (CSS), CSS1, CSS2 and CSS3 (Kitagawa et al., 2001; Gotoh, Sato et al., 2002; Gotoh, Yada et al., 2002; Uyama et al., 2002; Sato, Gotoh, Kiyohara, Akashima et al., 2003; Uyama et al., 2003; Yada, Gotoh et al., 2003; Yada, Sato et al., 2003). These enzymes have an activity by which GalNAc is transferred to glucuronic acid with ß1,4 linkage. None of the above five enzymes exhibited GalNAc transfer activity toward GlcNAc residues on acceptor substrates. Two members of the third group are LDN synthases, ß4GalNAc-T3 and ß4GalNAc-T4, that were cloned and characterized in our recent study (Sato, Gotoh, Kiyohara, Kameyama et al., 2003; Gotoh et al., 2004). These two enzymes can transfer GalNAc from UDP-GalNAc to GlcNAc residue with the ß1,4 linkage on both N- and O-glycans, resulting in the synthesis of the LDN structure. Though these two enzymes share 42.6% identity in their amino-acid sequence and exhibit a similar substrate specificity toward oligosaccharide acceptor substrates, their tissue distributions are quite different. ß4GalNAc-T3 mRNA was detected in the stomach and colon, whereas ß4GalNAc-T4 transcripts were predominantly detected in the brain, ovary, and mammary glands. Thus, ß4GalNAc-T3 is the most probable candidate for the enzyme that synthesizes LDN in the stomach.
Another study has described the possible presence of LDN in gastric mucosa by histochemical positive staining with WFA (Ota et al., 1991), although it made no mention whether WFA exclusively stained LDN. In the present study, we developed a monoclonal antibody (mAb) K1356 against human ß4GalNAc-T3. Using K1356 mAb, we examined the immunohistochemical localization of ß4GalNAc-T3 in conjunction with the WFA reactivity in human gastric mucosa. WFA did not react with GalNAc-o-Ser/Thr on the gastric surface cells but rather with the terminal GalNAc on gastric surface cells that express ß4GalNAc-T3 on the apical side of the nucleus. The frontal affinity chromatography (FAC) system demonstrated that the carbohydrate with the highest affinity to WFA is LDN among the carbohydrates examined. In the present study, we show that supra-nuclear localization of ß4GalNAc-T3 in gastric mucous cells is responsible for LDN expression detected by WFA.
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Results |
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K1356 mAb specifically binds to N-terminal fragment of ß4GalNAc-T3
The reactivity of K1356 mAb to ß4GalNAcT3 was examined using the recombinant ß4GalNAc-T3 with FLAG tag at N-terminus, which was prepared in our previous studies (Sato, Gotoh, Kiyohara, Kameyama et al., 2003
; Gotoh et al., 2004). The recombinant ß4GalNAc-T3 was truncated to lack its transmembrane domain. In a non-reducing condition, the recombinant ß4GalNAc-T3 migrated as a major band of 130 kDa (data not shown). However, in a reducing condition, Coomassie Brilliant Blue (CBB) staining (Lane 1 in Figure 1) showed four bands at 130, 65, 55, and 50 kDa. The 50 kDa band corresponds to an immunoglobulin (Ig) heavy chain because it appeared only with a secondary antibody. These results indicated that ß4GalNAc-T3 is proteolytically cleaved into N- and C-terminal fragments, and two fragments are covalently coupled. Anti-FLAG antibody specifically detected two bands, 130 and 55 kDa, indicating that the 55-kDa band is the N-terminal fragment (Figure 1). K1356 mAb also detected 130- and 55-kDa bands, indicating that K1356 recognized the N-terminal fragment of ß4GalNAc-T3.
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In parallel, we performed western blotting analysis using K1356 mAb for the cell lysates of ß4GalNAc-T3-transfected MKN45 cells and mock-transfected MKN45 cells. The full-length cDNA of ß4GalNAc-T3 was transfected into MKN 45 cells. A weak band at 140 kDa and a strong band at 70 kDa were detected in ß4GalNAc-T3-transfected MKN45 cells, although no bands were observed in mock-transfected MKN45 cells (Figure 2a). The sizes of the positive bands, 140 kDa and 70 kDa, corresponded to those of the full-length ß4GalNAc-T3 and the N-terminal fragment, respectively. These results indicated that ß4GalNAc-T3 was proteolytically cleaved in both truncated soluble form and Golgi-membrane bound form. Using these cells, we performed flow cytometry (FCM) analysis with WFA–fluorescent isothianate (FITC) which is a lectin-recognizing terminal GalNAc. In Figure 2b, MKN45- ß4GalNAc-T3 cells were more strongly stained with WFA–FITC than mock-transfected MKN45 cells, suggesting that LDN was newly synthesized on the surface of MKN45 cells by ß4GalNAc-T3 expression.
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Distribution of ß4GalNAc-T3 in normal and intestinal metaplastic human gastric mucosa
In our previous study, the transcripts for ß4GalNAc-T3 were detected in the human stomach. K1356 mAb was employed to investigate the localization of ß4GalNAc-T3 in the stomach. Among the human gastric tissues examined, the expression of ß4GalNAc-T3 was exclusively limited to the surface mucous cells of pyloric and fundic glands and to the cells in the glandular compartment of fundic glands (Figure 3a and b). The cells in the glandular compartment of pyloric glands exhibited few ß4GalNAc-T3-positive cells (Figure 3c). Intestinal metaplasia, which resembles normal small intestinal glands, commonly occurs in atrophic gastritis. We previously reported the sub-classifications of the metaplasia using both gastric and intestinal cell-specific differentiation markers (Niwa et al., 2005). The cells in intestinal metaplastic glands did not show any positive staining with K1356 mAb (Figure 3d), whereas they were strongly stained with anti-villin antibody which is one of the common intestinal markers (Figure 3e). These results indicate that ß4GalNAc-T3 expression is regulated by cell differentiation in a specific manner. Interestingly, the surface mucous cells exhibited ß3GalNAc-T3 in the supra-nuclear area, while it was detected in the basolateral nuclear area in the glandular compartment of both fundic and pyloric mucosa.
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Differential localization of ß4GalNAc-T3 between the cells in the surface mucous and in the glandular compartments
To confirm the differential sub-cellular localization between the cells in the glandular and foveolar compartments of fundic glands, we stained the nucleus with propidium iodide (Invitrogen, Carlsbad, CA) and evaluated the ß4GalNAc-T3 localization with K1356 mAb. As seen in Figure 4, ß4GalNAc-T3 was detected in the apical supra-nuclear area of the surface mucous cells in the foveolar cells (Figure 4a), but in the basolateral position of glandular compartment cells (Figure 4b).
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Double immunostaining was performed with K1356 mAb and Alexa 488-labeled secondary antibody, and Alexa 546-labeled anti ß1,4-galactosyltransferase I (ß4Gal-T1) and mAb 8628 (Uejima et al., 1992; Uemura et al., 1992). ß4Gal-T1 is ubiquitously expressed in epithelial cells in many tissues and widely used as a marker for the trans-Golgi apparatus. Upon fluorescence microscopy analysis, both ß4GalNAc-T3 and ß4Gal-T1 were expressed and detected on the apical side of the supra-nuclear region of surface mucous cells (Figure 5a and b, respectively). The merged image of the two enzyme signals showed a dominant yellow merged signal and residual green signals representing ß4GalNAc-T3 (Figure 5c). These results demonstrated that ß4GalNAc-T3 is mainly co-localized with ß4Gal-T1 in the trans-Golgi apparatus. The localization of ß4GalNAc-T3 was further explored on confocal microscopy analysis (Figure 5d, e and f). It was localized more on the apical side of the Golgi apparatus than ß4Gal-T1, indicating that the precise localization of ß4GalNAc-T3 is in the trans-Golgi network. Neither enzyme was found in stored mucin vesicles.
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Supra-nuclear localization of ß4GalNAc-T3 is responsible for LDN expression detected by WFA
To investigate whether LDN is synthesized by ß4GalNAc-T3 in the stomach, immunostaining was performed with K1356 mAb and WFA–FITC. As seen in Figure 6, ß4GalNAcT3 was again detected in the supra-nuclear region of surface mucous cells (white arrows in Figure 6b) and in the basolateral area in the cells below the neck zone (green arrows in Figure 6b). Although the WFA reactivity was seen near the surface mucous cells that express ß4GalNAc-T3, there was no signal of WFA reactivity around the cells below the neck zone (Figure 6c).
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To obtain insight into effects of the enzyme localization on the biosynthesis activity, we performed the following experiments. At first, we confirmed that WFA binds to a terminal GalNAc of LDN but not to Tn antigens (GalNAc-Ser/Thr, CD175) by comparing staining patterns of the two antigens using serial sections. Cytoplasms of the surface mucous cells were diffusely stained by WFA (Figure 7a), while the supra-nuclear region of the same cells was exclusively stained by anti-Tn-antibody (Figure 7b). On confocal microscopy analysis, the differential distributions of the two antigens were clearly observed as demonstrated in merged images, in which no yellow signals were seen (Figure 7c–h).
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Furthermore, we confirmed the histological findings by a sequential analysis with FAC that evaluates lectin–oligosaccharide interactions. FAC has significant advantages for the determination of association constants (Ka) because of its high sensitivity and reproducibility (Hirabayashi et al., 2003; Nakamura et al., 2005). As shown in Figure 8, LDN exhibited the highest Ka value in relation to WFA among the evaluated carbohydrates structures, of which some structures contain a GalNAc residue (Table 1). Weaker bindings were observed to the carbohydrates of glycolipids having a GalNAc residue on the non-reducing termini. These FAC results strongly supported that the supra-nuclear localization of ß4GalNAcT3 in the surface mucous cells determines the LDN expression that is histochemically detected by WFA.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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ß4GalNAc-T3 was identified to be a LDN synthase by in vitro assay in our previous study (Sato, Gotoh, Kiyohara, Kameyama et al., 2003
). We also reported the tissue distribution of the ß4GalNAc-T3 transcripts (Sato, Gotoh, Kiyohara, Kameyama et al., 2003). Among examined tissues, the stomach expressed the largest amount of the transcript followed by colon, testis, and adrenal glands. Small intestine did not express the ß4GalNAc-T3 transcripts.
In the present study, we established a specific mAb, K1356 mAb, specifically recognizing ß4GalNAc-T3, and performed an immunohistochemical analysis to pinpoint the precise localization of ß4GalNAc-T3 in the stomach. We detected the regulated expression of ß4GalNAc-T3, which depends on the cellular differentiation in gastric mucosa. In fundic glands, the surface mucous cells expressed a large amount of ß4GalNAc-T3, which was localized at the apical side of the supra-nuclear area, whereas the cells of the glandular compartment, such as parietal, chief, and accessory cells, partially expressed the ß4GalNAc-T3 in the basolateral area of the nucleus. In pyloric glands, the surface mucous cells also expressed abundant ß4GalNAc-T3, however, a few glandular compartment cells expressed only a small amount in the basolateral area of the nucleus (data not shown). Interestingly, ß4GalNAc-T3 disappeared in the intestinal metaplastic cells. This is consistent with our previous result which reported no ß4GalNAc-T3 transcripts in the small intestine (Sato, Gotoh, Kiyohara, Kameyama et al., 2003). Thus, ß4GalNAc-T3 expression is regulated in organ-specific and differentiation-specific manners in gastric mucosa. These results suggest that ß4GalNAc-T3 is a candidate for differentiation marker of fundic glands and surface mucous cells in the gastric mucosa.
One limitation of this study is the use of WFA for the histochemical analysis instead of the specific antibodies to LDN. In general, the specificities of lectins are uncertain compared with those of the antibodies. However, some histochemical studies have indicated that WFA preferentially binds to LDN on bovine pituitary glands and human and bovine mammary glands (Taka et al., 1996; Sato et al., 1997; Kitamura et al., 2003). In this study, preferential binding of WFA to LDN was confirmed by FAC. This indicated that WFA is sufficiently sensitive and specific to detect LDN in MKN45 cells and gastric mucosa. The wild-type MKN45 cells were weakly positive for the WFA binding. In the previous study, we observed a small amount of ß4GalNAc-T4 transcripts in MKN45 cells but no transcripts for ß4GalNAc-T3 (Sato, Gotoh, Kiyohara, Kameyama et al., 2003). The weak WFA binding to wild-type MKN45 cells may be carried by the endogenous ß4GalNAc-T4. The overexpression of ß4GalNAc-T3 resulted in the strongly positive WFA binding to MKN45 cells.
ß4GalNAc-T3 and ß4Gal-T1 share the same acceptor substrates such as bianntenary structure of N-glycans in in vitro experiments. ß4GalNAc-T3 was present in the supra-nuclear area of surface mucous cells, where ß4Gal-T1 was also present. However, precise detection using confocal microscopy demonstrated that ß4GalNAc-T3 is located more at the trans-side than ß4Gal-T1. The relative trans-side localization of ß4GalNAc-T3 may imply that the proteins carrying LDN are the minority compared with the proteins carrying lactosamine which is the product of ß4Gal-T1.
Results from lectin histochemistry demonstrated that the reactivity of WFA was exclusively restricted to secreted proteins. Results from the analysis using FAC indicated that the first binding partner of WFA was LDN, while the second and other candidates were carbohydrates on glycolipids (Figure 8). These results strongly suggest that WFA preferentially binds to LDN in gastric mucosa. In addition, WFA binding occurred around the surface mucous cells where ß4GalNAc-T3 was located in the apical side. However, the binding did not occur with the cells of the glandular compartment where the enzyme is located in the basolateral area. In light of the above, the intra-cellular localization of the enzyme seems to correlate with WFA binding. Taken together, the supra-nuclear expression of ß4GalNAc-T3 might be essential for the formation of LacdiNAc on the surface mucous cells.
ß4GalNAc-T3 was differentially localized in the upper and lower parts of the growth zone. This fact evoked an idea that ß4GalNAc-T3 in the apical side of surface mucous cells synthesizes LDN on proteins secreted into the stomach lumen, while the enzyme in the basolateral area in the cells of the glandular compartment serves to synthesize LDN on proteins secreted into blood. Proteins stained with WFA seem to secrete mucins which have O-glycans other than N-glycans (Ota et al., 1991; Niwa et al., 2005). LDN stained with WFA may be carried on O-glycans of mucin proteins, because ß4GalNAc-T3 could synthesize LDN on O-glycans in our previous study (Sato, Gotoh, Kiyohara, Akashima et al., 2003). Thus, the glycoproteins, probably mucins, having LDN in the stomach lumen were detected by WFA. The reason why the ß4GalNAc-T3-product of the cells of the glandular compartment could not be detected by WFA may be explained as follows: (1) LDN may be further modified by sialylation, fucosylation, or sulfation, which mask the LDN epitope from WFA recognition. In fact, fucosylated LDN (LDNF) showed no reactivity to WFA on the analysis of FAC. To confirm these possibilities, we tried WFA staining on pathological specimens of gastric samples after either sialidase or fucosidase treatment. The protocol of enzyme digestion employed was first confirmed to digest sialic acid and fucose of the sialyl-LacdiNAc and fucosyl-LacdiNAc oligosaccharides, which were enzymatically synthesized in vitro. We treated gastric specimens with this protocol. We observed apparent reduction of sialyl Lewis X but did not observe any change on the WFA reactivity (data not shown). These results suggested that LacdiNAc is not so largely sialylated or fucosylated in gastric tissues. (2) The amount of products carrying LDN is too small to be detected. In the stomach, many gastrointestinal hormones are secreted from the cells of the glandular compartment. Some gastric hormones may carry LDN, however, their LDN may be further modified or they may be present in only extremely small amounts. Thus, it is of interest that differential sub-cellular localization of ß4GalNAc-T3 is correlated with cellular differentiation between foveolar cells and glandular cells.
Some enzymes are known to be localized in the endocrine vesicles of various endocrine cells (Bendayan et al., 1986; Lang, 1999). ß4GalNAc-T3 was found in the basolateral area only in the cells of the glandular compartment. We speculated that ß4GalNAc-T3 may glycosylate specific hormonal proteins involved in an endocrine system of the gastric glandular cells.
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Materials and Methods |
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Cells and reagents
MKN45 (human gastric adenocarcinoma) and human embryonic kidney (HEK293T) cells were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD). The cell lines were maintained in monolayer culture in Dulbecco’s minimal essential medium (DMEM) containing 10% heat-inactivated fetal bovine serum at 37°C. Triton X-100 and bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO). The specificity of mAb 8628 antibody to ß1,4-galactosyltransferase I was described in our previous study (Uejima et al., 1992
; Uemura et al., 1992). A mAb against Tn-antigen, HB-Tn1, was purchased from Dako (A/S, Glostrup, Denmark).
mAb preparation
The specific mAb, K1356, was produced by immunizing BALB/c mice with the ß4GalNAc-T3 recombinant protein purified with FLAG tag which was prepared by the methods described in Sato, Gotoh, Kiyohara, Akashima et al., (2003). Hybridoma colonies were initially screened by enzyme-linked immunosolvent assay (ELISA) for the production of mouse IgG and then further screened directly if they reacted specifically to the purified ß4GalNAc-T3.
Western blotting
Approximately 0.5 µg of recombinant ß4GalNAc-T3 protein was separated by 10% SDS–PAGE under reducing or non-reducing conditions and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Amersham, UK). The membrane was probed with anti-FLAG M2 antibody (Sigma-Aldrich) or anti-ß4GalNAc-T3 antibody (K1356). Cells were lysed at 4°C in a lysis buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and complete Protease Inhibitor Cocktail from Roche Applied Science, Indianapolis, IN). Lysates were suspended in a Laemmli sample buffer and subjected to subsequent immune blotting either with K1356 or with anti-villin mAb. Whole cell lysates were separated by 10% SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. The signals were detected using peroxidase-conjugated anti-mouse IgG (Zymed Laboratories, South San Francisco, CA), Western Lightning Chemiluminescence Plus (PerkinElmer Life Sciences, Boston, MA) and high-performance chemiluminescence film (Amersham Biosciences).
ß4GalNAc-T3 expression in MKN45 cells
The ß4GalNAc-T3 cDNA was inserted in an expression vector pcDNA3.1(+) (Invitrogen) and named pcDNA3.1Neo/ ß4GalNAc-T3 (Sato, Gotoh, Kiyohara, Kameyama et al., 2003). The construct was then transfected into MKN45 cells using Lipofectamin 2000 (Invitrogen) and OptiMEM (Invitrogen), according to the manufacturer’s instructions. For a transient expression, the transfected cells were incubated at 37°C for 48 h and used for analysis. To establish independent clones stably expressing ß4GalNAc-T3, the transfected cells were plated at various dilutions in 12-well plates with a complete DMEM medium containing 0.5 mg/ml of G418. Multiple growing cell colonies were isolated and established as independent clones. The expression of ß4GalNAc-T3 was confirmed by the detection of the enzyme itself with western blotting using mAb K1356, by measuring the enzyme activity in cell homogenates, and by the detection of terminal GalNAc with FACS analysis using WFA.
FCM analysis
Expression of LDN epitopes on MKN45 cells transfected with ß3GalNAc-T3 was routinely evaluated by FCM analysis. The cells were directly stained with FITC-conjugated WFA (EY Laboratories, San Mateo, CA). MKN45 cells were washed twice with FCM buffer (phosphate-buffer saline [PBS] containing 5 mM EDTA and 5 mg/ml of BSA) and incubated on ice for 30 min with the FITC–WFA. After washing twice with FCM buffer, the cells were subjected to FCM analysis.
Immunohistochemistry with biotin-streptoavidine methods
Human gastric mucosa tissues containing either normal fundic gland or pyloric glands with metaplastic change were selected from the pathology files of the Central Clinical Laboratories (Aichi Cancer Center Hospital). Three-micrometer-thickness sections were prepared from the formalin fixed and paraffin-embedded tissue specimens and subjected to immunohistochemical analysis either with K1356 or with anti-villin mAb (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) using a Vector stain ABC elite kit (Vector Laboratories Inc., Burlingame, CA). Briefly, deparaffinized tissue sections were treated with 0.3% H2O2 in methanol and then autoclaved for 10 min at 120°C in distilled water. After cooling down to room temperature, sections were blocked with 1% normal goat serum in PBS. K1356 and anti-Villin mAbs were reacted overnight at 4°C. After washing out the primary antibody with PBS, they were incubated with biotinylated anti-rabbit IgG and then with horse-radish peroxidase (HRP)-labeled streptavidin, according to the manufacturer’s instructions. The antibody reaction was visualized with a diaminobenzidine (DAB)/H2O2 solution, and counterstaining was performed with hematoxylin. In control experiments, the primary antibody was replaced with a mouse antibody with the same isotype.
Immunohistochemistry with fluorescent probes
For double immunostaining with fluorescent probes, we used normal frozen stomach tissue samples at Aichi Cancer Center Hospital as described (Niwa et al., 2005). The samples were taken from normal areas >10 cm away from the cancer area. They were then frozen in Tissue-Tek Optimal Cutting Temperature Compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) with liquid nitrogen to be stored at –80°C. The frozen sections with thickness of 4 µm prepared with cryostat were fixed either in cold acetone (for double staining with K1356 and mAb 8628) or in 10% formalin (for double staining with WFA and either anti-Tn antigen antibody or K1356) for 10 min and then rehydrated in PBS for 15 min at room temperature. For the specific detection of the immune reactions of two mAbs, we employed Zenon Mouse IgG labeling kits to label the Mabs directly with Alexa flour 488 or Alexa flour 568 (Molecular Probes, Uugene, OR). The sections were incubated with blocking reagent (PBS containing 0.2% Triton-X 100, 0.2% BSA, and 5% heat-inactivated normal goat serum) for 30 min at room temperature and then reacted with a mixture of two primary antibodies labeled with either Alexa flour 488 or 568 for 2 h at room temperature. For glycosidase treatment, the sections were incubated with neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan) or -fucosidase from Almond meal (Glyco Inc., Novato, CA) for 18 h before blocking. After two washes with PBS containing 0.2% TritonX-100 for 15 min, the sections were incubated with 4',6-diamidino-2-phenylindole DAPI (Molecular Probes) to stain the nucleus for 1 min at a dilution of 1:20,000. After washing with PBS, the sections were mounted in ProLong Gold antifade reagent (Molecular Probes).
Immunofluorescence and confocal laser-scanning fluorescence microscopy
Tissues multicolor stained with Alexa flour 488, Alexa flour 568, and DAPI were observed using an Olympus BH5 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a xenon arc lamp and an appropriate filter set. Confocal laser scanning was performed with the Radiance 2100 K-3 system (BioRad, Hercules, CA; Clinisciences S.A., Montrouge, France) which employs an optical fiber both as the illumination source and as the detection aperture. This system was equipped with a 50-mW argon-ion laser and with filters allowing excitation with both a 488-nm and a 514-nm laser line. Two channels were available for simultaneous data acquisition: Channel 1 (displayed as green) could use a 510–550-nm bandpass filter and either a 515-nm or a 530-nm longpass filter, while Channel 2 (displayed in red) could employ either a 550-nm or a 590-nm longpass filter. All images were recorded by a digital video camera and converted to TIFF files. Merged images were made using Adobe Photoshop software (Adobe Systems, San Jose, CA).
FAC
FAC was performed with an automated FAC (FAC-1) system as previously described in Hirabayashi et al. (2003), Hirabayashi (2004), Nakamura et al. (2005). WFA-agarose (Vector Laboratories Inc.) was suspended in a 10 mM Tris–HCl buffer, pH 7.4, containing 1 mM CaCl2 and 1 mM MnCl2. The slurry was packed into a capsule-type miniature column (inner diameter, 2 mm; length, 10 mm; bed volume, 31.4 µL). The effective ligand contents (Bt) of the resulting columns were 0.20, 1.35, and 1.52 nmol, respectively. The lectin columns were slotted into a stainless holder and connected to FAC-1. The flow rate and the column temperature were kept at 0.125 mL/min and 25°C, respectively. After equilibrium of the miniature columns with 10 mM Tris–HCl buffer, pH 7.4, containing 0.8% NaCl (TBS), 1 ml of Pyridylaminated (PA)-oligosaccharides (2.5 nM) dissolved in TBS was injected into the lectin column by the auto-sampling system. Elution of PA-oligosaccharides was monitored by fluorescence (excitation and emission wavelengths of 310 and 380 nm, respectively). V–V0 (retardation of the elution front relative to that of an appropriate standard oligosaccharide) was calculated as previously described in Hirabayashi et al. (2003). Ka values were calculated from V–V0 and Bt values based on the basic equation of FAC, that is, Kd = Bt/(V–V0) (if Kd >> [A]0), where [A]0 is the initial concentration of PA-oligosaccharide. PA-oligosaccharides [Gb4, GM2, Leb, LNnT, LNT A(ABO)-tetra, and A(ABO)-hexa] were purchased from Takara Bio Inc. Non-labeled oligosaccharides (LeY tetra and T antigen) were purchased from Calbiochem (La Jolla, CA) and were pyridylaminated with GlycoTag (Takara Bio Inc., Ostu, Japan) before use. LDN, LDNF, Sda-type2, and Sda-type1 were synthesized from PA-oligosaccharides (PA-041 or PA-042, Takara Bio Inc.) after ß1,4 galactosidase (Calbiochem) treatment using recombinant enzymes (ß4GalNAc-T3, FUT4, ß4GalNAc-T2, and ST3GalI) expressed in HEK293T cells.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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This work was performed as part of the R&D Project of the Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization. Funding to pay the Open Access publication charges for this article was provided by
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Abbreviations |
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BSA, bovine serum albumin; CSGalNAc-T, chondroitin sulfate N-acetylgalactosaminyltransferases; FCM, flow cytometry; FITC, fluorescentisothianate; FSH, follicular stimulation hormone; FAC, frontal affinity chromatography; hCG, human chorionic gonadotropin; HEK, human embryonic kidney; Ig, immunoglobulin; LH, leutropin; mAb, monoclonal antibody; LacdiNAc or LDN, N,N'-diacetyllactosediamine, GalNAcß1,4GlcNAc; PBS, phosphate-buffer saline; TSH, thyrotropin; WFA, Wisteria floribunda agglutinin
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References |
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