Glycobiology Advance Access originally published online on August 3, 2005
Glycobiology 2005 15(12):1257-1267; doi:10.1093/glycob/cwj015
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Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products
2 Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan; 3 Department of Anatomy II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan; 4 CNRS UMR 1598, Institut Gustave Roussy, Villejuif cedex 94805, France; 5 Tokyo Blood Center, Japanese Red Cross, Hiroo 4-1-31, Shibuya-ku, Tokyo 150-0012, Japan; and 6 Department of Clinical Immunology, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan
1 To whom correspondence should be addressed; e-mail: koichi{at}med.nagoya-u.ac.jp
Received on December 13, 2004; revised on July 8, 2005; accepted on July 11, 2005
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Abstract |
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Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.
Key words: Gb3 / Gb4 / globoside / globotriaosylceramide / glycosphingolipid
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Introduction |
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It has been reported that carbohydrate structures conjugated to proteins and ceramides on the cell surface are involved in many biological processes. Rapid progress in cloning of glycosyltransferase cDNAs in recent years has provided innovative changes for experimental techniques to analyze the function of carbohydrates. Manipulation of these genes has enabled us to remodel carbohydrates at cellular and experimental animal levels. Consequently, new evidence for the functions of carbohydrates has been reported steadily. In particular, important findings for glycolipids are that the carbohydrate moiety of glycolipids is involved in the interaction between cells and cells or cells and pathogens. Namely, it has been demonstrated that many of the pathogens, such as viruses, bacteria, or their toxins, invade into cells using glycolipids as their receptors (Karlsson, 1995
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Glycosphingolipids are synthesized by the sequential action of glycosyltransferases starting from the glucosylation of ceramide (Hakomori, 1986). A ß1,4-galactosyltransferase, then, synthesizes lactosylceramide (LacCer), which is a common precursor structure for most glycosphingolipids present in mammals and birds. Three major series of glycolipids are synthesized starting from LacCer by the addition of ß1,3-N-acetylglucosamine (lacto/neolacto series), 
2,3-sialic acid (ganglio series), or 1,4-galactose (globo series). Moreover, the addition of N-acetylgalactosamine with a ß1,4-linkage leads to the synthesis of asialo–ganglio series.
Globo-series glycolipids are present in some of the human and many other mammalian tissues. Recently, cDNAs of the key enzyme to initiate the synthesis of the globo-series glycolipids, Gb3/CD77 synthase (1,4-galactosyltransferase [1,4Gal-T]) has been cloned by us (Kojima et al., 2000) and other groups (Keusch et al., 2000; Steffensen et al., 2000). The expression pattern of the gene indicated that globo-series glycolipids may be more widely expressed than previously believed, suggesting the importance of structures containing the globo-series backbone. Globotriaosylceramide (Gb3) is synthesized by 1,4Gal-T from LacCer (Taga et al., 1995). This glycolipid on the human red blood cells has been characterized as the Pk antigen of the P blood group system (Marcus et al., 1981). Since a monoclonal antibody (mAb) reactive with Burkitt’s lymphoma cells was reported by Wiels et al. (1981) and the recognized antigen was elucidated to be Gb3 (Nudelman et al., 1983), the expression and biological significance of Gb3 have been vigorously studied (Klein et al., 1983; Balana et al., 1985; Murray et al., 1985). Although Gb3 was clustered as CD77 (Dorken et al., 1989), we refer to this antigen as Gb3/CD77.
Although Gb3/CD77 was reported to be expressed at high levels on Burkitt’s lymphoma cells, it can be also found in some malignant tumors of B-cell lineage (Klein et al., 1983). Then, it is now considered to be a differentiation antigen expressed on a B-cell subset. Among normal leukocytes, it is expressed on a subset of tonsillar B cells in the germinal centers (Murray et al., 1985). Interestingly, germinal center B lymphocytes expressing Gb3/CD77 undergo rapid and spontaneous apoptosis when isolated and cultured in vitro (Mangeney et al., 1991). Gb3/CD77 has been recognized as a receptor for verotoxins (VTs) (Jacewicz et al., 1986; Lingwood et al., 1987), the Shiga-like toxin from the Escherichia coli O157 strain that can trigger serious cytotoxic effects (Endo et al., 1988). VT B subunit specifically binds to Gb3/CD77, and then the A subunit is incorporated into cells, resulting in the degradation of 28 S ribosomal RNA and cell death (Skinner and Jackson, 1997).
Globoside (Gb4) is the most prominent neutral glycosphingolipid in the human erythrocytes (Naiki and Marcus, 1975) and is an essential structure of blood group P antigen (Kijimoto-Ochiai et al., 1977). Gb4 is synthesized from Gb3/CD77 by the action of ß1,3-N-acetylgalactosaminyltransferase (ß1,3GalNAc-T). Therefore, Pk individuals lack ß1,3GalNAc-T activity and show the accumulation of the precursor Pk in their red cells. On the other hand, P individuals lack Gb3/CD77 synthase activity with intact ß1,3GalNAc-T activity (Lund et al., 1985), and they lack the expression of both Gb3/CD77 and Gb4. Although Gb4 has been considered to be an adhesion molecule on epithelial cells to various bacteria, such as uropathogenic E. coli (DeGrandis et al., 1989), and a receptor for pig edema disease toxin (Boyd et al., 1993; Song et al., 1998), its physiological function in vivo has never been elucidated.
In this study, we analyzed expression levels of 1,4Gal-T and ß1,3GalNAc-T genes and expression patterns of Gb3/CD77 and Gb4 in the mouse tissues with special focus on kidney and spleen. The results would provide essential information for the functional studies of globo-series glycolipids and contribute in studies of the human diseases in which these glycolipids are involved.
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Results |
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Isolation and identification of the mouse
1,4Gal-T and ß1,3GalNAc-T cDNAsWe have cloned the cDNA of the mouse
1,4Gal-T gene by a data base search with the sequence of the human 1,4Gal-T cDNA and reverse transcription–polymerase chain reaction (RT–PCR), as described in Materials and methods (Figure 1). This cDNA sequence predicts a type II membrane protein with 348 amino acids which consists of 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 303 amino acids. This protein has a molecular mass of 40,177 daltons and shows 84% homology to the human 1,4Gal-T. As a result of enzyme activity assay, this cDNA coded 1,4Gal-T and digalactosylceramide synthase activity. Then, we have identified the cDNA of the mouse ß1,3GalNAc-T gene by a data base search with the sequence of the human ß1,3GalNAc-T gene (Figure 2). This cDNA sequence predicts a type II membrane protein with 331 amino acids which consists of 19 amino acids of cytoplasmic domain, 23 amino acids of transmembrane region, and a catalytic domain with 289 amino acids. This protein has a molecular mass of 39,370 daltons and shows 88% homology to the human ß1,3GalNAc-T.
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Expression pattern of the mouse 1,4Gal-T gene in the mouse tissues
Expression levels of the mouse 1,4Gal-T gene in 12 mouse tissues were examined by northern blotting. Among tissues examined, strong gene expression was observed in kidney and lung; moderate expression was detected in heart, skin, spleen, stomach, and testis; and low level expression was observed in brain, small intestine, and thymus (Figure 3).
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Expression pattern of the mouse ß1,3GalNAc-T gene in the mouse tissues
Expression levels of the mouse ß1,3GalNAc-T gene in 12 mouse tissues were examined by northern blotting. Among tissues examined, strong gene expression was observed in brain, heart, kidney, liver, skin, and testis, and low level expression was observed in lung (Figure 3).
Expression of Gb3/CD77 in the mouse tissues as analyzed with immunohistochemistry
In kidney, Gb3/CD77 was expressed in renal tubules in the whole area of cortex and medulla (Figure 4). In cortex, Gb3/CD77 was expressed in tubules surrounding glomeruli and in the cortical labyrinths and the medullary rays. The medulla is composed of straight vessels, otherwise known as vasa recta, and renal tubules containing thin segments of Henle’s loops and collecting ducts. In this area, Gb3/CD77 was expressed in renal tubules extending straight toward renal papillae. Using serial sections, the expression pattern of Gb3/CD77 was compared with that of angiotensin-converting enzyme (ACE), a marker protein of the proximal convoluted tubule, and with that of Tamm–Horsfall protein (THP), a marker protein of the distal convoluted tubule (Figure 5). As the expression pattern of Gb3/CD77 coincided with that of ACE, it is suggested that Gb3/CD77 is mainly expressed in the proximal convoluted tubules. Gb3/CD77 was also found in the medulla, indicating that this antigen is present in collecting duct epithelial cells.
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Spleen is composed of two different regions, the so-called red and white pulps. Gb3/CD77 was expressed weakly in cells of the red pulp and strongly on cells in the peripheral region of the white pulp (Figure 6). The expression pattern of Gb3/CD77 was compared with that of CD10, a marker protein of the pre-B cell, and with that of CD20, a marker protein of the B cell. The expression pattern of Gb3/CD77 was prone to coincide with that of the CD10, suggesting that Gb3/CD77 is expressed in the pre-B cells (Figure 6), although two-color flow cytometry (FACS) for simultaneous staining of Gb3/CD77 and CD10 was not successful.
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As the expression pattern of Gb3/CD77 was similar to that of CD34, a marker protein of the endothelium (Figure 7), it is suggested that Gb3/CD77 was expressed on the endothelia of the alveolar capillaries in lung. In dermis, Gb3/CD77 was expressed on the sebaceous ducts aside of the hair follicle (data not shown).
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Expression of Gb4 in the mouse tissues
In kidney, Gb4 was expressed on renal tubules in the whole area of cortex and medulla (Figure 8). In cortex, Gb4 was expressed on tubules surrounding glomeruli and in the cortical labyrinths and the medullary rays. In medulla, Gb4 was expressed on renal tubules extending toward renal papillae. The expression pattern of Gb4 was compared with that of ACE and THP using serial sections (Figure 9). As the expression pattern of Gb4 coincided with that of ACE, Gb4 appeared to be expressed mainly in the proximal convoluted tubule. Gb4 was stained more densely in the cortex than in the medulla in kidney, when compared with the staining of Gb3/CD77, which was stained almost equally between two regions.
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In lung, Gb4 was expressed on cilia of the ciliated pseudostratified columnar epithelial cells, the epithelium of the bronchus. Besides cilia, the expression pattern of Gb4 was very similar to that of CD34, suggesting that Gb4 was also expressed on the endothelia (Figure 10). In testis, Gb4 was expressed in some cells in the seminiferous tubules except for spermatogonia, which located directly above the basement membrane. In particular, it was expressed in tails of the spermatozoa as well as blood vessels in stroma (data not shown). In brain, Gb4 was expressed on blood vessels of choroid plexus, area postrema (data not shown), and endothelial cells widely present in the tissue (data not shown). In liver, Gb4 was expressed in the central veins and the hepatoportal veins (data not shown).
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Discussion |
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We defined the expression levels of the mouse
1,4Gal-T gene and the mouse ß1,3GalNAc-T gene in 12 tissues with northern blotting and analyzed the expression patterns of Gb3/CD77 and Gb4 mainly in the tissues which express these synthase genes. In northern blotting, the 1,4Gal-T gene was expressed strongly in kidney and lung and moderately in heart, skin, spleen, stomach, and testis (Figure 3). The expression pattern is almost similar to that in the human tissues, as reported previously (Kojima et al., 2000), although there are some differences in the expression patterns between humans and mice.
In the immunohistostaining by anti-Gb3 Ab, kidney, spleen, and lung strongly expressed Gb3/CD77, dermis did weakly, and intestine and muscle did not. In the past, a study that the expression of Gb3/CD77 in the mouse lymphoid tissues was detected by anti-Gb3 polyclonal Ab reported that Gb3/CD77 was expressed in cortex of thymus, peripheral region of the white pulp, cortex and medulla of lymph node (Jacewicz et al., 1986). Furthermore, a study was reported in which the depositions of VTs injected from tail vein were detected by radioisotope-labeled anti-VT Ab (Rutjes et al., 2002). VTs deposited mainly on collecting tubules, proximal and distal convoluted tubules in kidney, and capillary vessels in alveoli, as well as nasal cavity, long bone, and ribs. Although these results partly agree with the results of our study with regard to kidney and spleen, our results showed slightly more restricted expression of Gb3/CD77. One possible reason might be because we used a specific heterophile (not mice) mAb, resulting in more specific detection of Gb3/CD77.
The results of the northern blotting and the immunohistostaining for Gb3/CD77 are summarized in Table I. There are differences between the expression patterns of 1,4Gal-T gene and Gb3/CD77 antigen in some tissues. Generally, cells forming tubules, blood vessels, renal tubules, and so on expressed Gb3/CD77, whereas a distinctive cell lineage expressed the antigen in the spleen.
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In kidney, Gb3/CD77 was expressed on renal tubules in the cortex and medulla (Figure 4). By comparing the expression pattern of Gb3/CD77 with that of ACE and THP using serial sections (Figure 5), it is suggested that the proximal convoluted tubule expresses Gb3/CD77 based on the similarity of its distribution with that of ACE. However, there are some regions where the expression of Gb3/CD77 did not coincide with those of ACE and THP in the cortical labyrinths and medulla. These regions contain collecting tubules, Henle’s loops, and vasa recta besides proximal and distal convoluted tubules. Consequently, collecting tubules and Henle’s loops seemed to express Gb3/CD77 also. In the morphological analysis, vasa recta appeared not to express Gb3/CD77. The proximal convoluted tubules reabsorb the filtered glucose and amino acids and some of the sodium, chloride, and bicarbonate ions. This active process enhanced by the numerous microvilli results in an osmotic gradient, which causes the passive reabsorption of water. Gb3/CD77 might be implicated in these processes, resulting in the maintenance of the body homeostasis.
In the human spleen, it is known that the germinal B cells express Gb3/CD77. The result of this study in the mouse spleen agrees with those reports, that is, Gb3/CD77 was expressed weakly on cells in the red pulp and strongly on cells in the peripheral region of the white pulp (Figure 6). Similar expression pattern of CD10 with that of Gb3/CD77 suggested that the pre-B cells express Gb3/CD77. Although Gb3/CD77 was previously found on the cell surface of spleen cells with flow cytometry (Kovacic et al., 2000), two-color FACS was not successful in our study because of unknown reason.
In lung, Gb3/CD77 was expressed in the whole area except for bronchia (Figure 7). The alveolar wall is composed of basal lamina, endothelial cells, and two types of epithelial cells, Type I and Type II cells. Type I cells, squamous alveolar cells, constitute large percent of the respiratory epithelium, and the remainder is Type II cells which are cuboidal. These epithelial cells rest on a basal lamina, which is, in turn, intimately associated with capillaries of the pulmonary vascular system. As the expression patterns of Gb3/CD77 and CD34 were very similar, it is suggested that Gb3/CD77 is expressed on the endothelia of the blood vessels. Although two-color staining should be desirable, it could not be achieved because of natural fluorescence.
Besides Gb3/CD77, we analyzed the expression levels of the ß1,3GalNAc-T gene, showing the expression in brain, heart, kidney, liver, skin, and testis (Figure 3). As we previously reported that the human ß1,3GalNAc-T gene was expressed strongly in brain and heart and moderately in lung, placenta, testis, lowly in kidney, liver, spleen, and stomach (Okajima et al., 2000), there are some differences in the expression patterns between humans and mice. In the immunohistostaining of the mouse tissues by anti-Gb4 Ab, Gb4 was expressed strongly in kidney, lung, and testis, and weakly in brain and liver. These results are summarized in Table I. The expression of the mouse ß1,3GalNAc-T gene was almost consistent with that of Gb4 antigen with some exceptions. Gb4 is the most prominent neutral glycosphingolipid in the human erythrocytes (Suzuki et al., 1980) and is an essential structure of blood group P antigen (Naiki and Marcus, 1975). Although Gb4 has been considered to be a receptor for parvo-B 19 virus (Weigel-Kelley et al., 2001, 2003) and an initiator of signal transduction, real function of Gb4 is not known.
In kidney, Gb4 was expressed on renal tubules in the whole area of cortex and medulla (Figure 8). Comparison of the expression pattern of Gb4 with those of ACE and THP (Figure 9) suggested that the proximal convoluted tubules express Gb4. However, there are some regions where the Gb4 expression did not coincide with those of ACE and THP in the cortical labyrinths and medulla, suggesting that Gb4 was also expressed on collecting tubules and Henle’s loops.
In lung, Gb4 was expressed on cilia of the ciliated pseudostratified columnar epithelial cells (Figure 10). Furthermore, like Gb3/CD77, Gb4 was expressed on membranes surrounding the epithelial cells, and the expression patterns of Gb4 and CD34 were very similar, suggesting that the endothelia express Gb4. In testis, Gb4 was expressed on cells in the seminiferous tubules and in tails of the spermatozoa as well as blood vessels in stroma (data not shown), suggesting that Gb4 is involved in the spermatogenesis and fertility.
Furthermore, it is interesting that the Gb4-expressing regions were broader than that of Gb3/CD77. As described above, Gb4 was expressed on blood vessels in brain, liver, and testis. Although the blood vessels did not express Gb4 in the other tissues, it is thought that Gb4 is involved in the specific function of the blood vessels in these particular tissues.
Although the expression patterns of the mouse Gb3/CD77 and Gb4 are similar in kidney and lung, it is suggested that the functions of these glycolipids are very close. However, as there are some differences in the expression patterns in kidney, only Gb4 is expressed in the ciliated pseudostratified columnar epithelial cells. Furthermore, the expression patterns of Gb4 and ß1,3GalNAc-T fairly match in contrast with those between 1,4Gal-T gene and Gb3/CD77. This may be partly because Gb3/CD77 is a precursor of Gb4 and the Gb4 synthesis is restricted with that of Gb3/CD77. Other explanations are also possible, that is, Gb4 can not be rapidly elongated to other glycolipids in the tissues examined, degradation rates of Gb3 and Gb4 are different, or Gb4 synthase is more active than Gb3 synthase resulting in the less amount of Gb3.
The tissues expressing these glycolipids, such as kidney, lung, B lymphocytes, and testis, are all vital tissues, suggesting that these glycolipids might play important roles in the regulation of homeostasis. Consequently, it is very interesting to establish gene knockout mice that lack all globo-series glycolipids and to analyze their phenotypes.
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Animals
C57BL/6 mice were used in all experiments. For immunohistochemistry, animals were perfused with 0.1 M phosphate-buffered saline (PBS), pH 7.4, under anesthosis by an intraperitoneal injection of sodium pentobarbital (Nembutal) (Dainippon Pharmaceutical, Osaka, Japan), and then perfused with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. After perfusion, the tissues were removed and postfixed overnight in 4% paraformaldehyde in 0.1 M PBS, pH 7.4, at 4°C. Thereafter, the tissues were cryoprotected overnight in 0.1 M PBS, pH 7.4, containing 20% sucrose at 4°C. The tissues were embedded in O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan) and snap frozen at –80°C.
Antibodies
A rat anti-Gb3 mAb 38.13 (Immunotech, a Beckman–Coulter Company, Marseille, France), a human anti-Gb4 mAb 9H6 generated at the Japanese Red Cross Society in the Central Blood Center, a goat anti-ACE (Santa Cruz Biotechnology, Santa Cruz, CA), a sheep anti-THP polyclonal Ab (Chemicon International, Temecula, CA), a rat anti-CD34 mAb (HyCult biotechnology b.v., Uden, The Netherlands), a rabbit anti-CD10 Ab (Santa Cruz Biotechnology), and a goat anti-CD20 Ab (Santa Cruz Biotechnology) were used as a first Ab for immunohistostaining. As secondary antibodies, a goat anti-rat IgM Ab (Jackson Immuno Research Laboratories, West Grove, PA), a goat anti-human IgM Ab, a horse anti-goat IgG Ab, a rabbit anti-sheep IgG Ab, a rabbit anti-rat IgG Ab, and a goat anti-rabbit IgG Ab (Vector Laboratories, Burlingame, CA) were used in the biotin-conjugated form.
Immunohistostaining
About 10-µm thick tissue sections were cut on a cryomicrotome (Leica Microsystems AG, Wetzlar, Germany) at –20°C, mounted on chrome alum gel-coated glass slides, and air-dried at room temperature. Nonspecific Ab binding was blocked by incubating the sections with 10% serum (a normal serum from an animal species same as the species origin of the secondary Ab) in 0.1 M PBS, pH 7.4, for 1 h at room temperature. Blocking solution was poured off, and each section was incubated overnight with the first Ab diluted in 0.1 M PBS supplemented with 5% normal serum and 0.05% Tween 20 same as the blocking solution at 4°C. Preimmune serum, derived from the same animal species in which the first Ab was raised, was used as a negative control at the same dilution as the first Ab. The sections were then rinsed in 0.1 M PBS containing 0.05% Tween 20 and incubated for the next 1 h with a biotinylated second Ab diluted at 1 µg/mL in PBS. The sections were rinsed and treated with 3% H2O2 in 0.1 M PBS to quench endogenous peroxidase activity. Thereafter, the sections were incubated with an avidin–biotin complex (Vector Laboratories) for 40 min at room temperature. Then the sections were rinsed, and the color reaction was performed with 0.1% 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich Japan, Tokyo, Japan) solution containing 0.03% H2O2. Counter staining was performed with 2% cresyl violet (Sigma-Aldrich Japan) for nerve tissues and 4% methyl green (ICN Biomedicals, Aurora, OH) for other tissues. Finally, the sections were cover slipped with EUKITT mounting medium (Kenis, Osaka, Japan), following dehydration with graded alcohols and xylene.
Extraction of the mouse RNA
Fifty milligrams of the mouse tissues were homogenized in 5 mL TRIzol reagent® (Invitrogen, Carlsbad, CA) to facilitate homogenization. One milliliter of chloroform was added, then, the sample was centrifuged at 5800 rpm for 20 min at 4°C. The upper aqueous phase containing the RNA was transferred to a fresh tube, and 2.5 mL isopropanol (Sigma-Aldrich Japan) was added. The sample was centrifuged at 7500 rpm for 20 min, and the pellet was washed with 75% ethanol. The RNA pellet was air-dried and dissolved in 50 µL of distilled water (DW).
Isolation of cDNA clones
cDNA was synthesized using 1 µL of RNA solution (3 µg/µL), oligo dT primer, and murine leukemia virus reverse transcriptase (Invitrogen). Mice EST data base (dbEST) at The National Center for Biotechnology Information (NCBI) was searched with the coding sequence of the human 1,4Gal-T gene (GenBankTM Accession number AB037883
[GenBank]
) using tBLASTn algorithms. Although three mouse EST clones were found, the downstream sequences were lacked. Then, the 3' side sequence of the 1,4Gal-T gene was obtained by PCR using mouse fibrosarcoma cDNA library (CMS5j/CDM8) as a template. The primers used were as follows, a sense primer 5'-ACACAGACTTCATCGTCCT-3' and an antisense primer (AS-1, in pCDM8) 5'-CTGCAGGCGCA GAACTGGT-3'. Based on these sequences, we performed RT–PCR using the mouse kidney cDNA as a template to obtain a cDNA clone containing the whole open reading frame sequence. The primers used here were as follows, a sense primer 5'-AGGAGACCATGTCCAAGC-3' and an antisense primer 5'-GTCACAAGTACATCTTCATG-3'. Then, the insert was cloned into the expression vector pMIKneo. The 1,4Gal-T/pMIK neo was transfected into L cells, and the galactosyltransferase assay was performed as described (Kojima et al., 2000).
By a data base search with the coding sequence of the human ß1,3GalNAc-T gene (GenBankTM Accession number AB050855 [GenBank] ) using tBLASTn algorithms at NCBI, we obtained a mouse cDNA which showed 88% homology with the human ß1,3GalNAc-T gene. Based on this sequence, a 437-bp DNA fragment was amplified by PCR by using primers 5'-TGAACTTCTCTGCTGCTGTG-3' and 5'-AGAACGTGCTCATCCTCCAA-3' and was subcloned into the TOPO vector and sequenced to confirm their identity.
Northern blotting
cDNA probes of the mouse 1,4Gal-T (nucleotides 1–1047) and the mouse ß1,3GalNAc-T (nucleotides –21 to 416) were radiolabeled with [32P]dUTP (PerkinElmer, Wellesley, MA) and used for northern blotting. Multiple ChoiceTM northern blot membrane (OriGene Technologies, Rockville, MD) with 2 µg/lane of poly(A)+ RNA from the mouse brain, thymus, lung, heart, muscle, stomach, small intestine, liver, kidney, spleen, testis, and skin was used. It was rehydrated in 4 x SSC for 10 min with shaking. The membrane was placed into a sealable plastic bag, and 20 mL of prewarmed UltraHybTM solution was added, then the bag was incubated overnight at 42°C. The radiolabeled probe was added into the bag, mixed well, and hybridized overnight at 42°C. The membrane was washed twice with wash buffer 1 (2 x SSC, 0.1% sodium dodecyl sulphate [SDS]) for 15 min at 42°C each. Then, the membrane was washed twice with wash buffer 2 (0.25 x SSC, 0.1% SDS) for 15 min at 65°C. Thereafter the membrane was exposed to an imaging plate. To strip the blot for rehybridization, we dipped the membrane into boiling water for 2 min and into boiling 0.1% SDS for the next 2 min. Then, the membrane was stored at –20°C until needed.
Transfection
cDNA expression vectors in pCDM8 (1,4Gal-T) or in pSPROT6 (ß1,3GalNAc-T) were transfected into L cells or LVTR-1, respectively, with DEAE–Dextran method. Expression of Gb3/CD77 or Gb4 was analyzed with flow cytometry after 2 days of the transfection using mAb 38.13 or mAb 9H6, respectively, as described previously (Iwamura et al., 2003).
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We thank Tetsuya Okuda, Manabu Ogiso, Noriyo Tokuda at the Department of Biochemistry II, Nagoya University School of Medicine for helpful discussion. This study was supported by the grants-in-aid for Scientific from the Ministry of Education, Culture, Sports, and Science of Japan (MEXT).
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Abbreviations |
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Ab, antibody; ACE, angiotensin-converting enzyme; FACS, flow cytometry; Gb3, globotriaosylceramide or CD77; Gb4, globoside or globotetraosylceramide; LacCer, lactosylceramide; mAb, monoclonal Ab; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS, sodium dodecyl sulphate; THP, Tamm–Horsfall glycoprotein; VT, verotoxin;
1,4Gal-T, 1,4-galactosyltransferase or Gb3/CD77 synthase; ß1,3GalNAc-T, ß1,3-N-acetylgalactosaminyltransferase or Gb4 synthase |
References |
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