3Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan; 4Department of Pathology, Beijing Medical University, Beijing 100083, P.R. China; and 5Nisshin Flour Milling Co., Ltd., Saitama 356-8511, Japan
Received on April 19, 2001; revised on June 20, 2001. accepted on June 21, 2001.
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Abstract |
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Key words: acceptor specificity/in vivo ß-1,4-galactosylation/Km value/N-glycan
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Introduction |
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Because the Galß14GlcNAc structure on N-linked oligosaccharides from several tissues of ß-1,4-GalT I-knockout mouse was detected (Asano et al., 1997
; Kido et al., 1998
, 2000), some of ß-1,4-GalTs II-VI could be really involved in the galactosylation of N-linked oligosaccharides in vivo. To examine which of ß-1,4-GalTs beside ß-1,4-GalT I are involved in N-linked oligosaccharide biosynthesis in vivo, individual human ß-1,4-GalT cDNAs were introduced into Sf-9 cells, which appeared to lack ß-1,4-GalT but to contain UDP-Gal in the Golgi apparatus and the acceptor oligosaccharides in the endogenous glycoproteins (Hollister et al., 1998
; Yoshimi et al., 2000
), and the galactosylation of membrane glycoproteins were examined by lectin blot analysis using RCA-I, which preferentially interacts with oligosaccharides terminated with the Galß1
4GlcNAc group (Baenziger and Fiete, 1979
). To establish this further, antisense ß-1,4-GalT cDNAs were introduced into human colorectal adenocarcinoma SW480 cells and the galactosylation of membrane glycoproteins was examined by lectin blot analysis. The present study showed that ß-1,4-GalTs II, III, IV, V, and VI can galactosylate N-acetylglucosamine terminated on N-linked oligosaccharides.
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Results |
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To determine the nature of sugar-acceptor molecules of ß-1,4-GalTs II-VI in vivo, the human ß-1,4-GalT I to VI cDNAs were independently cloned into a pVL1393 vector, and the resultant plasmids were separately transfected into Sf-9 cells. As described already, a very weak ß-1,4-GalT activity toward p-nitrophenyl-N-acetyl-1-thio-ß-D-glucosaminide (GlcNAcß-S-pNP) was detected in the homogenates from untransfected Sf-9 cells, which was slightly increased on transfection of a vector (less than 0.1 pmol galactose-transferred/min-mg protein) as described previously (Sato et al., 1998a, 2000a), while significantly higher transferase activities toward GlcNAcß-S-pNP were detected in the homogenates from the cells transfected with the ß-1,4-GalT I, II, III, IV, V, and VI cDNAs (0.431.11 nmol galactose-transferred/min-mg protein as obtained by three independent experiments), in which the titers of the viruses used for infection of the plasmids were adjusted. When [3H]-galactosylated GlcNAcß-S-pNP isolated from the assay mixtures of all samples was digested with diplococcal ß-1,4-galactosidase, which specifically cleaves the Galß1
4GlcNAc linkage but not the Galß1
3GlcNAc linkage (Glasgow et al., 1977
), and then digests were applied to a Sep-Pak C18 cartridge, all of the radioactivities were recovered in the pass-through fraction of the column (data not shown), indicating that galactose is transferred to the acceptor in a ß-1,4-linkage. Kinetic studies showed that human recombinant ß-1,4-GalTs possess apparent Km values of 42105 µM toward UDP-Gal and of 63238 µM toward GlcNAcß-S-pNP (Table I). The Vmax values were also determined using GlcNAcß-S-pNP as a sugar acceptor to obtain kinetic efficiencies for individual ß-1,4-GalTs, which showed that ß-1,4-GalTs I and II can galactosylate the acceptor more effectively than other ß-1,4-GalTs (Table I). These results indicate that human ß-1,4-GalTs I, II, III, IV, V, and VI can catalyze transfer of galactose from UDP-Gal to N-acetylglucosamine-terminated N-linked oligosaccharides with different efficiencies.
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Because ß-1,4-GalT activities were detected in Sf-9 cells transfected with individual ß-1,4-GalT cDNAs, whether or not each ß-1,4-GalT is involved in the galactosylation of N-linked oligosaccharides was examined in the gene-transfected cells by lectin blot analysis of endogenous membrane glycoprotein samples that contained acceptor-substrates for ß-1,4-GalT. No significant differences were observed in Coomassie Brilliant Blue (CBB) staining patterns among the samples except for 50 kDa and 53 kDa bands, which appeared in those from the cells transfected with the ß-1,4-GalT II cDNA and with ß-1,4-GalT III cDNA (lanes C and D, respectively, in Figure 1 CBB). When blots were incubated with peroxidase-conjugated RCA-I and the lectin reactivity was determined by color development within 1 min in the presence of 4-chloro-1-naphthol, a band with an apparent molecular weight of 60 kDa reacted strongly with the lectin in the samples from those transfected with ß-1,4-GalT I, II, III, and IV cDNAs (lanes B, C, D, and E, respectively, in Figure 1 RCA-I [a]) but weakly with the lectin in the samples from those transfected with ß-1,4-GalT V and VI cDNAs (lanes F and G, respectively, in Figure 1 RCA-I [a]). The molecular weights of RCA-I-positive bands appeared to be identical to those of PVL-positive bands as reported previously (Yoshimi et al., 2000). The results indicate that ß-1,4-GalTs I, II, III, and IV can galactosylate the endogenous glycoprotein acceptors, most probably hybrid- and/or monoantennary complex-type acceptor oligosaccharides more effectively than ß-1,4-GalTs V and VI. Prolonged incubation (10 min) of blots with peroxidase-conjugated RCA-I resulted in the strong color development on several protein bands whose color intensities were not discriminated among the samples (Figure 1 RCA-I [b]). However, differences in the number of lectin-positive bands and their reactivities with the lectin became more clearly (lanes B to G in Figure 1 RCA-I [b]). Very weak lectin-reactive bands were also detected in mock-transfected Sf-9 cells (lane A in Figure 1 RCA-I [a]), indicating that Sf-9 cells may contain a ß-1,4-GalT gene that could be activated on viral infection. No lectin binding was observed when blots were treated with diplococcal ß-1,4-galactosidase or N-glycanase prior to incubation with lectin (the results from ß-1,4-GalT III cDNA-transfected cells are shown as representatives in lanes D1 and D2 of Figure 1 RCA-I [b], respectively). These results indicate that human ß-1,4-GalTs I, II, III, IV, V, and VI expressed as membrane-bound forms in Sf-9 cells can galactosylate endogenous N-linked oligosaccharides.
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Discussion |
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Analysis of acceptor-specificity of ß-1,4-GalT IV expressed in a soluble form showed no capability of galactosylating glycoprotein acceptors (Schwientek et al., 1998). Similarly, in a purified chimeric protein of human ß-1,4-GalT V with protein A, the transferase also failed to galactosylate glycoprotein acceptors (unpublished data). However, the present study clearly demonstrated that ß-1,4-GalTs IV and V expressed as membrane-bound forms in Sf-9 cells can galactosylate the endogenous N-linked oligosaccharides. Because some lipid-rich environment stimulated ß-1,4-GalT I activities (Mitranic and Moscarello, 1980
; Mitranic et al., 1983
), most of these ß-1,4-GalTs may require such an environment for maintaining proper tertial structures that affect their acceptor specificities. It is of interest to investigate whether or not the purified recombinant human ß-1,4-GalTs in membrane-bound forms can galactosylate glycoprotein acceptors effectively in the presence of some types of lipids. Similarly, several studies showed that ß-1,4-GalTs III, IV, and VI are involved in the biosynthesis of glycolipids such as lactosylceramide and lacto-N-neotetraosylceramide in vitro (Almeida et al., 1997
; Nomura et al., 1998
; Schwientek et al., 1998
; Takizawa et al., 1999
), but the present study showed that they can also galactosylate N-linked oligosaccharides in vivo. Because they are supposed to be in the trans-Golgi (Roth and Berger, 1982
) in which the galactosylation of N-linked oligosaccharides but not of glucosylceramide takes place, ß-1,4-GalTs III, IV, and VI may not be involved in the biosynthesis of lactosylceramide in vivo. Concerning to this issue, it is important to elucidate the fine localization of individual ß-1,4-GalTs within the Golgi apparatus once antibodies specific to individual ß-1,4-GalTs are available, and it is of interest to investigate whether the galactosylation of glycolipids is also enhanced in Sf-9 cells on transfection of individual ß-1,4-GalT cDNAs if appropriate endogenous precursor substrates such as glucosylceramide and lacto-N-triaosylceramide are accumulated in Sf-9 cells.
The present study also demonstrated that ß-1,4-GalT I prefers to galactosylate the GlcNAcß12Man branch and ß-1,4-GalTs IV, V, and VI galactosylate the GlcNAcß1
6Man branch predominantly, whereas no preference is observed for ß-1,4-GalTs II and III. In the case of ß-1,4-GalT V, prolonged incubation showed no galactosylation of another N-acetylglucosamine residue of oligosaccharide 5, indicating that other ß-1,4-GalTs could galactosylate it. Using oligosaccharides with the Galß1
4GlcNAcß1
2(GlcNAcß1
6)Man and Galß1
4GlcNAcß1
6(GlcNAcß1
2)Man structures, ß-1,4-GalT I was shown to galactosylate the GlcNAcß1
6Man branch more effectively than the GlcNAcß1
2Man branch of the monogalactosylated oligosaccharides (Ujita et al., 1999
), indicating that the initial galactosylation affects the subsequent galactosylation of the branched oligosaccharides. Therefore, it is worthy of determining the order of ß-1,4-GalTs for the galactosylation of all of N-acetylglucosamine residues of highly branched oligosaccharides. In the case of ß-1,4-GalT I-knockout mouse, polysialic acid, which is supposed to be expressed on highly branched N-linked oligosaccharides (Kudo et al., 1996
), was expressed without reduction when compared to that of wild-type mouse (Kido et al., 1998
), suggesting that ß-1,4-GalTs II to VI are involved in the galactosylation of the highly branched oligosaccharides. In support of our findings in the present study, some or all of ß-1,4-GalTs II, III, IV, V, and VI have been recently shown to be invovled in the galactosylation of highly branched N-linked oligosaccharides using Chinese hamster ovary mutant cell lines (Lee et al., 2001
).
The tissue-specific expression of ß-1,4-GalTs II, III and IV in human tissues (Almeida et al., 1997; Lo et al., 1998
; Sato et al., 1998b
; Schwientek et al., 1998
; Takizawa et al., 1999
) suggests that they are contributing to tissue-specific galactosylation of N-linked oligosaccharides. Interestingly, the gene expression level of ß-1,4-GalT V but not ß-1,4-GalTs IV and VI, whose transcripts were under detection levels was increased on malignant transformation of NIH3T3 cells (Shirane et al., 1999
; Sato et al., 2000b
), indicating that ß-1,4-GalT V is mainly involved in the galactosylation of the GlcNAcß1
6Man branch, on which a variety of tumor antigens are expressed and involved in metastasis (Dennis et al., 1987
; Dennis and Laferte, 1989
; Ohyama et al., 1999
). Further investigation is necessary as to whether suppression of the ß-1,4-GalT V gene expression will result in the reduction of the tumor formation and/or tumor metastatic processes to show functions of the carbohydrates. Finally, to raise and to analyze phenotypes of individual ß-1,4-GalT-knockout mice will contribute to our real understandings how individual ß-1,4-GalTs are important for our lives as already shown in ß-1,4-GalT I-knockout mouse (Asano et al., 1997
; Lu et al., 1997
).
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Materials and methods |
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Isolation of human ß-1,4-GalT II, III, and IV cDNAs
The total RNA preparation was obtained from human erythroleukemic K562 cells with RNeasy Total RNA System (QIAGEN) from which single-strand cDNAs were prepared using a hexadeoxyribonucleotide mixture [pd (N)6, TAKARA] as a primer and Ready-To-GoTM You-Prime First Strand Beads (Pharmacia Biotech). The cDNAs encoding the full length of human ß-1,4-GalTs II, III, and IV were amplified by polymerase chain reaction (PCR) (95°C, 10 min [94°C, 0.5 min; 60°C, 0.5 min; and 72°C, 1.5 min] x 40, and 72°C, 15 min) using AmpliTaq GoldTM (Perkin Elmer), the single-strand cDNAs as a template and 5'- and 3'-primers specific for each ß-1,4-GalT. The following 5'- and 3'-primer pairs were used (the newly synthesized BamH I, EcoR l and Xba I sites in the primers are underlined):
TS49 (5'-CGGGATCCTTGCGGGATGAGCAGACTG-3') and TS50 (5'-CGGAATTCCATTAGTGTCAGCCCCGAG-3') for ß-1,4-GalT II
TS51 (5'-CGGGATCCAGGATGTTGCGGAGGCTG-3') and TS52 (5'-GCTCTAGAGGAGTCAGTGTGAACCTC-3') for ß-1,4-GalT III
TS53 (5'-CGGGATCCAACATGGGCTTCAACCTGAC-3') and TS54 (5'-CGGAATTCAGGGTCATGCACCAAACCAG-3') for ß-1,4-GalT IV
PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced. The nucleotide sequences of human ß-1,4-GalT II, III, and IV cDNAs isolated in the present study were deposited in the DDBJ/GenBank/ EMBL data base (accession numbers: AB024434, AB024435, and AB024436, respectively).
Plasmid construction and expression of ß-1,4-GalT cDNA in Sf-9 and SW480 cells
Plasmid containing each ß-1,4-GalT cDNA was digested with BamH l and EcoR l or BamH l and Xba l, and the fragment was ligated into the BamH lEcoR l or BamH lXba l site of baculovirus transfer vector pVL1393 (Pharmingen). The resultant plasmids, pVL1393/ß-1,4-GalT II, pVL1393/ß-1,4-GalT III, and pVL1393/ß-1,4-GalT IV, were transfected independently with BaculoGoldTM Linearized Baculovirus DNA into Sf-9 cells by the method described in BaculoGoldTM systems protocol (Pharmingen). Recombinant baculoviruses were obtained after two successive amplification in Sf-9 cells. Similarly, plasmids, pVL1393/ß-1,4-GalT I, pVL1393/ß-1,4-GalT V and pVL1393/ß-1,4-GalT VI were also transfected independently into the cells as described previously (Sato et al., 1998a, 2000a; Sato and Furukawa, 1999
). The titers of the viruses to be used for infection to Sf-9 cells (9 x 106 cells/10 cm
) were determined with monitoring the enzyme activities of individual ß-1,4-GalTs.
Human colorectal adenocarcinoma cell line, SW480, was obtained from the Institute of Development, Aging and Cancer, Tohoku University, Sendai, and grown at 37°C in RPMI1640 containing 10% fetal calf serum, 50 units/ml penicillin, and 50 mg/ml streptomycin. Fragments containing a partial coding sequence of human ß-1,4-GalT II (5 to 500) and a partial coding sequence of human ß-1,4-GalT V (5 to 500) were subcloned in an antisense orientation into a mammalian expression vector, pcDNA3.1 (Invitrogen), and the expression vectors were named pcDNA3.1/GTII AS and pcDNA3.1/GTV AS, respectively. For transfection, 1 x 105 SW480 cells were seeded in tissue culture dishes with a diameter of 35 mm and cultured 37°C for 24 h in a CO2 incubator. Two micrograms of pcDNA3.1, pcDNA3.1/GTII AS, or pcDNA3.1/GTV AS formulated with a FuGENE reagent (Boehringer Mannheim, Mannheim, Germany) was added to the cells grown at the 6070% confluent stage and cultured for 72 h. The plasmid-transfected cells were selected with a medium containing geneticin (700 µg/ml G418 sulfate, Sigma) for 2 weeks.
Determination of ß-1,4-GalT activities
The harvested cells were washed with phosphate buffered saline (pH 7.2) three times, suspended in 100 mM 2-(N-morpholino)ethansulfonic acid (MES) buffer (pH 7.0) containing 1.25% Triton X-100, and then sonicated. The cell homogenates were used for transferase assays as an enzyme source. ß-1,4-GalT activities were determined by the method described previously (Sato et al., 1998b). In all instances the reaction mixture contained 100 mM MES buffer (pH 7.0) containing 4 mM 5'-AMP, 250 mM UDP-[3H]Gal, 1 mM appropriate acceptor, 20 mM MnCl2, and enzyme preparation in a total volume of 50 µl. After incubation the product was isolated by using a Sep-Pak C18 cartridge or an AG x1 column (Cl form), and radioactivity incorporated into the product was determined. To diminish or inhibit ß-N-acetylhexosaminidase activity possibly present in the cell homogenates (Kubelka et al., 1994
), pH of the assay mixture was adjusted to 7.0 rather than 6.0, at which maximal activities of ß-1,4-GalT (Furukawa and Roth, 1985
) and ß-N-acetylglucosaminidase were obtained (Altman et al., 1995
), and, in some assays, 2 mM N-acetylglucosamine was included in the reaction mixture (Bendiak and Schachter, 1987
).
Lectin blot analysis of membrane glycoproteins
Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells and gene-transfected cells was performed as described previously (Sato et al., 1993). Membrane glycoproteins were separated by 7.5% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to the method of Laemmli (1970)
, and separated proteins were electrophoretically transferred to polyvinylidenedifluoride membranes by the method described previously (Towbin et al., 1979
). Blotted membranes were blocked with 1% bovine serum albumin and incubated with peroxidase-conjugated RCA-I. In some experiments, blotted membrane blocked with bovine serum albumin was treated with 3.5 U N-glycanase in 150 µl of 0.1 M phosphate buffer (pH 8.2), 1.0 U of jackbean ß-N-acetylhexosaminidase in 150 µl of 0.3 M citrate-phosphate buffer (pH 5.0), or 40 mU of diplococcal ß-1,4-galactosidase in 150 µl of 0.3 M citrate-phosphate buffer (pH 6.0) per one lane at 37°C for 24 h before incubation with lectin.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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