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J. Biol. Chem., Vol. 275, Issue 51, 40498-40503, December 22, 2000

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Expression Cloning of Human Globoside Synthase cDNAs

IDENTIFICATION OF 3Gal-T3 AS UDP-N-ACETYLGALACTOSAMINE:GLOBOTRIAOSYLCERAMIDE 1,3-N-ACETYLGALACTOSAMINYLTRANSFERASE^*

Tetsuya Okajima, Yoko Nakamura, Makoto Uchikawa^§, David B. Haslam^¶, Shin-ichiro Numata, Keiko Furukawa, Takeshi Urano, and Koichi Furukawa^**

From the  Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Nagoya 466-0065, the ^§ Japanese Red Cross Central Blood Center, Hiroo 4-1-31, Shibuya-ku, Tokyo 150, Japan, and the ^¶ Department of Pediatrics, Child Health Research Center of Excellence in Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, August 1, 2000, and in revised form, September 13, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (1,3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported 3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually 1,3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation 3GalNAc-T1 for the cloned globoside synthase gene.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycosphingolipids are synthesized by the sequential action of glycosyltransferases starting from the glucosylation of ceramide (1). 1,4-Galactosyltransferase then synthesizes lactosylceramide (LacCer),¹ which is a common precursor structure of the majority of glycosphingolipids present in mammals and birds. Three major series of glycolipids are synthesized starting from LacCer by addition of 1,3-N-acetylglucosamine (lacto/neolacto-series), 2,3-sialic acid (ganglio-series), or 1,4-galactose (globo-series). Moreover, addition of N-acetylgalactosamine in a 1,4-linkage leads to the synthesis of asialo-ganglio series.

Globo-series glycolipids are ubiquitously present in human and many other mammalian tissues, whereas some tissues such as kidney, placenta, testis, erythroid cells, heart, and spleen express them at high levels. Recently, the key enzyme to initiate the synthesis of the globo-series glycolipid, Gb3/CD77 synthase (1,4-galactosyltransferase, 1,4Gal-T), gene has been cloned by us (2) and other groups (3, 4). The expression pattern of the gene also indicated that globo-series glycolipids may be more widely expressed than previously believed, suggesting the importance of structures containing the globo-series backbone.

Globoside is the most prominent neutral glycosphingolipid in human erythrocytes (5) and is an essential structure of blood group P antigen (6). Globoside is synthesized from globotriaosylceramide (Gb3, P^k antigen) by the action of 1,3-N-acetylgalactosaminyltransferase (1,3GalNAc-T). Therefore, P^k individuals lack 1,3GalNAc-T activity and accumulate the precursor P^k. On the other hand, p individuals lack Gb3/CD77 synthase activity with essentially intact 1,3GalNAc-T activity (7), and they lack the expression of both Gb3/CD77 and globoside.

Recently, a large number of glycosyltransferase genes responsible for the synthesis of glycoproteins and glycolipids have been isolated. Many of them could be classified into several families based on their similarities in primary structures, e.g. there have been 9 fucosyltransferase genes (8), 18 members of sialyltransferase genes (9), 7 4-galactosyltransferase genes (10), 5 3-galactosyltransferase genes (11), and 7 peptide N-acetylgalactosaminyltransferase genes (12) isolated to date. However, no glycosyltransferases responsible for 1,3GalNAc linkages have been isolated so far.

In this study, we have isolated cDNAs of 1,3GalNAc-T responsible for the synthesis of globoside using a eukaryocytic cell expression cloning system and taking advantage of the previously cloned Gb3/CD77 synthase. To our surprise, the cloned cDNAs of globoside synthase turned out to be identical with 1,3Gal-T3 (3Gal-T3) which was considered to be a galactosyltransferase responsible for the formation of Gal1,3GlcNAc-R structures, although no enzymatic activity was reported for the expressed cDNA (11). These results suggest that glycosyltransferases that seem to be members of a transferase family do not necessarily catalyze enzyme reactions with either the same sugar donor or the same acceptor structure. We propose here the name 3GalNAc-T1 for the cloned globoside synthase gene.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- UDP-GalNAc, LacCer, globotriaosylceramide (Gb3), and globoside (Gb4) were purchased from Sigma. GM3 and GD3 were purchased from Snow Brand Milk Products Co. (Tokyo, Japan). UDP-[³H]GalNAc was obtained from New England Nuclear. Culture supernatant of anti-Forssman glycolipid monoclonal antibody (mAb) M1/22.25.8.HL was prepared from a hybridoma line obtained from American Type Culture Collection. pBS-SVT, an expression vector for SV40 large T antigen, was obtained from the Japanese Cancer Research Resources Bank (Tsukuba, Japan). An expression vector of Forssman antigen synthase (FS) (13) was constructed by inserting HindIII/XhoI-digested fragment from pFS-35 (14) into pCDM8. An expression vector of Gb3 synthase pCDNA3.1/VTR-1 was prepared by inserting XhoI fragment from pVTR-1 (2) into the XhoI site of pCDNA3.1 (Invitrogen).

Cell Lines-- A mouse fibroblast L cell was kindly provided by Dr. A. P. Albino (Sloan-Kettering Cancer Center, New York) and was maintained in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum. A mouse fibroblast line, designated 1B9, used as a recipient cell in the transient expression system was prepared by co-transfection of L cell with pBS-SVT (SV40 large T Ag) and pCDNA3.1/VTR-1 (2). 1B9 was established from neo-resistant transfectant cells by screening the expression of Gb3 and SV40 large T Ag using rat anti-Gb3 mAb 38.13 (15) and mouse anti-SV40 large T Ag mAb Pab101 (Santa Cruz Biotechnology, Inc.), respectively. The expression of SV40 large T Ag and Gb3 was detected by an indirect immunofluorescence assay and flow cytometry, respectively. Stable transfectants were maintained in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum and G418 (300 µg/ml).

Expression Cloning of Human Globoside Synthase cDNA-- Plasmids of the human adult kidney cDNA library (Invitrogen) were transfected into 1B9 cells together with pCDM8/FS using DEAE-dextran as described (16). After 48 h, the transfected cells were detached by trypsinization and incubated with a rat mAb M1/22.25.8.HL on ice for 1 h. After washing, cells were plated on dishes coated with goat anti-rat IgM (ICN) as described (17). Plasmid DNA was rescued from the panned cells by preparing Hirt extracts and transformed into Escherichia coli XL-1 Blue (Stratagene). The same procedure was repeated four times. By using microscale transfection and immunofluorescence assays, cDNA clones that determined the Forssman glycolipid expression were isolated.

Sequencing Analysis-- The nucleotide sequence of the cloned cDNAs was determined by dideoxynucleotide termination sequencing using the PRISM dye terminator cycle sequencing kit and a model 310 DNA sequencer (Applied Biosystems). Amino acid sequence and hydropathy analyses were performed with Genetyx-Mac software, version 8.0 (Software Development, Tokyo). Genomic organization was determined by comparison between the cDNA sequence and the genomic one from the Human Genome Project.

Preparation of the Membrane Fraction-- L cells at 80% confluency were transfected with expression vectors using the DEAE-dextran method. After 80 h, the cells were collected and lysed in ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride using a nitrogen cavitation apparatus as described previously (18). Nuclei were removed by low speed centrifugation, and the supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. The pellet was resuspended in ice-cold 100 mM MES buffer (pH 6.5) and used as an enzyme source.

Enzyme Assay-- The N-acetylgalactosaminyltransferase assay was performed in a mixture containing 10 mM MnCl₂, 0.3% Triton X-100, 100 mM MES buffer (pH 6.5), 0.1 mM UDP-[³H]GalNAc (160 dpm/pmol), 200 µg of the membrane extracts, and 20 µg of substrates in a total volume of 50 µl. After incubating at 37 °C for 3 h, the reaction was terminated by the addition of 0.5 ml of water. The products were isolated with a C18 Sep-Pak cartridge (Waters, Milford, MA), spotted onto aluminum-backed silica gel-60 high performance TLC plates (Merck), and developed with a solvent system of chloroform/methanol/water (65:25:5). The plates were air-dried and sprayed with En³Hance^TM (PerkinElmer Life Sciences), and radiolabeled products were visualized by autofluorography.

Extraction of Glycolipids-- Glycolipids were isolated as described previously (19). Briefly, lipids were extracted from about 0.24 ml of packed volume of transfectant cells using chloroform/methanol (2:1, 1:1, 1:2) sequentially. After acetylation, the glycolipid fraction was isolated using a Florisil column. After deacetylation and desalting, the total glycolipids were dissolved in chloroform/methanol (2:1) and spotted on TLC plates for further analysis.

TLC Immunostaining-- TLC immunostaining was performed as described (19) according to the method of Taki et al. (20). In brief, the TLC plate was heat-blotted onto a polyvinylidene difluoride membrane after chromatography of the glycolipids. The membrane was incubated with human anti-Gb4 mAb 9H6 at a 1:100 dilution for 1 h, washed, and incubated with biotinylated goat anti-human IgM (Sigma) for 1 h. The antibody binding was revealed with ABC-PO^TM (Vector, Burlingame, CA) and HRP-1000^TM (Konica, Tokyo, Japan) according to the manufacturers' instructions.

Flow Cytometry Analysis-- 1B9 cells were transfected with expression vectors using the DEAE-dextran method. Two days later, cells were analyzed by flow cytometry with mAb M1/22.25.8.HL on a FACSCalibur with Cell Quest^TM version 3.1f software (Becton Dickinson) as described (21).

Northern Blotting-- Multiple Choice^TM Northern blot membrane (OriGene Technologies, Rockville, MD) with 2 µg of poly(A)⁺ RNA from human brain, colon, heart, kidney, liver, lung, muscle, placenta, small intestine, spleen, stomach, and testis was used. It was hybridized with a [³²P]dCTP-labeled cDNA probe of 1,3GalNAc-T-1 (nucleotides 108-710 in Fig. 2A) or of control actin probe according to the manufacturer's instructions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strategy of Expression Cloning of Globoside Synthase cDNA-- To prepare an appropriate recipient host cell line to isolate globoside synthase gene, L cells originally expressing LacCer were stably transfected with Gb3 synthase cDNA, pCDNA3.1/VTR-1, together with pBS-SVT containing SV40 large T antigen for extrachromosomal replication of the transfected plasmids. A transfectant line designated 1B9 contained abundant Gb3 and a negligible level of Gb4 and Forssman antigen (data not shown). Moreover, the nuclei of 1B9 line were strongly stained by anti-SV40 large T antigen antibody under fluorescence microscopy (data not shown). Thus, we could expect the expression of Forssman antigen after transfection of pCDM8/FS and Gb4 synthase cDNA which should have been contained in the human kidney cDNA library (Fig. 1, A and B). Because the extracted plasmids from panned cells were amplified in E. coli XL-1 Blue in the presence of ampicillin, only the plasmids from the library could be rescued.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Strategy of cDNA cloning of globoside synthase using anti-Forssman mAb. A, synthetic pathways of Gb4 and Forssman antigens. B, a scheme of expression cloning using 1B9. Recipient cell line 1B9 expressing SV40 large T Ag and Gb3 was transfected with human kidney cDNA library in pCDNA3.1 together with Forssman synthase (1,3GalNAc-T) cDNA. When globoside synthase (1,3GalNAc-T) and Forssman synthase cDNAs were introduced together into a single cell, Forssman glycolipid expression could be expected. C, flow cytometry of Forssman glycolipid expression on 1B9 cells after transfection. 1B9 cells were transiently transfected with pCDNA3.1 alone, pCDNA3.1/1,3GalNAc-T-1, pCDM8/FS, or pCDNA3.1/1,3GalNAc- T-1 together with pCDM8/FS. Cells were incubated with mAb M1/22.25.8.HL followed by staining with fluorescein isothiocyanate-conjugated goat anti-rat IgM (gray lines). Black lines represent the controls with the second antibody alone.

Isolation of cDNA Clones of Globoside Synthase Gene-- Following four rounds of enrichment by transfection of the cDNA library, panning with anti-Forssman mAb M1/22.25.8.HL, and rescue of plasmids by Hirt extraction, a pool of approximately 1000 bacterial colonies was identified to be positive in microscale immunofluorescence assays. These colonies were subdivided until three independent clones were identified to direct the expression of Forssman antigen when cotransfected with Forssman synthase cDNA into the 1B9 cell. Consequently, two clones of putative globoside synthase gene (designated type 1 and 2) with different 5'-untranslated regions were identified (Fig. 2B). These two clones contained alternatively spliced transcripts, i.e. type 1 transcript contained a single exon and type 2 consisted of five exons (Fig. 2B). All intron sequences at the exon-intron junctions conform to the GT-AG consensus (data not shown). Since the nucleotide sequence of the open reading frame was essentially same, type 1 clone was selected for further analysis and named 1,3GalNAc-T-1. As shown in Fig. 1C, only 1B9 cells cotransfected with 1,3GalNAc-T-1 and Forssman synthase gene expressed a definite amount of Forssman antigen, whereas those transfected with either 1,3GalNAcT-1 or Forssman synthase gene plasmids did not. These data indicated that 1,3GalNAc-T-1 is responsible for the expression of globoside.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   Nucleotide and deduced amino acid sequences of human 1,3GalNAc-T-1 and hydropathy plot of the protein. A, sequencing of type 1 of 1,3GalNAc-T-1 revealed an insert of 1897 base pairs in size encoding a single open reading frame in the sense orientation with respect to the cytomegalovirus promoter. The deduced amino acid sequence is shown below the nucleotide sequence. The putative transmembrane hydrophobic domain is underlined, and five potential N-linked glycosylation sites are boxed. Polyadenylation signal is also underlined. B, the nucleotide sequence of the 5'-untranslated region of type 2 transcript is shown. Exon-intron junctions are indicated. C, the hydropathy plot was calculated by the method of Kyte and Doolittle (23) with a window of 17 amino acids.

Amino Acid Sequence Analysis of 1,3GalNAc-T-1-- The open reading frame predicted a protein of 331 amino acids in length with a calculated molecular mass of 39,511. Unexpectedly, when this amino acids sequence was compared with other cDNAs in the data base, it was found to be identical to human 3GalT-3 reported by Amado et al. (11, 34). Although human 3GalT-3 was believed to belong to 3GalT gene family, no galactosyltransferase activity was reported. 1,3GalNAc-T-1 contained five potential N-linked glycosylation sites. The position of the AUG start codon was determined according to the Kozak consensus sequence (22). Hydropathy analysis (23) indicated one prominent hydrophobic segment of 23 residues in length in the amino-terminal region, predicting that the protein had the type II transmembrane topology characteristic of many other glycosyltransferases cloned to date.

A comparison between the 1,3GalNAc-T-1 isolated here and the previously characterized 3GalT proteins revealed that various sequence motifs in the putative catalytic domains were conserved (Fig. 3). In contrast to 3GnT, the four conserved cysteine residues that are considered to be essential for maintenance of the tertiary structures of 3GalTs are aligned with those of 1,3GalNAc-T-1 gene (Fig. 3).

View larger version (72K): [in this window] [in a new window]   Fig. 3.   Multiple amino acid sequence alignment of human 1,3GalNAc-T-1, 3GalTs and 3GnT. The sequences are from human 3GalT-1, 3GalT-2, 3GalT-4 (11), 3GalT-5 (36), 3GnT (41), and human 1,3GalNAc-T-1/3GalT-3 (this article). Identical residues are boxed in black. Gray boxes indicate conserved residues, and introduced gaps are shown as hyphens. Four cysteine residues highly conserved between six human genes are indicated by arrowheads.

N-Acetylgalactosaminyltransferase Activity of 1,3GalNAc-T-1-- To confirm the N-acetylgalactosaminyltransferase activity of 1,3GalNAc-T-1, L cells were transiently transfected with control pCDNA3.1 vector or pCDNA3.1/1,3GalNAc-T-1, and the membrane extracts were assayed for N-acetylgalactosaminyltransferase activity using UDP-[³H]GalNAc as a donor. The enzyme catalyzed the addition of [³H]GalNAc efficiently onto Gb3 (79 pmol/h/mg of protein) resulting in the synthesis of a new component with the same migration as standard Gb4, whereas LacCer, GM3, GD3, and Gb4 were not utilized as an acceptor (Fig. 4B), indicating that this enzyme is different from GA2/GM2/GD2 synthase or Forssman glycolipid synthase. No activity was detected in the extracts prepared from mock-transfected cells (Fig. 4A).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Thin layer chromatography of glycosphingolipids. A, N-acetylgalactosaminyltransferase activity of 1,3GalNAc-T-1 in vitro. The membrane extracts from L cells transfected with control pCDNA3.1 vector or with pCDNA3.1/1,3GalNAc-T-1 were incubated with or without Gb3 glycolipid acceptors. B, various glycosphingolipids were used as acceptors. The enzyme products were separated on a TLC plate with a solvent system of chloroform/methanol/water (65:25:5). The plate was sprayed with En3Hance, and radiolabeled products were visualized by autofluorography. The migration of the standard glycolipids LacCer, Gb3, and Gb4 are indicated on the left.

Synthesis of Gb4 in the Transfectant Cells-- To investigate the expression of Gb4 by 1,3GalNAc-T-1 in vivo, glycolipids were extracted from 1B9 cells transfected with pCDNA3.1 or pCDNA3.1/1,3GalNAc-T-1 and then separated on TLC. As shown in Fig. 5A, 1B9 cells transfected with pCDNA3.1/1,3GalNAc-T-1 showed definite Gb4 bands in TLC, whereas the transfectant cells with pCDNA3.1 alone showed no Gb4 band. In order to confirm the neo-synthesis of Gb4, TLC-immunostaining was conducted using a human anti-P mAb² prepared from lymphoid cells from an individual with p phenotype. As shown in Fig. 5B, the glycosphingolipids extracted from the transfectant cells with 1,3GalNAc-T-1 clearly gave bands like the control Gb4 at the same migration site. None of the other neutral glycolipids were stained, confirming the specificity of the mAb. Thus, the product was confirmed to be Gb4.

View larger version (48K): [in this window] [in a new window]   Fig. 5.   TLC analysis of glycosphingolipids isolated from transfected cells. Glycolipids were extracted from 240 µl of 1B9 cells transfected with pCDNA3.1 or pCDNA3.1/1,3GalNAc-T-1 as described under "Experimental Procedures" and then separated on TLC. A, orcinol (left) or primulin spray (right) was performed to detect the bands. Lane 1, neutral glycolipids extracted from human B red blood cells; lane 2, Gb4 (2 µg); lanes 3 and 5, extracts from 1B9 transfectant with pCDNA3.1 alone (derived from 50 µl of cells); lanes 4 and 6, extracts from 1B9 transfectants with pCDNA3.1/1,3GalNAc-T-1 (50 µl of cells). B, TLC immunostaining of globoside. TLC was prepared as in A, and glycolipids were blotted and then stained by mAb 9H6 as described under "Experimental Procedures." Lane 1, neutral glycolipids extracted from human B red blood cells; lane 2, Gb4 (0.5 µg); lane 3, LacCer (0.5 µg); lane 4, Gb3 (0.5 µg); lane 5, extracts from 1B9 transfectant cells with pCDNA3.1 alone (derived from 5 µl of cells); lane 6, extracts from 1B9 transfectants with pCDNA3.1/1,3GalNAc-T-1 (5 µl of cells).

Expression of the 1,3GalNAc-T-1 Gene-- To determine the expression pattern of the 1,3GalNAc-T-1 mRNA, Northern blotting was performed. Among 12 tissues examined, strong gene expression was observed in brain and heart as reported previously, and moderate expression was detected in lung, placenta, and testis, and low level expression was observed in kidney, liver, spleen, and stomach (Fig. 6).

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Expression pattern of the 1,3GalNAc-T-1 gene in various human tissues. Northern blots with 2 µg of poly(A)+ RNA from 12 adult human tissues were probed with 32P-labeled 1,3GalNAc-T-1 cDNA as described under "Experimental Procedures." The same filters were probed with the -actin cDNA after removing the radioactivity. The sizes of the markers are indicated at the left, and those of bands are at the right.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Globoside was defined as a major sugar-contained lipid of human blood stroma that formed perfectly round globules (spherocrystals) as viewed under microscope, and its name was chosen to reflect its property (24). The main glycolipid structure from hog erythrocyte stroma was also determined to be -N-acetylgalactosaminyl-(13)-galactosyl-(14)-galactosyl-(14)-glucosyl-ceramide, namely globoside (25). The synthetic pathway of globoside has been recognized in the studies of rare blood group types P^K and p (26). Since P and P^k structures were absent in the p individuals, and P structure was absent in P^k individuals; P (globoside) was considered to be synthesized from P^k (Gb3) independently from P1 antigen structure (27), although there were some ambiguous interpretations and remaining issues for the relationship between these structures (6, 26, 28). Enzymes responsible for the synthesis of globo-series glycolipids were studied by Kijimoto-Ochiai et al. (7), Hilderbrand and Hauser (30), and Ishibashi et al. (31). Their results demonstrated that cells from P^k individuals lacked 1,3-GalNAc-T activity, and those of p individuals were deficient in 1,4-Gal-T activity in accordance with the predicted synthetic pathway of these glycolipid structures.

Chien et al. (32) and Taniguchi and Makita (33) purified globoside synthase from embryonic chicken brain or canine spleen, respectively. These results showed good agreement with the nature of the enzyme expected from the isolated cDNA reported here, i.e. Mn²⁺ requirement, pH optimum at 6.9, and substrate specificity. The molecular masses they determined (64 and 57 kDa) differed from those predicted from the cloned cDNAs (~40 kDa); however, this discrepancy might be due to glycosylation modifications that would increase the predicted molecular mass or to a difference in species studied.

The goal of this study was to isolate 1,3GalNAc-T cDNA responsible for the synthesis of globoside from Gb3. Surprisingly, the cloned cDNA insert contained a primary structure similar to those of 1,3Gal-T gene family (3Gal-T family) and was identical to 3Gal-T3 (11). This 3Gal-T family now consists of 5 published members and several additional ones that have not yet been defined (34). Although 3Gal-T1, 3Gal-T2 (11, 35), 3Gal-T3 (11), and 3Gal-T5 (36, 37) have all been reported to catalyze the transfer of 1,3-galactosyl onto GlcNAc1,3-R residue, expressed human 3Gal-T3 showed no activity (11), and mouse 3Gal-T2 and 3Gal-T3 exhibited only 3-4% of the activity compared with that of mouse 3Gal-T1 (38). 3Gal-T4 was shown to be GM1/GD1b/GA1 synthase, i.e. the 1,3-galactosyltransferase responsible for GalNAc1,4-R (39). These findings suggest that the 3Gal-T family is heterogenous and contains glycosyltransferases that utilize a variety of sugar donors or acceptors. Our results also indicated that authors (11, 38) of the previous studies failed to determine the exact substrate specificity of the so-called "3Gal-T3" and how EST approach is misleading and how the authors of the previous two papers (11, 38) incorrectly interpreted the results.

Zhou et al. (37) found that 3Gal-T5, which was reported to be involved in the synthesis of sialyl-Lewis, an antigen in gastrointestinal and pancreatic epithelia and tumor cells derived therefrom, was a stage-specific embryonic antigen-3 (SSEA-3) synthase (i.e. a 1,3-galactosylgloboside synthase) (40). Although they did not exclude the existence of other 3Gal-T which could be responsible for the formation of SSEA-3, it seems clear that a member of 3Gal-T family certainly shows dual activity toward GlcNAc and GalNAc-based acceptors (40). Furthermore, Zhou et al. (41) cloned a -1,3-N-acetylglucosaminyltransferase (3Gn-T) capable of both initiating and elongating poly-N-acetyllactosamine chains based on the sequence similarity with mouse 3Gal-T1-3. This cDNA product exhibited inverted donor and acceptor specificities (1,3GlcNAc-transfer onto Gal1,4-R), whereas it shared the conserved sequence motifs among 3Gal-Ts except for the majority of conserved cysteine residues. Together with our results, these data indicate that the 3Gal-T family contains diverse glycosyltransferases that use various nucleotide sugars and acceptors, and this family might represent enzymes responsible for the catalysis of glycosidic 1,3-linkages.

Recently, a function of globoside as an initiator of signal transduction through AP1 and CREB associated with cell adhesion was reported (42). Although globoside has been considered to be an adhesion molecule on epithelial cells to various bacteria such as uropathogenic E. coli (43), and a receptor for pig edema disease toxin (44-46), its physiological function in vivo has never been elucidated. If signals transduced via globoside regulate transcription factors like AP1 and CREB, the control of the gene expression of globoside synthase would be very critical in the development and differentiation, and the availability of globoside synthase gene would strongly promote researches in these fields.

	ACKNOWLEDGEMENT

We thank Dr. K. O. Lloyd at Memorial Sloan-Kettering Cancer Center for carefully reading the manuscript.

	FOOTNOTES

^* This work was supported by Grants-in-aid for Scientific Research 10470029 and 12670111, for Priority Areas 10178104, 12215058, and 12204055, and for Center of Excellence 10CE2006 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB050855 and AB050856.

Research Fellow of the Japan Society for the Promotion of Science.

^** To whom correspondence should be addressed: Dept.of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi@med.nagoya-u.ac.jp.

Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M006902200

² M. Uchikawa, T. Toyoda, Y. Suzuki, K. Shinozaki, K. Nakajima, and T. Juji, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: LacCer, lactosylceramide; Gb3, globotriaosylceramide, Gal1,4Gal1,4Glc-Cer; Gb4, globoside, GalNAc1,3Gal1,4Gal1,4Glc-Cer; Forssman (antigen), GalNAc1,3GalNAc1,3Gal1,4Gal1,4Glc-Cer; Gb3/CD77 synthase, 1,4-galactosyltransferase, 1,4Gal-T; mAb, monoclonal antibody; Ag, antigen; FS, Forssman (glycolipid) synthase; MES, 4-morpholineethanesulfonic acid, the nomenclature of gangliosides is based on that of Svennerholm (29).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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