Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase.

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 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 beta3Gal-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 beta1, 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 beta3GalNAc-T1 for the cloned globoside synthase gene.

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 Nacetylgalactosaminyltransferase 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 Gal␤1,3GlcNAc-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.
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 g/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 ϫ 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 MnCl 2 , 0.3% Triton X-100, 100 mM MES buffer (pH 6.5), 0.1 mM UDP-[ 3 H]GalNAc (160 dpm/pmol), 200 g of the membrane extracts, and 20 g of substrates in a total volume of 50 l. 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 En 3 Hance (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 (Vector, Burlingame, CA) and HRP-1000 (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 FAC-SCalibur with Cell Quest version 3.1f software (Becton Dickinson) as described (21).
Northern Blotting-Multiple Choice Northern blot membrane (Ori-Gene Technologies, Rockville, MD) with 2 g of poly(A) ϩ RNA from human brain, colon, heart, kidney, liver, lung, muscle, placenta, small intestine, spleen, stomach, and testis was used. It was hybridized with a [ 32 P]dCTP-labeled cDNA probe of ␤1,3GalNAc-T-1 (nucleotides Ϫ108 -710 in Fig. 2A) or of control actin probe according to the manufacturer's instructions. 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.

Strategy of Expression Cloning of Globoside
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 5Ј-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 ␤1,3Gal-NAcT-1 or Forssman synthase gene plasmids did not. These data indicated that ␤1,3GalNAc-T-1 is responsible for the expression of globoside.
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 posi- tion 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).
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-[ 3 H]GalNAc as a donor. The enzyme catalyzed the addition of [ 3 H]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).
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 mAb 2 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.
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). 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 En 3 Hance, and radiolabeled products were visualized by autofluorography. The migration of the standard glycolipids LacCer, Gb3, and Gb4 are indicated on the left.

DISCUSSION
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-(133)-galactosyl-(134)-galactosyl-(134)-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 globoseries 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. Mn 2ϩ 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 (ϳ40 kDa); however, this discrepancy might be due to glycosylation modifications that would increase the predicted molecular mass or to a difference in species studied.
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-Nacetylglucosaminyltransferase (␤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 Gal␤1,4-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.