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J Biol Chem, Vol. 275, Issue 20, 15152-15156, May 19, 2000

Molecular Cloning of Globotriaosylceramide/CD77 Synthase, a Glycosyltransferase That Initiates the Synthesis of Globo Series Glycosphingolipids^*

Yoshinao Kojima^§, Satoshi Fukumoto^§¶, Keiko Furukawa, Tetsuya Okajima, Joelle Wiels, Keiko Yokoyama^**, Yasuo Suzuki, Takeshi Urano, Michio Ohta^**, and Koichi Furukawa

From the  Department of Biochemistry II and ^** Department of Bacteriology, Nagoya University School of Medicine, Tsurumai, Nagoya 466-0065, ^¶ Department of Pediatric Dentistry, Nagasaki University School of Dentistry, Sakamoto, Nagasaki 852-8588,  Department of Biochemistry, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan,  CNRS UMR 1598, Institut Gustave Roussy, Villejuif Cedex, 94805 France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed 1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycosphingolipids are amphipathic molecules (1) that are synthesized by sequential actions of glycosyltransferases (2). The addition of one of three different sugars onto lactosylceramide (LacCer)¹ results in the synthesis of one of three major glycolipid series, i.e. ganglioside series (2,3-sialic acid), lacto/neolacto series (1,3-N-acetylglucosamine), and globo series (1,4-galactose). Although a number of genes coding for enzymes responsible for the synthesis of the carbohydrate moiety of glycosphingolipids have been recently isolated (3), no glycosyltransferase genes specific for the synthesis of globo series glycolipids have been isolated to date.

Globotriaosylceramide (Gb3) is synthesized by 1,4-galactosyltransferase (1,4Gal-T) from LacCer (4). This glycolipid has been characterized on red blood cells as the P^k antigen of the P blood group system (5). Since a monoclonal antibody (mAb) reactive with Burkitt's lymphoma-associated antigen was reported by Wiels et al. (6) and the recognized antigen was elucidated to be Gb3 (7), the expression and biological significance of Gb3 have been vigorously studied (8-10). Since Gb3 was clustered as CD77 (11), we refer to this antigen as Gb3/CD77.

Gb3/CD77 was reported to be expressed in high amounts on Burkitt's lymphoma cells. However, it is now considered to be a differentiation antigen expressed on B cells and can also be found in some malignant tumors of B cell lineage (8). Among normal leukocytes, it is only expressed on a subset of tonsillar B cells in the germinal centers (10). Interestingly, germinal center B lymphocytes expressing Gb3/CD77 undergo rapid and spontaneous apoptosis when isolated and cultured in vitro (12). Furthermore, Burkitt's lymphoma cells with Gb3/CD77 antigen were also easily induced to enter apoptosis upon culture at low serum concentration or cross-linking by anti-immunoglobulin M antibodies (13).

Gb3/CD77 has been recognized as a receptor for verotoxins (VTs) (14, 15), the Shiga-like toxin from the Escherichia coli O157 strain that can trigger serious cytotoxic effects (16). 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 (17). However, only the B subunit is also able to induce apoptosis when cross-linked (18). These results indicate that Gb3/CD77 is a critical molecule in mediating apoptosis signals, although the precise mechanisms remain to be investigated.

In this study, we have cloned the cDNA of the Gb3/CD77 synthase gene by a eukaryotic cell expression cloning. The isolated cDNA revealed that Gb3/CD77 synthase is a type II membrane protein with no significant homology with any other galactosyltransferase genes cloned so far. Transfection of the cloned cDNA into L cells could constitute a functional receptor for VTs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Library-- A cDNA library was prepared from poly(A⁺) RNA of a human melanoma cell line SK-MEL-37 as described (19). The library contained 5 × 10⁶ independent colonies. The strain of bacteria was MC1061/P3 (20).

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. Stable transfectants of L cells 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 Gb3/CD77 Synthase cDNA-- Plasmids of the cDNA library were transfected into L cells together with pdl3027 (polyoma T gene, provided by Dr. C. Basilico (New York University, New York)) using DEAE-dextran as described (20). After 48 h, the transfected cells were detached and incubated with a rat mAb 38.13 (6) on ice for 45 min. After washing, cells were plated on dishes coated with rabbit anti-rat IgM (Zymed Laboratories Inc.) as described (19). Plasmid DNA was rescued from the panned cells by preparing Hirt extracts and transformed into MC1061/P3. The same procedure was repeated five times. Using microscale transfection and immunofluorescence assay, cDNA clones that determined the CD77 expression were isolated.

DNA Sequencing-- The DNA sequence was determined by dideoxynucleotide termination sequencing using the PRISM dye terminator cycle sequencing kit and a model 310 DNA sequencer (Applied Biosystems).

Enzyme Assay-- The enzyme activity of 1,4Gal-T was measured as described previously (21). Briefly, membrane fractions were prepared as described (22). The reaction mixture for the assay contained the following in a volume of 50 µl: 50 mM sodium cacodylate-HCl (pH 6.0), 10 mM MgCl₂, 5 mM galactonolactone (Sigma), 0.3% Triton X-100 (Sigma), 0.4 mM LacCer, 2.9 mM phosphatidylglycerol (Sigma), 0.2 mM UDP-Gal (Sigma), UDP-[¹⁴C]Gal (2.5 × 10⁵ dpm) (NEN Life Science Products), and membrane fraction containing 50 µg of protein. The protein concentration was determined by the methods of Lowry et al. (23). The products was isolated by a C₁₈ Sep-Pak cartridge (Waters, Milford, MA) and analyzed by TLC and autoradiograph using a Bio-Imaging Analyzer BAS2000 (Fuji Film, Tokyo).

Northern Blotting-- Multiple Choice Northern blot membranes (OriGene Technologies, Rockville, MD) were used. They were hybridized with [³²P]dCTP-labeled cDNA probe of pVTR1 or control -actin as described (20).

Glycolipid Extraction and TLC-Immunostaining-- Glycolipids were extracted as described (24). Briefly, glycolipids were extracted from about 400 µl of packed cells using chloroform/methanol (2:1, 1:1, 1:2) sequentially. TLC was performed on a high performance TLC plates (MERCK, Darmstadt) using the solvent system chloroform, methanol, 0.22% CaCl₂ (60:35:8) and sprayed by orcinol. For standards, bovine brain ganglioside mixture (Wako, Tokyo), neutral glycolipids from human erythrocytes, and Gb3 (Sigma) were used.

The identity of Gb3/CD77 was confirmed by TLC-immunostaining using an aluminum-backed silica plate (Merck) as described (24). After TLC, the plate was blotted onto polyvinylidene difluoride membrane as described (25). After blocking, the plate was incubated with mAb, and then antibody binding was detected with the ABC kit (Vector Laboratories, Burlingame, CA) and Konica Immunostaining HRP-1000 (Konica, Tokyo).

Homology Search-- Nucleotide and amino acid sequence homology search was carried out using the Internet program BLAST (National Center for Biotechnology Information). Amino acid sequence and hydropathy analyses were performed with GENETYX-MAC software, version 8.0 (Software Development, Tokyo).

Flow Cytometry-- Cell Surface expression of CD77 was analyzed by flow cytometry (Becton Dickinson) as described (22). mAb 38.13 or TU-1 (26) was used with Fluorescein isothiocyanate-conjugated goat anti-rat IgG or anti-mouse IgM (Zymed Laboratories Inc.), respectively.

Stable Transfection-- To prepare stable transfectants, pVTR1 and pSV2neo were co-transfected into L cells using Lipofection kit (TOYOBO, Tokyo, Japan). To select transfectants, the cells were cultured in Dulbecco's modified Eagle's minimal essential medium containing fetal bovine serum (7.5%) and G418 (300 µg/ml). G418-resistant cells were cloned by limiting dilution. Clones transfected with pSV2neo alone were prepared for control.

MTT Assay-- To compare the reactions of L-VTR1 and L-neo to VTs, MTT assay was performed using cells prepared in 48-well plates (1 × 10⁴ cells/well) and cultured in the presence of VT1 or VT2. The assay was performed by triplicate samples. To quantify the cell proliferation, 50 µl of 5 mg/ml MTT (Sigma) in phosphate-buffered saline was added to each well. After incubation for 5 h at 37 °C, the supernatants were aspirated, and 100 µl of n-propyl alcohol containing 0.1% Nonidet P-40 and 4 mM HCl was added. The color reaction was quantitated using automatic plate reader IMMUNO-MINI NJ-2300 (Nihon InterMed, Tokyo, Japan) at 590 nm with a reference filter of 620 nm.

DNA Fragmentation Assay-- Cells were cultured in the presence of VT2 (200 ng/ml). After 24 h, cells were collected and the pellets were lysed in 100 µl of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.5% Triton X-100) for 10 min at 4 °C. After centrifugation, the supernatants were collected, and 2 µl of RNase (10 mg/ml) and 2 µl of Proteinase K (10 mg/ml) were added. After incubation for 1 h at 37 °C, the fragmented DNA was 2-propanol-precipitated. Electrophoresis was conducted using DNA derived from 1.5 × 10⁶ cells in a 2% agarose gel containing 0.2 µg/ml ethidium bromide in TEA buffer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of 1,4Gal-T cDNA That Determines Surface Expression of CD77-- L cells do not contain 1,4Gal-T activity and not express CD77, while they expressed abundant LacCer as described (22). These properties of L cells made them excellent host recipient cells for cDNA cloning of 1,4Gal-T. A cDNA library in pCDM8 constructed from mRNA of SK-MEL-37 was used for transfection, since SK-MEL-37 expressed a fairly high level of 1,4Gal-T, although it lacked surface expression of CD77. Finally, two clones were isolated that showed positive reactions in microscale immunofluorescence assays. Since the two clones were essentially same, one of them was selected for further analysis and named pVTR1.

Synthesis of CD77 in the Transfectant Cells-- As shown in Fig. 1C, L cells transfected with pVTR1 expressed a definite amount of CD77, whereas those transfected with the vector alone did not. Glycosphingolipids extracted from the transfectant cells showed definite Gb3 bands in TLC, although the transfectant cells with pCDM8 alone showed no Gb3 band (Fig. 2A), suggesting that the cloned pVTR1 was derived from the 1,4Gal-T gene. TLC-immunostaining revealed strong bands of Gb3 only in the extracts from the cDNA-transfected cells (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Strategy of cDNA cloning of 1,4Gal-T using anti-CD77 mAb. A, synthetic reaction of Gb3/CD77. B, a scheme of transfection into L cells. L cells with the LacCer+ phenotype were transfected with a cDNA library in pCDM8. L cell transfectants (Tr.) transiently expressing 1,4Gal-T express CD77. C, flow cytometry of CD77 expression on L cells after transfection of pCDM8 alone (left) or of pVTR1/CDM8 (right). Cells were incubated with mAb 38.13 followed by staining with fluorescein isothiocyanate-conjugated rabbit anti-rat IgG (thick lines). Thin lines represent the controls with the second antibody alone.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   TLC of glycolipids extracted from transiently- transfected cells. A, glycolipids were extracted from L cells transfected with pCDM8 alone (VC) or pVTR1/CDM8 (TF) and then separated in TLC. RBC, neutral glycolipids extracted from human B red blood cells. Solvent used was chloroform, methanol, 0.22% CaCl2 (60:35:8). Orcinol spray was performed to detect the bands. B, TLC-immunostaining of Gb3/CD77. TLC was prepared as in A and then blotted and stained by mAb 38.13 as described under "Experimental Procedures."

Sequence of the Insert of pVTR1-- Fig. 3 shows the entire sequence of the insert of pVTR1. The initiation codon is embedded within a sequence similar to the Kozak consensus initiation sequence (27, 28). This open reading frame predicts a 353-amino acid protein with a molecular mass of 40,498 daltons. No cDNAs or proteins in the data base showed significant homology to the product of VTR1. A single hydrophobic segment with 26 amino acids was present near the amino terminus (Fig. 3A). This putative signal anchor sequence would place 19 residues within the cytoplasm and 308 amino acids within the Golgi lumen. The presence of two potential N-glycosylation sites is indicated. A relatively high frequency of proline (10/31) was detected at the C'-side of the transmembrane domain.

View larger version (77K): [in this window] [in a new window]   Fig. 3.   Nucleotide sequence of cloned 1,4Gal-T pVTR1. A, the nucleotide and the deduced amino acid sequences of pVTR1 insert. The putative transmembrane region is double underlined. Potential N-glycosylation sites are marked by thick underlines. A polyadenylation signal is marked by a thin underline. B, hydropathy analysis according to Kyte and Doolittle (38).

Products of the 1,4Gal-T cDNA Can Transfer Galactose onto LacCer and Galactosylceramide-- L cells transfected with pVTR1/CDM8 showed high Gb3 synthase activity (7,012 units (pmol/h/mg of protein)) when LacCer was used as an acceptor. On the other hand, L cells transfected with pCDM8 alone were completely negative. Thus, this cDNA determined 1,4Gal-T activity and the surface expression of CD77, indicating that this cDNA encodes the Gb3/CD77 synthase. Enzyme activity toward other potential acceptors was also examined. None of the acceptors examined except LacCer and galactosylceramide showed significant levels of [¹⁴C]galactose incorporation (Fig. 4B). K_m values for these two substrates were 54.5 µM (LacCer) and 132 µM (galactosylceramide). The P1 antigen in the P blood group system is also formed by 1,4-galactose transfer (acting on paragloboside), but it was confirmed that this enzyme is not responsible for the synthesis of P1 antigen (Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   1,4Gal-T activity in the extracts from transient transfectants of pVTR1. A, enzyme activity using LacCer as an acceptor. Membrane fractions were prepared from L cells transfected with pCDM8 alone or with pVTR1/CDM8. Enzyme activity was determined using LacCer as described under "Experimental Procedures." As shown in the inset, a Gb3 band was observed in the TLC of the products. B, 1,4Gal-T activity for various acceptors. The conditions for the enzyme assay were as described for A. PG, paragloboside.

Northern Blot Analysis-- Expression levels of the 1,4Gal-T gene in various human tissues were examined by Northern blotting. Among tissues examined, heart, kidney, spleen, liver, testis, and placenta strongly expressed the gene (Fig. 5).

View larger version (66K): [in this window] [in a new window]   Fig. 5.   Northern blotting of 1,4Gal-T gene. A, hybridization with 32P-labeled probe derived from pVTR1 (upper) was performed as described (22) using membranes as described under "Experimental Procedures." The same membranes were probed with a -actin cDNA probe (lower) for control. B, relative expression levels of mRNA of 1,4Gal-T gene among human tissues measured by Bio-Imaging Analyzer BAS2000 (Fuji Film) are presented as a percentage of the value of heart after correction with the control.

High Sensitivity to VTs of the Transfectant Cells of pVTR1-- A stable transfectant of pVTR1 (L-VTR1) clearly demonstrated strong expression of Gb3/CD77, while L-neo expressed did not (Fig. 6A). The sensitivity of these two cell lines to VTs were compared using the MTT assay. L-VTR1 in VT (+) medium showed marked growth suppression compared with that cultured in the absence of VT, while L-neo showed no effects of VT (Fig. 6B).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Sensitivity of stable transfectant cells to VTs. A, flow cytometry of the stable transfectants. pSV2neo alone (L-neo, left) or cloned pVTR1 and pSV2neo (L-VTR1, right) were performed as described under "Experimental Procedures." Cells were incubated with mAb 38.13 followed by fluorescein isothiocyanate-conjugated rabbit anti-rat IgG (thin lines). Thick lines indicate the profile of the controls stained with the second antibody alone. B, MTT assay of L-neo and L-VTR1. L-neo and L-VTR1 cultured without VT2 () or with VT2 (+) (100 ng/ml) were served for MTT assay. The absorbances (590 nm) were measured on day 1-3 as described under "Experimental Procedures." C, dose effects of VTs on the cell proliferation. Absorbances on day 3 were plotted for L-neo and L-VTR1, which were exposed to various concentrations of VT1 or VT2 as indicated. The results on day 2 were essentially the same. D, fragmented DNA prepared from L-neo (lane 1) and L-VTR1 (lane 2) after cultivation with VT2 (200 ng/ml) for 24 h.

MTT Assay of L-VTR1 and L-neo after the exposure to various concentrations of VTs revealed marked growth suppression of L-VTR1 even at 0.01 ng/ml but not of L-neo (Fig. 6C). To confirm the mechanisms for the death of VT-treated L-VTR1, DNA fragmentation was examined. Agarose gel electrophoresis of cytoplasmic DNA extracted from L-VTR1 revealed a clear pattern of DNA fragmentation characteristic of apoptosis (Fig. 6D). In contrast, the L-neo sample did not show any ladder formation. Thus, it was confirmed that Gb3/CD77 generated by the cDNA serves as a functional receptor for VTs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Gb3/CD77 antigen is sometimes cryptic, depending on co-expressed sialosyl components on the cell surface (29). The SK-MEL-37 melanoma line, which was the source of the cDNA library used, was also negative for CD77 expression despite showing a definite 1,4Gal-T activity. Nevertheless, we could isolate cDNA clones of the Gb3/CD77 synthase gene from the cDNA library.

The cDNA clones obtained here are very valuable, because this enzyme initiates the synthesis of globo series glycolipids. Results of a homology search indicated that no significantly homologous cDNA has been reported to date. Although a number of members of 1,3-galactosyltransferases or 1,4Gal-Ts have been identified (30-33), this gene is the first and only 1,4Gal-T gene isolated so far. Moreover, no homologous genes to this gene were detected in the data base of Caenorhabditis elegans or Drosophila melanogaster genes, although many 1,4Gal-T- and 1,3-galactosyltransferase-related genes have been identified. These facts may indicate that the 1,4Gal-T gene evolved relatively later than other galactosyltransferase genes, and globo series glycolipids synthesized through Gb3 are playing more precise roles compared with glycolipids of the other series.

Gb3/CD77 seems to be unusual because it can mediate various apoptotic signals in both normal cells and malignant tumor cells, although it does not contain the cytoplasmic domain (18, 34). The observed rapid death of CD77⁺ germinal center B cells in vitro suggests that endogenous ligand molecules interact with Gb3/CD77 to bring about the physiologic selection of immature B cells (12, 35). Furthermore, the capability of the B subunit of VT to induce apoptosis of Gb3/CD77⁺ cells (18) strongly encourages the investigation of Gb3/CD77-associating cytoplasmic molecules (34). Investigations of these ligands and signal transducers relevant to Gb3/CD77 might contribute in further understanding of the B cell selection and of the pathogenesis of hemolytic uremic syndrome caused by E. coli O157 infection. In particular, the tissue specificity of the syndrome, such as renal failure, hemolysis, and neurological disorders, might be clarified by gene manipulation of the cloned Gb3/CD77 synthase.

Furthermore, it has recently been reported that Gb3/CD77 and ganglioside GM3 may function as alternative cofactors for the entry of human immunodeficiency virus type 1 in CD4-induced interactions between gp120 and glycosphingolipid microdomains (36, 37). If this is the case, Gb3/CD77 may be a receptor not only for bacterial toxins but for viruses, and the regulation of Gb3/CD77 expression could be a key target for the therapeutic approaches of viral infections such as human immunodeficiency virus type 1.

	ACKNOWLEDGEMENT

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

	FOOTNOTES

^* This work was supported by grants-in-aid for the Center of Excellence Research, Scientific Research and Scientific Research of Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and also a Research Grant on Human Genome and Gene Therapy from the Ministry of Health and Welfare 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 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) AB 037883.

^§ These authors contributed equally to this work.

To whom correspondence and reprint requests should be addressed. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi@med.nagoya-u.ac.jp.

Published, JBC Papers in Press, March 9, 2000, DOI 10.1074/jbc.M909620199

	ABBREVIATIONS

The abbreviations used are: LacCer, lactosylceramide; Gb3 or Gb3/CD77, globotriaosylceramide (Gal1,4Gal1,4Glc-Cer); 1, 4Gal-T, 1,4-galactosyltransferase; mAb, monoclonal antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Ganglioside nomenclature is based on that of Svennerholm (39).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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