©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression Cloning of a Human G Synthase
G AND G ARE SYNTHESIZED BY A SINGLE ENZYME (*)

(Received for publication, August 16, 1995; and in revised form, November 6, 1995)

Jun Nakayama (1) Michiko N. Fukuda (1) Yoshio Hirabayashi (2) Akiko Kanamori (1) (2) Katsutoshi Sasaki (3) Tatsunari Nishi (3) Minoru Fukuda (1)(§)

From the  (1)Glycobiology Program, La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037, the (2)Frontier Research Program, the Institute of Physical and Chemical Research (RIKEN), Wako 351-01, Japan, and (3)Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Machida 194, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Gangliosides of the C series such as G are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of G synthase that adds the second alpha-2,8-sialic acid to G, NeuNAcalpha28NeuNAcalpha23Galbeta14GlcCer, thus forming G, NeuNAcalpha28NeuNAcalpha28NeuNAcalpha23Galbeta1 4GlcCer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes G synthase. The newly identified enzyme was therefore named G/G synthase (G/GST). G/GST synthesized G most efficiently when G, NeuNAcalpha23Galbeta14GlcCer, was incubated as an acceptor, indicating that G/GST is a polysialyltransferase that can transfer more than one sialic acid residue via alpha-2,8 linkage to gangliosides. Moreover, a longer period of incubation of G with G/GST produced a significant amount of G and higher polysialogangliosides. Among various cell lines expressing G/GST, higher polysialogangliosides including G were detected only in cell lines where the amount of G/G mRNA is sufficiently high. The expression of G/GST mRNA among human tissues is highly restricted to fetal and adult brains. The G/GST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, G/GST, that is specifically expressed in neural tissues.


INTRODUCTION

Glycoconjugates are major components of the plasma membrane of mammalian cells, and their carbohydrate structures change dramatically during development. Specific sets of carbohydrates are expressed in different stages of differentiation, and many of those carbohydrates are recognized by specific antibodies, thus providing differentiation antigens (Feizi, 1985; Fukuda, 1985). During the course of development, expression of distinct carbohydrates is eventually restricted to specific cell types, and aberrations in these cell surface carbohydrates are frequently observed in malignant cells (Hakomori, 1984). The functional significance of these cell type-specific carbohydrates and their alterations in malignancy is not well understood, although various reports suggest that some of these carbohydrates are involved in cell adhesion processes (Fukuda, 1992; Lowe, 1994).

Among glycosphingolipids, gangliosides comprise a structurally diverse set of sialylated species and are enriched in nervous tissues. Gangliosides have been found to act as receptors for growth factors, toxins, and viruses and are apparently involved in cell adhesion. For example, cholera toxin binds to G, (^1)Galbeta13GalNAcbeta14(NeuNAcalpha23)Galbeta14Glcbeta1Cer, before its entry into cells (Spiegel and Fishman, 1987). Influenza A virus binds to sialylparagloboside, NeuNAcalpha23Galbeta14GlcNAcbeta13Galbeta14Glcbeta1Cer (Higa et al., 1985; Suzuki et al., 1986). In addition, there have been reports suggesting that gangliosides, G in particular (see Fig. 1for its structure) may play roles in cell-cell interaction. Cheresh et al.(1986) found that G and G facilitate the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. Epithelial-mesenchymal interactions in embryonic kidney formation were perturbed by anti-G antibody, which reacted with G on the mesenchymal cells (Sariola et al., 1988). Gangliosides also modulate enzymatic activities. For example, G was found to inhibit epidermal growth factor receptor-mediated phosphorylation (Bremer et al., 1986), and G was shown to inhibit ADP-ribosyltransferases (Hara-Yokoyama et al., 1995).


Figure 1: Synthetic pathways of C series polysialogangliosides. alpha-2,8-sialic acid residues are shown in boldface type. The rest of the sialic acid residues are alpha-2,3-linked. In MeWo cells, the synthesis of G and G does not take place, since beta-1,4-N-acetylgalactosaminyltransferase is absent. G and STVI are newly proposed in the present study. STII and STIII were found to be the same enzyme in the present study, and STVI is probably the same enzyme as STII.



Among gangliosides, increasing attention has been directed to the so-called C series polysialogangliosides, which have unique trisialosyl residues, NeuNAcalpha28NeuNAcalpha28NeuNAcalpha23GalR (Fig. 1). C series polysialogangliosides were found to be major constituents in adult fish brain. In higher vertebrates the C series polysialogangliosides comprise a minor proportion of total gangliosides present in the brain (Ando and Yu, 1979). However, a substantial amount of C series polysialogangliosides are present in fetal brain of higher vertebrates including human. They are also found in various neuroectodermal tumors, such as melanoma and glioma (Yates, 1988; Nakayama et al., 1993). In the early stages of neural development, G is predominantly expressed in the neural tube that consists of progenitor cells for neurons and macroglial cells. During the later stage of development, progenitor cells migrate and extend processes and finally differentiate to postmitotic neurons. In this developmental period, G decreases, and C series polysialogangliosides, such as G, increase (Rösner et al., 1985).

It has been generally accepted that each glycosyltransferase involved in the synthesis of gangliosides transfers only one sugar residue to form a specific linkage (Pohlentz et al., 1988) (Fig. 1). Until recently, the studies of C series polysialogangliosides have been limited to their structural analysis, since the enzyme responsible for G synthesis (STIII) was not purified or cloned. In order to understand the roles and synthesis of C series polysialogangliosides, it is critical to isolate a cDNA clone of STIII that forms G.

In this report, we describe the cloning of cDNA encoding G synthase, STIII, using a mammalian expression cloning with a newly devised modification. Surprisingly, the newly isolated cDNA was found to be identical to that of G synthase, STII. By transfecting the newly isolated cDNA into HeLa and MeWo cells and assaying the activity of the soluble form of the enzyme, we demonstrated that a single enzyme encoded by the isolated cDNA forms both G and G. We also found that G synthase transcripts are expressed exclusively in neural tissues and that its gene is located at chromosome 12, region p12. These results, taken together, indicate that polysialogangliosides are synthesized by a single enzyme, G/G synthase, which is specifically expressed in neural tissues.


EXPERIMENTAL PROCEDURES

Antibodies

Monoclonal antibodies M6703 and M6704 were shown to react with G having NeuNAcalpha28NeuNAcalpha28 NeuNAcalpha23Galbeta1R structure of C series polysialogangliosides (Hirabayashi et al., 1988). Monoclonal antibodies R24 (Pukel et al., 1982) and KM641 (Ohta et al., 1993) were shown to react with G, NeuNAcalpha28NeuNAcalpha23Galbeta14GlcCer. R24 antibody was obtained from American Type Culture Collection, and KM641 antibody was kindly provided by Drs. Nobuo Hanai and Kenya Shitara at the Kyowa Hakko Kogyo, Japan.

Construction of Stably Transfected COS-1 Cells Expressing G

COS-1 cells were transfected with pAMo-GD3 (Sasaki et al., 1994b) and selected for G418 resistance. The transfected COS-1 cells expressing G were selected by immunofluorescent staining with anti-G antibody, R24, and one clone named COS-1bulletG cells was isolated.

Expression Cloning of a Human GST cDNA

A mammalian expression vector, pcDNAI-based cDNA library, pcDNAI-SK-MEL-28 constructed from poly(A) RNA isolated from human melanoma SK-MEL-28 cells, was purchased from Invitrogen (San Diego, CA). SK-MEL-28 cells express a significant amount of G (Dubois et al., 1986). COS-1bulletG cells (1.2 times 10^7) were transfected with 20 µg of pcDNAI-SK-MEL-28 using lipofectamine (Life Technologies, Inc.). After 62 h, the transfected cells were detached at 37 °C in Hanks'-based cell dissociation solution (Specialty Media, Lavallette, NJ). The detached cells were pooled and resuspended in cold phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin and were reacted with mouse monoclonal antibody M6703 at 1:200 dilution. After a 30-min incubation on ice, the cells were washed, and then fluorescein isothiocyanate (FITC)-conjugated (Fab`)(2) fragment of goat anti-mouse IgG (Cappel, Durham, NC) was added. After a 30-min incubation on ice, the cells were washed and subjected to fluorescence-activated cell sorting (FACS) using FACStar (Becton-Dickinson, San Jose, CA). The sorting region was set where only strongly positive COS-1 cells were recovered. Plasmid DNAs were rescued from the positive cells (Hirt, 1967) and transformed into the host Esherichia coli MC1061/p3 cells by electroporation using Cell-Porator (Life Technologies, Inc.). The transformed cells were placed into 20 plates, each containing about 500 colonies. Plasmid DNAs prepared from each plate were separately used for transfection by lipofectamine into HeLa cells, and the transfected HeLa cells were examined by immunofluorescent staining using M6703 antibody. Sibling selection with sequentially smaller active pools identified a single plasmid, pcDNAI-GST, that determined the expression of G at the cell surface.

Immunofluorescence Microscopy

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline and stained with mouse monoclonal antibodies R24 (anti-G), KM641 (anti-G), or M6703 (anti-G), followed by FITC-conjugated (Fab`)(2) fragment of goat anti-mouse IgG (Cappel). The cells were then examined under a Zeiss Axioplan microscope, as described previously (Williams and Fukuda, 1990).

Nucleotide Sequence Analysis

The cDNA insert of pcDNAI-GST was sequenced by the dideoxy nucleotide chain termination method (Sanger et al., 1977) using oligonucleotide primers, as described (Bierhuizen et al., 1993). The sequencing was initially carried out by using DyeDeoxy terminator cycle sequencing kit and DNA autosequencer (Applied Biosystems, Foster City, CA). The sequence was confirmed by using [S]dATP and a Sequenase sequencing kit (Amersham Corp.).

Construction of a Truncated Form of G/GST, G/GST-S

The cDNA encoding a truncated form of G/GST was prepared by polymerase chain reaction (PCR) using pcDNAI-GST as a template. Upstream and downstream primers used were 5`-cccaagcttGAGGGGCC-3` (HindIII site shown by underline) and 5`-atagtttagcgggcgcCCATTGTTCC-3` (NotI site shown by underline), respectively. The PCR product encompasses the sequence from nucleotide 38 to nucleotide 1,080. The nucleotide 38 is 8 nucleotides upstream from the second initiation methionine, and the nucleotide 1080 resides 9 nucleotides downstream from the stop codon. PCR was performed in a final volume of 100 µl using the primers (0.5 pmol each) for 30 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min. The amplified DNA fragment was digested with HindIII and NotI and cloned into the same sites of pcDNAI.

Establishment of Stable Transfectants Expressing G

Human cervical epitheloid carcinoma HeLa cells and human melanoma MeWo cells were cotransfected with pcDNAI-G/GST and pSV2neo (10:1) and selected by G418 resistance. The transfected cells expressing G were screened by immunofluorescent staining with M6704 antibody, and two clones, named HeLabulletG and MeWobulletG, were established.

Thin-layer Chromatography of Glycosphingolipids

Analytical thin-layer chromatography was carried out on precoated high performance thin-layer chromatography (HPTLC) plates (Si-HPF, J.T. Baker, Inc., Phillipsburg, NJ). The solvent systems used were chloroform, methanol, 14 mM MgCl(2) in water (5:4:1 by volume) for the first development and chloroform, methanol, water, 15 M NH(4)OH (50:40:8:2 by volume) for the second development. Gangliosides were visualized by resorcinol/HCl reagent.

Isolation of gangliosides from cells and TLC-immunostaining were performed as reported previously (Hirabayashi et al., 1988). The purified gangliosides were applied onto a plastic plate (Poligram Sil G, Nagel, Doren, Germany) and developed under the same conditions as described above. The plate was subjected to immunostaining with R24 or M6703 antibody, followed by peroxidase-conjugated goat anti-mouse IgG antibody (Cappel). The peroxidase activity was visualized with 4-chloro-1-naphthol/H(2)O(2).

In Vitro Sialyltransferase Assays and Product Characterization

The expression vector for protein A-G/GST fusion protein was constructed using pAMoA vector as described (Sasaki et al., 1994b). The cDNA in this pAMoA-GD3 was excised by SalI and Asp718, filled in by the Klenow fragment, and subcloned into the EcoRV site of pcDNAI, resulting in pcDNA-proA-G. After confirming the correct orientation by sequencing, pcDNAI-proA-G or pPROTA (Kukowska-Latallo et al., 1990) as a control was transfected into COS-1 cells. The protein A-G/GST fusion protein secreted into the culture medium was adsorbed to equilibrated IgG-Sepharose 6FF (Pharmacia Biotech Inc.) containing 0.05% Tween 20, washed nine times with 50 mM Tris-HCl buffer, pH 7.5, containing 1% bovine serum albumin and then two times with 20 mM Tris-HCl buffer, pH 7.5, containing 5 mM CaCl(2) and 0.05% Tween 20, and finally suspended in Dulbecco's modified Eagle medium containing 10% fetal calf serum, as detailed previously (Kukowska-Latallo et al., 1990; Bierhuizen and Fukuda, 1992).

Sialyltransferase activity was measured as described previously (Sasaki et al., 1994b). Briefly, after a 1-min sonication of 25 µl of 0.1 M sodium cacodylate buffer (pH 6.0) containing 20 mM MgCl(2), 1% Triton CF-54, 2.4 nmol of CMP-[^14C]NeuNAc, and 10 µg of a substrate with or without a competing substrate, 25 µl of the enzyme solution was added and incubated for 4, 12, or 24 h at 37 °C. At the end of the incubation period, 200 µl of phosphate-buffered saline was added to the incubation mixture, and the contents were applied to an Aspec Pak tC18 cartridge (M & S, Tokyo, Japan), according to the procedure described (Williams and McCluer, 1980). After washing the column with water, glycosphingolipids were eluted with 3 ml of chloroform-methanol (2:1 by volume). The sample was dried under nitrogen stream and then subjected to chromatography using an HPTLC plate under the same conditions as described above. Radioactive materials were visualized by fluorography after spraying an autoradiography enhancer (DuPont NEN). Standard and acceptor gangliosides were visualized by the resorcinol/HCl method.

Quantitation of G/GST Transcripts Using Competitive PCR

The level of G/GST transcript was measured by the competitive PCR using the cDNAs, which were prepared by reverse transcription of total RNA, as detailed in the previous report (Sasaki et al., 1994a). For distinction of a target cDNA from its competitor DNA, G/GST cDNA was truncated by deleting a 125-base pair EcoT22I-PvuII fragment of the cDNA from pUC-GD3R (Sasaki et al., 1994b). The 5` and 3` primers were 5`-ACAGTTACATCTACATGCCTGCCTT-3` and 5`-CATGAAACAACTTGACCATTCCCTC-3`, respectively. The amount of amplified cDNAs was calculated from the respective standard curves, converted into the values of molar numbers. As a control, the beta-actin transcript was measured in the same cDNA samples.

Northern Blot Analysis of Various Human Tissues

Poly(A) RNA from human fetal (19-23 gestational weeks) and adult brains purchased from Clontech (Palo Alto, CA) were electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a nylon filter (Micron Separation, Westboro, MA). Human multiple-tissue Northern blots of poly(A) RNA were purchased from Clontech, and these blots were hybridized with a gel-purified cDNA insert of pcDNAI-GST after labeling with [alpha-P]dCTP by random oligonucleotide priming (Feinberg and Vogelstein, 1983) (Prime-It II labeling kit, Stratagene, San Diego, CA).

Fluorescence in Situ Hybridization Analysis of GST Gene

Human genomic P1 plasmid library was screened by PCR as described (Onda and Fukuda, 1995). The 5` and 3` primers for PCR correspond to the sequence of the nucleotides 1184-1203 and that of nucleotides 1424-1443 of the GST sequence (see Sasaki et al., 1994b).

Purified DNA from one of the isolated P1 clones, clone 5459, was labeled with digoxigenin-dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate and 2 times SSC. Specific hybridization signals were detected by incubating the hybridized slides in FITC-labeled anti-digoxigenin antibody followed by counterstaining with propidium iodide for one color experiment. Probe detection for two-color experiments was accomplished by cohybridizing the slides with a biotin-labeled probe, D12Z1-specific for centromere of chromosome 12 and the digoxigenin-labeled clone 5459. After incubating these slides with Texas Red-labeled avidin and FITC-labeled anti-digoxigenin antibody, they were counterstained with 4`,6-diamidino-2-phenylindole (Rouquier et al., 1995).


RESULTS

Isolation of a cDNA Clone Encoding G Synthase

In order to clone G synthase, it was necessary to employ cells expressing a precursor ganglioside G but lacked G itself as recipients for transfection. The parent COS-1 cells did not react with M6703 (anti-G) or R24 (anti-G) monoclonal antibodies, indicating that G and G are not synthesized by COS-1 cells. Therefore, we transfected COS-1 cells with pAMo-G, which harbors cDNAs encoding the G synthase and G418 resistance gene (Sasaki et al., 1994b), and isolated COS-1bulletG cells that were strongly stained by R24.

When the COS-1bulletG cells were tested for the presence of G by M6703 antibody, however, 3.5% of COS-1bulletG cells showed a strong positive signal for G judged in FACS analysis. We thus isolated COS-1bulletG cells, which barely reacted with M6703 antibody by FACS. The freshly sorted COS-1bulletG cells expressed only G and were expanded once up to 1.2 times 10^7 cells in culture. Although a few of them still expressed G (Fig. 2C), they were used as recipient cells for expression cloning of G synthase.


Figure 2: Expression of polysialylated gangliosides by pcDNAI-GST. COS-1bulletG cells (A-D) and HeLa cells (E-J) were transfected with pcDNAI-GST (B, D, F, H, J) or with control pcDNAI vector (A, C, E, G, I). Sixty-two h after transfection, the cells were fixed and stained with monoclonal antibodies R24 (A, B, E, F), M6703 (C, D, I, J), and KM641 (G, H), followed by FITC-conjugated (Fab`)(2) fragment of goat anti-mouse IgG antibody. COS-1bulletG cells are positive for the M6703 antibody before transfection with pcDNAI-GST (C), while HeLa cells were negative for the M6703 antibody before transfection with the same vector (I). Bar, 50 µm.



COS-1bulletG cells were transfected with the SK-MEL-28 cDNA library in pcDNAI. Sixty-two h after transfection, COS-1bulletG cells expressing G were enriched by FACS using M6703 antibody under the conditions where only highly positive cells were selected. From 2.6 times 10^6 COS-1bulletG cells applied, 4,044 cells were sorted. Plasmid DNAs were rescued from these M6703-positive cells.

When COS-1bulletG cells were transiently transfected with a mixture of the above isolated plasmids, it was not possible to distinguish the cells that newly became G-positive from the cells that were endogenously G-positive by immunofluorescent staining with M6703 antibody. We reasoned that this failure was due to the high background expression of G in COS-1bulletG cells (see Fig. 2C). In order to overcome this problem, the plasmid DNAs were transfected into HeLa cells. The wild-type HeLa cells expressed detectable amounts of G as judged by immunofluorescent staining using another anti-G antibody, KM641 (Ohta et al., 1993) but were completely negative for G (Fig. 2, G and I).

The transformed bacteria obtained after the Hirt procedure were thus divided into 20 pools, and the plasmid DNA from each plate was transfected separately into HeLa cells. The transfectants were screened by immunofluorescent staining using antibody M6703. Because of no background staining for M6703 in HeLa cells, we could identify two out of 20 plasmid pools that directed the expression of G in HeLa cells. One of the plasmid pools, which produced strongly positive cell staining by M6703, was selected, and subsequent rounds of sibling selection with sequentially smaller, active pools identified a single plasmid, pcDNAI-GST, that directed the expression of G at the cell surface of HeLa cells.

COS-1bulletG and HeLa cells were transiently transfected with pcDNAI-GST, and the transfected cells were examined for the expression of G and G by immunofluorescent staining. Fig. 2, D and J, show that the expression of G, detected by M6703, was clearly seen on both COS-1bulletG and HeLa cells after the transfection. The expression of G was also notably increased in some of the transfected HeLa cells (Fig. 2, F and H).

Sequencing of the isolated cDNA in pcDNAI-GST revealed an insert of 1622 base pairs in size encoding a single open reading frame in the sense orientation with respect to the pcDNAI promoter. The open reading frame predicts a protein of 356 amino acids in length with a calculated molecular mass of 40,517. When this cDNA sequence was compared with other cDNAs in the data base, it was found to be identical to that encoding another alpha-2,8-sialyltransferase, G synthase (Haraguchi et al., 1994; Nara et al., 1994; Sasaki et al., 1994b) (Fig. 3A). The newly isolated sequence is, however, shorter in the 5`-flanking sequence and starts with 11 base pairs upstream from the initiation methionine (Fig. 3A). The 3`-flanking sequence just before poly(A) was shorter by 3 base pairs compared with the reported G synthase (Sasaki et al., 1994b). The deduced amino acid sequence predicts that this protein has a type II membrane topology, which has been found in all mammalian glycosyltransferases cloned (Schachter, 1994). These results indicate that G synthase (STII) and G synthase (STIII) are the same enzyme and suggests a possibility that a single enzyme catalyzes the reactions for the formation of disialosyl and trisialosyl groups. The newly identified enzyme is thus called G/G synthase or G/GST hereafter.


Figure 3: The nucleotide sequence of GST and schematic representation of G/GST protein and its derivatives. A, the nucleotide sequence of the 5`-terminal region of the cDNA insert of pcDNAI-GST is shown. The translated amino acid sequence is shown below the nucleotide sequence. The possible translation initiation methionines are at residues 1 and 16, and they are shown in boldface type. The putative transmembrane/anchor domain is underlined by a solid line. The translation product starting from the second methionine is underlined by a dotted line. The rest of the sequence is shown by Sasaki et al. (1994b). B, the schematic representation of two G/GST translation products and proA-G/GST chimeric protein are shown. G/GST and G/G-S start the translation in residues 1 and 16 shown in A, respectively. The cytoplasmic (Cyt), transmembrane/anchor (Tm), tentative stem (Stem), and catalytic (Cata) domains are shown. Sialylmotif L (residues 137-182) and S (residues 273-295) are shown by cross-hatched boxes. The signal peptide sequence (Sig) of the human granulocyte colony-stimulating factor and IgG-binding domain of S. aureus protein A (ProA) was fused with a catalytic domain of G/GST. The catalytic domain encompasses residue 57 (shown by the arrow in A) to the COOH terminus of the G/GST.



The above cDNA sequence shows a second initiation methionine, which resides 16 codons from the first initiation methionine. We synthesized the shorter cDNA encoding nucleotides 37-1,080 of the cDNA sequence by PCR, allowing the translation initiation only from the second initiation methionine (Fig. 3A). This truncated cDNA encoding G/GST-S (Fig. 3B) was cloned into pcDNAI and expressed in both COS-1bulletG and HeLa cells. The results obtained by immunofluorescent staining of the transfected cells clearly indicate that this truncated cDNA is also capable of G and G expression (data not shown). In fact, the nucleotide sequence surrounding the second initiation methionine, GCCATGG is consistent with the consensus sequence, (A/G)CCATGG for optimal translation initiation (Kozak, 1991). In contrast, the nucleotide sequence surrounding the first methionine GCGATGA does not conform with the Kozak sequence. Moreover, the size of the cytoplasmic sequence in this shorter translated product is reasonably short (12 residues), which is characteristic for all glycosyltransferases cloned to date (Schachter, 1994). These results suggest that actual initiation of translation starts probably from this downstream methionine coding for a protein of 341 amino acids with a molecular mass of 38,901.

Synthesis of G, G, and Possibly Higher Polysialogangliosides by G/G Synthase

In order to determine whether or not G/GST catalyzes the formation of G and G, HeLa and MeWo cells were transfected with pcDNAI-G/GST, and the resultant transfected cells, termed HeLabulletG and MeWobulletG, were analyzed for the synthesis of gangliosides. The results shown in Fig. 4, C and D, indicate that the HeLabulletG cells synthesized not only G but also G, although the expression of G was stronger. Similarly, the MeWobulletG cells synthesized a significant amount of G together with G (Fig. 4, G and H). Since the parent MeWo cells barely synthesize G even though a substantial amount of G is synthesized (Fig. 4, E and F), the expression of G in the MeWobulletG cells should be solely due to the newly introduced G/GST cDNA, but not the accumulation of newly synthesized G. On the other hand, the enhanced expression of G and the new synthesis of G in the HeLabulletG cells were due to the newly introduced G/GST cDNA.


Figure 4: Expression of polysialylated gangliosides in stably transfected HeLa and MeWo cells. A-H, the parent HeLa (A, B) and MeWo (E, F) cells and those stably transfected HeLabulletG (C, D) and MeWobulletG (G, H) cells were examined by immunofluorescent staining using R24 (anti-G) antibody (A, C, E, G) or M6703 (anti-G) antibody (B, D, F, H). Bar, 50 µm. I-K, gangliosides expressed in HeLabulletG and MeWobulletG cells were analyzed by TLC. Gangliosides were extracted from HeLa (lane 1), HeLabulletG (lane 2), MeWo (lane 3), and MeWobulletG (lane 4) cells and subjected to HPTLC as described under ``Experimental Procedures.'' Lane 5 represents ganglioside standards. The plate was then visualized by resorcinol/HCl (I), by R24 (anti-G) antibody (J), or by M6703 (anti-G) antibody (K) followed by a peroxidase-conjugated goat anti-mouse IgG. L, G/GST transcripts in various cells were analyzed by competitive PCR analysis. Single-stranded cDNAs reverse-transcribed from total RNA of a variety of cells labeled on the top or standard G/GST cDNAs (50, 500, 2,500, and 5,000 fg) were mixed with 500 fg of the competitor cDNA (truncated G/GST cDNA) and subjected to 23 cycles of PCR as described under ``Experimental Procedures.'' The amplified products were separated by electrophoresis in 1.8% agarose gel and visualized by staining with ethidium bromide. Size markers, from the top were 4.3, 1.8, 1.1, 0.68, 0.38, 0.25, and 0.12 kb.



In order to confirm that the transfected cells synthesize both G and G, gangliosides were isolated from the parent HeLa and MeWo cells and their stable transfectants. The thin-layer chromatogram of the gangliosides, detected by resorcinol reaction, which reacts with sialic acid, showed that the HeLabulletG cells contained both G and G, while the parent HeLa cells contained no G (Fig. 4I, lanes 1 and 2). Similarly, the MeWobulletG cells contained G, whereas the parent MeWo cells did not contain G (see lanes 3 and 4 in Fig. 4I). These results were confirmed by immunostaining of gangliosides after separation by thin-layer chromatography. The parent HeLa cells express a very small amount of G, but HeLabulletG cells express a substantially increased amount of G, detected by R24 antibody (Fig. 4J, lanes 1 and 2). The parent MeWo cells, on the other hand, express a significant amount of G (Fig. 4J, lane 3), but no G was detected by M6703 antibody (Fig. 4K, lane 3). In contrast, both HeLabulletG and MeWobulletG cells express a large amount of G (Fig. 4K, lanes 2 and 4). These results, taken together, clearly indicate that G/GST transfers an alpha-2,8-linked sialic acid to G and G, forming G and G, respectively. The immunofluorescent stainings of HeLabulletG and MeWobulletG cells by M6703 were completely abolished by pretreatment of chloroform-methanol (2:1) extraction (data not shown), indicating that all of the newly formed trisialosyl groups are attached to glycosphingolipids. If some of them were attached to glycoproteins, some staining should remain because M6703 also reacts with trisialosyl residues in glycoproteins (Nakayama et al., 1993).

In order to formally prove if G synthase also has G synthase activity, a putative catalytic domain of this protein was expressed as a protein fused with the IgG-binding domain of Staphylococcus aureus protein A preceded by a signal peptide sequence (Sasaki et al., 1993) (see ProA-G/GST in Fig. 3B). The cDNA encoding this chimeric protein was cloned into pcDNAI and expressed in COS-1 cells. The fusion protein secreted into the culture medium was absorbed to IgG-Sepharose and then incubated with G or G and the donor substrate CMP-[^14C]NeuNAc. As shown in Fig. 5, lane 3, the soluble form of G/GST synthesized both G and G when incubated with G. These results establish that the newly identified enzyme, G/GST, is a polysialyltransferase that adds more than one sialic acid residue in alpha-2,8-linkage.


Figure 5: In vitro sialyltransferase assay using the protein A-G/GST chimeric protein. The chimeric soluble G/GST was incubated for 4 h with CMP-[^14C]NeuNAc and 10 µg of G (lane 3) or 10 µg of G (lane 5), and the reaction products were subjected to HPTLC followed by fluorography. Lanes 4 and 6 represent the experiments that used a control vector, pPROTA, that lacks G/GST cDNA, and G (lane 4) or G (lane 6). The products are shown for those experiments after 12 h (lane 7) and 24 h (lane 8) incubation using G as the acceptor. Lane 9 represents the experiment under the same conditions as lane 8 except that pPROTA was used. Lane 10 shows the products when 10 µg of G was incubated for 4 h with CMP-[^14C]NeuNAc together with 50 µg of G, and lane 11 shows the products when 10 µg of G was incubated for 24 h with CMP-[^14C]NeuNAc together with 50 µg of G. Lanes 1 and 2 represent the G and G used as acceptors, and lane 12 is a mixture of standard gangliosides, detected by resorcinol/HCl.



The above experiments also suggested that G/GST added sialic acids much less efficiently when G was used as an acceptor (Fig. 5, lane 5). However, the enzyme added sialic acid residues to G (136 µM final concentration) after a longer period of incubation (Fig. 5, lanes 7 and 8). Under these conditions, G/GST also synthesized higher polysialogangliosides, which presumably have more than three sialic acid residues, as shown in lanes 7 and 8 of Fig. 5.

We also tested if the product or an intermediate inhibits the enzymatic reaction as competing substrates. The results shown in Fig. 5indicate that G (680 µM final concentration) inhibits the formation of G and G from G (Fig. 5, lane 10), while G (567 µM final concentration) inhibits the formation of G and higher polysialogangliosides from G (Fig. 5, lane 11). These results taken together support the above conclusion that G/GST synthesizes G from G and then G/GST utilizes G as an acceptor to form G.

The above results also suggested that the amount of G/GST mRNA transcript may be proportional to the amount of polysialylated gangliosides synthesized. In order to test this hypothesis, G/GST transcript was quantitated in the parent HeLa, HeLabulletG, parent MeWo, and MeWobulletG cells. Fig. 4L shows that the HeLabulletG cells express a significant amount of the G/GST transcript (330 fg), while the parent HeLa cells scarcely express it. The MeWobulletG cells express approximately the same amount (370 fg) of the transcript as the HeLabulletG cells and about 15 times more than that in the parent MeWo cells (23 fg). As shown in Fig. 4, I, J, and K, the parent MeWo cells express G but barely express G, while the MeWobulletG and HeLabulletG cells express both G and G. These results clearly indicate that G is synthesized only when G/GST is abundantly present.

G/GST Is Expressed in both Fetal and Adult Brains

To determine the tissue distribution of G/GST mRNA, Northern blots of poly(A) RNA derived from various human tissues were examined. As shown in Fig. 6, a band of 2.3 kb was detected in the poly(A) RNA isolated from the fetal and adult brains. The transcript was also detected in fetal lung. In adult tissues, the G/GST transcripts were detected in brain and very weakly in lung. Among different parts of the adult brain, a substantial amount of G/GST mRNA was detected invariably in different parts of brain (Fig. 6, right side). In some regions, a band of 9.5 kb was also detected. These two different sizes of the transcript might be produced due to the alternate usage of polyadenylation sites. The expression pattern of G/GST is different from that of the neural cell adhesion molecule (N-CAM)-specific polysialyltransferase (Nakayama et al., 1995), and G/GST expression is more restricted to brain.


Figure 6: Northern blot analysis of G/GST in various human tissues. Each lane contained 2 µg of poly(A) RNA. The blot for the first two lanes at the far left was made separately and apparently contained less poly(A) RNA than the other Northern blots. The same blots were probed by P-labeled G/GST cDNA (G/GST) or beta-actin cDNA (beta-actin).



G/GST Gene Is Mapped to Chromosome 12p12

The previous studies showed that the GST gene is present in chromosome 12, but no precise chromosomal location of this gene was reported (Sasaki et al., 1994b).

In order to localize precisely the G/GST gene, we utilized fluorescence in situ hybridization (FISH) procedures. First, P1 plasmid harboring G/GST gene, named clone 5459 was isolated, and genomic DNA was prepared from this P1 clone. Using this genomic DNA as a probe, the initial experiment resulted in specific labeling of the short arm of a group C chromosome. A second experiment was conducted in which a biotin-labeled probe (D12Z1) specific for the centromere of chromosome 12 was cohybridized with the digoxigenin-labeled clone 5459. This experiment resulted in the specific labeling of the centromere of chromosome 12 in red and the short arm of the same chromosome in green (Fig. 7A). Measurements of 10 specifically hybridized chromosome 12 demonstrated that the clone 5459 is located at a position that is 43% of the distance from the centromere to the telomere of short arm, an area that corresponds to 12p12 (Fig. 7B). This location is close to that of the c-Ki-ras protooncogene (Muleris et al., 1993) and human islet amyloid polypeptide gene (Christmanson et al., 1990).


Figure 7: Chromosomal localization of G/GST gene as revealed by FISH. A, two-color FISH analysis on metaphase chromosomes using a digoxigenin-labeled P1 clone 5439 encoding GST (stained as green signal) and biotin-labeled D12Z1, a specific probe for the centromere of chromosome 12 (stained as red signal). A discrete signal is discernible on all chromosome 12 chromatids for each probe. DNA was counterstained with 4`,6-diamidino-2-phenylindole. B, the specific hybridization occurs on chromosome 12, region p12.




DISCUSSION

The present study describes the isolation of a cDNA clone encoding G synthase, the key enzyme responsible for C series polysialogangliosides using expression cloning with a newly devised modification. We utilized COS-1 cells as recipient cells for the enrichment of plasmids that directed the expression of G. Since the large T antigen is synthesized in COS-1 cells, plasmids such as pcDNAI that contain the replication origin of SV40 are amplified in the cells. In order to test whether or not plasmids isolated from sorted COS-1bulletG cells could convert G to G, COS-1bulletG cells were not suited as recipient cells because of a substantial background of G expression. In contrast, HeLa cells do not express G but synthesize a small amount of G. Therefore HeLa cells should be able to synthesize G once a cDNA encoding G synthase is introduced. By using HeLa cells, we thus could identify a pool of plasmids that directed G expression. This is the first report where recipient cells for enriching plasmids differ from cells used for testing the enriched plasmids that direct the expression of a desired gene.

It was generally accepted that G synthase (STII) produced the first disialosyl linkage, forming NeuNAcalpha28NeuNAcalpha23Galbeta14GlcCer, and then another enzyme, G synthase (STIII), added one more sialic acid, forming NeuNAcalpha2 8NeuNAcalpha28NeuNAcalpha23Galbeta14GlcCer (Fig. 1). The present study, however, demonstrates that the same enzyme can form disialosyl and trisialosyl residues.

It was also noted that the stable transfectants of both HeLa and MeWo cells acquired gangliosides larger than G (see the arrowhead in Fig. 4K). Since MeWo cells lack beta-1,4-N-acetylgalactosaminyltransferase (Yamashiro et al., 1995), G, or G can not be formed in MeWobulletG cells (see Fig. 1for the structure of G and G). In addition, M6703 recognizes not only trisialosyl residues but also tetrasialosyl residues. (^2)When G was incubated with the soluble G/GST, slow migrating gangliosides were also produced in addition to G (Fig. 5, lanes 7 and 8). Since this assay was performed in the absence of other enzymes, these higher gangliosides are most likely polysialogangliosides having more than three sialic acid residues. The results, taken together, strongly suggest that the slow migrating band may represent G shown in Fig. 1. It is possible that G is present as a very minor component so that it has escaped attention. Further studies are necessary to confirm the presence of this glycosphingolipid.

The present study indicates that the same enzyme is apparently capable of adding all of the alpha-2,8-linked sialic acid, forming disialosyl, trisialosyl, and possibly tetrasialosyl residues in gangliosides. This finding is very similar to those reported for polysialylation of N-CAM. We and others have recently cloned a polysialyltransferase, which is responsible for polysialylation of N-CAM (Eckhardt et al., 1995; Nakayama et al., 1995). Although it has been suggested that the first disialosyl linkage is separately formed by an initiation enzyme (see Kitazume et al., 1994), the results obtained on mutant Chinese hamster ovary cells lacking polysialylation strongly suggest that polysialyltransferase carries out all reactions that form alpha-2,8-linked sialic acid polymer in N-CAM (Eckhardt et al., 1995). Moreover, it has been demonstrated recently that polysialyltransferase can add all alpha-2,8-linked sialic acid residues necessary for polysialic acid formation (Nakayama and Fukuda, 1996). These studies, taken together, strongly suggest that the polysialylation is catalyzed by single enzymes in both N-CAM glycoprotein (polysialyltransferase) and glycosphingolipids (G/GST).

The present study demonstrated that G/GST synthesizes G more efficiently from G than from G. It is tempting to speculate that G/GST first binds to G and then continuously adds sialic acid residues until the binding of the enzyme to the product is weakened. In fact, the excess amount of G inhibited the formation of G from G, confirming that G is an intermediate in the polysialylation. Once the enzyme is released from the enzyme-acceptor complex, it is likely that the enzyme has much less affinity with the product, which would be an acceptor for another reaction. Similarly, N-CAM-specific polysialyltransferase was shown to scarcely add a sialic acid on the polymerized sialic acid residues such as colomic acid (McCoy et al., 1985). These results suggest that termination of polysialylation takes place when the enzyme no longer binds to the polysialylated acceptor. It is apparent that G/GST terminates its reaction early, most likely due to its inefficiency in binding to trisialosyl or tetrasialosyl residues.

It was shown recently that Neuro2a cells exhibited better neurite extension after the cells were stably transfected to express G/GST (Kojima et al., 1994). Although these authors thought that this effect was due to the synthesis of G and B series gangliosides, the effect may be due to the synthesis of G and C series polysialogangliosides in the transfected Neuro2a cells. We have shown recently that neurite outgrowth in substratum cells is enhanced by the presence of polysialic acid in N-CAM (Nakayama et al., 1995). Previous studies showed that the presence of polysialic acid not only exhibits antiadhesive properties on homophilic N-CAM interaction but also influences cell-cell interactions carried by other cell surface receptors (Edelman, 1985; Rutishauser et al., 1988; Jessell et al., 1990). Further studies are thus needed to determine if the presence of polysialylated gangliosides influence the cell-cell interaction carried by other adhesive molecules.

Together with previously cloned polysialyltransferase cDNA that forms polysialic acid in glycoproteins, the cDNA cloned in the present study will be a powerful tool to dissect the intricate and complex roles of polysialic acids attached to glycoproteins and glycosphingolipids in cell-cell interactions during development.


FOOTNOTES

*
This work was supported by Grants RO1 CA33895 (to M. F.) and DK 37016 (to M. N. F.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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) L43494[GenBank].

§
To whom correspondence should be addressed: Tel.: 619-455-6480 (ext. 3689); Fax: 619-450-2101.

(^1)
The abbreviations for gangliosides are according to Svennerholm nomenclature (Svennerholm, 1964). The abbreviations used are: FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; PCR, polymerase chain reaction; HPTLC, high performance thin-layer chromatography; N-CAM, neural cell adhesion molecule; FISH, fluorescence in situ hybridization.

(^2)
Y. Hirabayashi, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Kenneth Lloyd for the kind gift of MeWo cells, Nobuo Hanai and Kenya Shitara for KM641 antibody, Joseph Trotter for carrying out the flow cytometry analysis, David Small for carrying out FISH analysis, and Yu Yamaguchi for critical reading of the manuscript. We also thank Susan Greaney for secretarial assistance and the members of the laboratories for useful discussion.


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