Molecular Cloning and Expression of Mouse GD1α/GT1aα/GQ1bα Synthase (ST6GalNAc VI) Gene*

A novel member of the mouse CMP-NeuAc:β-N-acetylgalactosaminide α2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of α2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1α, GQ1bα, and GT1aα. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of α-series gangliosides in the nervous tissues and many other tissues.

For the biosynthesis of sialyl compounds containing sialic acids in their carbohydrate moiety, a number of sialyltransferases should be present depending on the individual linkages and on the individual acceptor structures. Actually, more than 16 species of sialyltransferase genes utilizing glycoproteins and/or glycolipids as an acceptor have been cloned (2), i.e. 6 genes for ␣2,3Gal (ST3Gal), 1 gene for ␣2,6Gal (ST6Gal), 5 genes for ␣2,8Sia (ST8Sia), and 5 genes for ␣2,6GalNAc (ST6GalNAc). Many of these sialyltransferase genes were isolated by polymerase chain reaction based on similar sequences named sialyl motifs, which were first identified by Paulson and co-workers (3) in purified sialyltransferases, and in all sialyltransferases isolated thereafter (2).
Among gangliosides, ␣-series gangliosides that were defined as a new series of gangliosides containing NeuAc linked to the C6 position of GalNAc of the gangliotetraosyl backbone (4,5) have been considered to be a minor component (6). Compared with O-glycan, ␣2,6-sialylated GalNAc structures are rarely detected in glycosphingolipids, and little is known about their expression and significance. Recently, we have isolated a unique member of the ST6GalNAc family designated ST6GalNAc V expressed in brain tissues in a restricted manner. ST6GalNAc V encoded by this gene utilized exclusively GM1b 2 as an acceptor, resulting in the synthesis of GD1␣ but not of other ␣-series gangliosides such as GT1a␣ and GQ1b␣. Furthermore, ␣-series gangliosides have been reported to be expressed not only in brain but in lymphocytes (7), macrophages (8), mammary glands (9), and fibroblasts (10).
In the present study, we have isolated a novel member of the ST6GalNAc gene subfamily designated ST6GalNAc VI. This enzyme is specific for glycolipid acceptors and can synthesize all ␣-series gangliosides so far defined. The expression pattern of the gene is much broader than that of ST6GalNAc V and is distinct from that of other members of the ST6GalNAc subfamily.
Materials-CMP-NeuAc, LacCer, asialo-GM2 (GA2), GM2, GM1, GD1a, GD1b, GT1b, fetuin, asialofetuin, bovine submaxillary mucin (BSM), and bovine submaxillary asialomucin (asialo-BSM) were purchased from Sigma. GM3 and GD3 were purchased from Snow Brand Milk Products Co. (Tokyo, Japan). [␣-32 P]dCTP was from ICN (Costa * This work was supported by Grants-in-aid for Scientific Research on Priority Areas 10178104 and 11140228, Grant-in-aid for Scientific Research 10470029, and a grant-in-aid of Center of Excellence Research from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported by 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. This 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 /EBI Data Bank with accession number(s) B035123.
EST Data Base Search-A data base search was performed with the coding sequence of the ST6GalNAc IV gene (GenBank TM accession number Y15780) using tBLASTn algorithms against the EST data base (dbEST) at The National Center for Biotechnology Information (NCBI). A mouse EST clone (GenBank TM accession number AA790409) was found, and the sequence (nucleotide number 804-1121) was obtained by the reverse transcription polymerase chain reaction (PCR) using mouse brain cDNA as a template.
Cell Culture-Mouse fibroblast L cells and human melanoma SK-MEL-37 cells were grown in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 7.5% fetal calf serum at 37°C in a 5% CO 2 atmosphere. L cells were kindly provided by Dr. A. Albino at Memorial Sloan Kettering Cancer Center.
Construction of the Expression Vector-A cDNA fragment encoding the open reading frame of ST6GalNAc VI was prepared by PCR using a 5Ј primer, 5Ј-TGAGAGCGGCGCGGACCC-3Ј (nucleotides 1-18) and a 3Ј primer containing an XbaI site, 5Ј-TGGACTCTAGAACTAGGTC-CAGGAGGGGTG-3Ј (nucleotides 1121-1102) and mouse brain cDNA as a template. The PCR product was digested with XhoI and XbaI and introduced into these sites of pMIKneo vector (kindly provided by Dr. K. Maruyama at Tokyo Medical and Dental University). A truncated form of ST6GalNAc VI lacking 24 amino acids from the amino terminus was prepared by PCR using a 5Ј primer containing an EcoRI site, 5Ј-ATC-CTCGAATTCTACAGCTCCAACAGTGCC-3Ј (nucleotides 292-309), and a 3Ј primer containing a XhoI site, 5Ј-TGGACACTCGAGCTAG-GTCCAGGAGGGGTG-3Ј (nucleotides 1117-1098). The product was digested with EcoRI and XhoI and subcloned into these sites of pCDSA vector (kindly provided by Dr. Tsuji, RIKEN Institute, Wako, Japan).
Preparation of the Membrane Fraction-Mouse fibroblast L cells were grown in DMEM supplemented with 7.5% fetal calf serum. L cells at 80% confluency were transfected with expression vectors using the DEAE-dextran method (19). After 48 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 (20). Nuclei were removed by low speed centrifugation, and supernatant was centrifuged at 100,000 ϫ g for 1 h at 4°C. The pellet was resuspended in ice-cold 100 mM sodium cacodylate buffer (pH 6.0) and used as an enzyme source.
Preparation of Soluble Forms of ST6GalNAc VI-L cells were transfected with pCDSA-ST6GalNAc VI by the DEAE-dextran method and cultured for 16 h in DMEM containing 7.5% fetal calf serum. The medium was replaced with DMEM containing ITS TM culture supplement (Becton Dickinson, Bedford, MA), and the cells were cultured for another 32 h. The culture medium was then collected, concentrated 100-fold, and dialyzed against 100 mM sodium cacodylate buffer (pH 6.0) as described previously (21).
Sialyltransferase Assay-The sialyltransferase assay was performed in a mixture containing 10 mM MgCl 2 , 0.3% Triton CF-54, 100 mM sodium cacodylate buffer (pH 6.0), 0.66 mM CMP-NeuAc (Sigma), 4,400 dpm/l CMP-[ 14 C]NeuAc (Amersham Pharmacia Biotech), the enzyme solution, and substrates in a total volume of 50 l for glycolipid acceptors or 25 l for glycoproteins. The reaction mixture was incubated at 37°C for 1 h. For glycolipid acceptors, the reaction was terminated by the addition of 1 ml of water. The products were isolated with a C 18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by thin layer chromatography (TLC) with a solvent system of chloroform, methanol, 12 mM MgCl 2 (40:50:10). High performance TLC plates (Merck) were used. For glycoprotein acceptors, the reaction was terminated by the addition of 25 l of SDS-polyacrylamide gel electrophoresis loading buffer, and the mixtures were directly subjected to SDS-polyacrylamide gel electrophoresis. The radioactivity on each plate and gel was visualized with a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).
TLC-Immunostaining-TLC-immunostaining was performed as described (22) according to the method of Taki et al. (23). In brief, the TLC plate was heat-blotted onto a polyvinylidene difluoride membrane after chromatography of the glycolipids. The membrane was incubated with mouse anti-GD1␣ monoclonal antibody (mAb) KA-17 (24) at a 1:100 dilution or mouse anti-GQ1b␣ mAb GGR41 (25) at a 1:2 dilution for 90 min, washed, and incubated with biotinylated horse anti-mouse IgG for 1 h. The antibody binding was revealed with ABC-PO TM (Vector, Burlingame, CA) and HRP-1000 TM (Konica, Tokyo, Japan) as described previously (26).
Stable Transfection-SK-MEL-37 cells (10 6 cells/6-cm plate) were transfected with 5 g of pMIKneo-ST6GalNAc VI using 20 l of Super-Fect TM (Qiagen) according to the manufacturer's instructions. Approximately 24 h after the beginning of the transfection, the cells were trypsinized, and cell suspensions were transferred to 10-cm plates and cultured for 30 days with G418 (750 g/ml).
Flow Cytometric Analysis-The cell surface expression of GQ1b␣ was analyzed on a FACSCalibur with Cell Quest TM version 3.1f software (Becton Dickinson) using mouse anti-GQ1b␣ mAb GGR41 at a dilution of 1:2. The cells were incubated with mAbs for 45 min on ice and stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (ICN/ Cappel) at a 1:200 dilution. Control samples were prepared without mAbs.
Immunofluorescence Assay-SK-MEL-37 cells were transiently transfected with 5 g of pMIKneo-ST6GalNAc VI or pMIKneo-ST6GalNAc V using SuperFect TM as described above. After trypsinization, cells were resuspended in complete medium, transferred onto cover glasses, and incubated at 37°C for 24 h. The cells were fixed, with cold acetone for 10 min, air-dried, and processed for indirect immunofluorescence analysis as described (27).
Northern Blot Analysis-mRNA was isolated using mRNA isolation kit (Miltenyi Biotec, Bergisch, Germany) according to the manufacturer's instructions from C57BL/6 mouse tissues. Two g of poly(A) ϩ RNA was separated on 1.2% agarose, 2% formaldehyde gel, then transferred onto a GeneScreen Plus membrane (DuPont). After baking, the filter was prehybridized for 2 h at 42°C in a solution consisting of 5ϫ saline/sodium phosphate/EDTA, 50% formamide, 5ϫ Denhardt's solution, 1% SDS, and 10% dextran sulfate. Hybridization was carried out for 16 h at 42°C in the same solution containing 5 ϫ 10 5 dpm/ml of the 32 P-labeled probes. Alternatively, a mouse Multiple Choice TM Northern blot was obtained from OriGene Technologies Inc. (Rockville, MD) and hybridized according to the manufacturer's instructions. The filters were washed then exposed to the imaging plate to be analyzed in a FUJIX BIO-Imaging Analyzer BAS2000 (Fuji Photo Film).

RESULTS
Isolation of ST6GalNAc VI cDNA-To clone GT1a␣ and GQ1b␣ synthase, the NCBI data bank of EST cDNA clones was probed with the deduced amino acid sequence of the mouse ST6GalNAc IV. A mouse EST clone (GenBank TM accession number AA790409) was found, and the sequence (nucleotide number 804-1121 in Fig. 1A) was obtained by reverse transcription PCR using mouse brain cDNA as a template. The sequence was similar to but distinct from that of the previously cloned sialyltransferases, suggesting that the clone encoded a novel member of the sialyltransferase gene family.
To obtain the 5Ј-end, the cloning strategy of 5Ј-RACE was employed. The revealed sequence of the overlapping cDNA fragments indicated a single open reading frame of 999 base pairs coding for a protein of 333 amino acids containing three potential N-glycosylation sites (Fig. 1A). The deduced amino acid sequence corresponds to a 38,166-Da polypeptide. The position of the AUG start codon was determined according to the Kozak consensus sequence (18). Hydropathy (28) indicated one prominent hydrophobic segment of 21 residues in length in the amino-terminal region, predicting that the protein has type II transmembrane topology characteristic of many other glycosyltransferases cloned to date (Fig. 1B). The primary structure of the identified cDNA showed 29, 30, and 40% amino acid sequence identity to mouse ST6GalNAc III, ST6GalNAc IV, and ST6GalNAc V, respectively (Fig. 2). The newly cloned gene was designated ST6GalNAc VI based on results described below.
Sialyltransferase Activity of the Newly Cloned Enzyme-To analyze the sialyltransferase activity of ST6GalNAc VI, a fusion gene consisting of the IgM signal peptide sequence, the protein A IgG binding domain, and the putative active domain of ST6GalNAc VI (residue number 25-333) was constructed and transfected into L cells. In this system, the soluble enzyme (ProtA-ST6GalNAc VI) would be secreted. Using this soluble form of ProtA-ST6GalNAc VI as the enzyme source, we examined the sialyltransferase activity toward various glycolipids. As shown in Fig. 3, [ 14 C]NeuAc was incorporated into GM1b, GD1a, and GT1b commonly containing the NeuAc␣2,3Gal␤1,3GalNAc sequence at the nonreducing end. ProtA-ST6GalNAc VI exhibited little activity toward GA1 and GD1b and no activity toward GM1, indicating that a sialic acid linked to galactose at the nonreducing end by an ␣2,3 linkage was required for the substrate activity. Then we determined the acceptor specificity of the enzyme toward glycoproteins. As summarized in Table I, ProtA-ST6GalNAc VI showed minimal activity toward fetuin and no activity toward asialofetuin, BSM, and asialo-BSM. Similar results were obtained using the L cell extracts transfected with pMIKneo-ST6GalNAc VI (data not shown).
Characterization of the Enzyme Product-As shown in Fig. 3, the TLC mobilities of the enzyme products synthesized from GM1b and GD1a were identical to those of GD1␣ and GT1a␣, respectively. In addition, when GT1b was used as a substrate, the enzyme product was detected at a position slightly lower than authentic GQ1b, suggesting that the band is GQ1b␣.
To confirm the chemical structures of the synthesized prod-ucts, these products were used for TLC-immunostaining with KA-17 and GGR-41 specific for GD1␣ and GQ1b␣, respectively. The complete conversion of each substrate to a less migrating compound was confirmed by TLC followed by visualization with premulin reagent. As shown in Fig. 4A, GM1b sialylated with ST6GalNAc VI was stained with KA-17 (lane 4) at the same level as GD1␣ in the standard (lane 2). Moreover, GT1b sialylated with ST6GalNAc VI (lane 5) was detected using GGR-41 (Fig. 4B), indicating that this band is GQ1b␣. GD1a sialylated with ST6GalNAc VI (lane 3) as well as GT1a␣ in the standard (lane 1) were weakly stained with mAb GGR-41 as reported previously (24), indicating that they had an identical structure.
Determination of the Enzymatic Activity in Cell Culture-To explore the enzymatic activity of the ST6GalNAc VI gene in vivo, we transiently transfected a ST6GalNAc VI expression vector into SK-MEL-37 cells, which synthesize GT1b but not GQ1b␣ (data not shown). As shown in Fig. 5B, indirect immunofluorescence assays using mAb GGR-41 revealed that the overexpression of the ST6GalNAc VI cDNA in SK-MEL-37 cells resulted in the expression of a novel antigen localized in the cytoplasm and probably in Golgi apparatus. This staining was specific for GQ1b␣ (or possibly GT1a␣), since no significant immunofluorescence was observed in cells transfected with the vector alone (Fig. 5A). On the other hand, SK-MEL-37 cells transiently transfected with an expression vector of ST6GalNAc V (GD1␣ synthase) cDNA exhibited no immunofluorescence, confirming that the enzyme could not utilize GD1a nor GT1b as substrates (Fig. 5C).
To test the ability of the ST6GalNAc VI gene to determine GQ1b␣ expression on the cell surface, we attempted to establish stable transfectant lines of SK-MEL-37 cells. As shown in Fig. 5E, SK-MEL-37 cells stably transfected with ST6GalNAc VI (designated SK-MEL-37/ST6GalNAc-VI) gave a positive peak with mAb GGR-41. Then glycosphingolipids extracted from the stable line were analyzed by TLC. Because we could not visualize the newly synthesized gangliosides with resorcinol/HCl reagent, TLC-immunostaining was conducted using GQ1b␣ synthesized in vitro from GT1b as a control. As shown in Fig. 5F, the acidic glycolipids extracted from SK-MEL-37/ ST6GalNAc-VI clearly gave a band like the control GQ1b␣ at the same migration site, indicating that the newly synthesized gangliosides contained GQ1b␣.
Expression of the ST6GalNAc VI Gene-To determine the size of ST6GalNAc VI mRNA and its expression pattern, Northern blot analysis was conducted using the full-length cDNA as a probe. As shown in Fig. 6, a transcript of 2.5 kilobase pairs was detected in all the tissues examined. The gene expression is abundant in colon, brain, liver, and heart. An additional band at approximately 7.5 kilobase pairs was detected only in mouse colon.
We previously reported that the ST6GalNAc V (GD1␣ synthase) gene is expressed exclusively in the brain tissue and is involved in the synthesis of GD1␣ in the nervous tissues (15). To compare the transcription levels of ST6GalNAc VI and ST6GalNAc V in mouse brain, poly (A) ϩ RNA prepared from adult cerebrum, cerebellum, or 16-day postcoitum mouse embryo was analyzed by Northern blot hybridization. As shown in Fig. 7, both ST6GalNAc V and VI are expressed in cerebrum   3. Thin layer chromatography of sialylated glycosphingolipids in the enzyme assay. Glycolipids (10 nmol) were sialylated with ProtA-ST6GalNAc VI (5 l) for 1 h and purified by C 18 Sep-Pak cartridge, dried, and subjected to TLC with a solvent system of chloroform, methanol, 12 mM MgCl 2 (40:50:10). Under this assay condition, the enzymatic reactions for GM1b and GT1b were saturated. The plate was exposed to a BAS imaging plate and then analyzed with a BAS 2000 radioimage analyzer. and 16-day postcoitum mouse embryo. In contrast, only the ST6GalNAc VI transcript was found in cerebellum.

DISCUSSION
Among five ST6GalNAc cDNAs isolated to date, ST6GalNAc I and ST6GalNAc II were cloned as sialyltransferases that mainly utilize O-glycans as an acceptor. ST6GalNAc I prefers GalNAc-Ser/Thr, and ST6GalNAc II acts on Gal␤1,3GalNAc-Ser/Thr. ST6GalNAc III, ST6GalNAc IV, and ST6GalNAc V showed high homology in their primary structures (15), and a similar substrate specificity, i.e. a terminal sialic acid with an ␣2,3 linkage on galactose was essential as an acceptor structure. The amino acid sequence alignment of these six ST6GalNAc demonstrated that ST6GalNAc VI is closer to ST6GalNAc III, IV, and V in the primary structure (Fig. 2).
However, ST6GalNAc IV preferred O-glycans as acceptors (14), whereas ST6GalNAc III and V better utilize glycolipid acceptors. Therefore, the ST6GalNAc VI reported in this study is more similar to ST6GalNAc III and V in terms of the preferred substrate structure than ST6GalNAc I, II, and IV.
In the fine substrate specificities of ST6GalNAc III, V, and VI, ST6GalNAc III utilized both O-glycan and glycolipids as an acceptor when analyzed by cDNA of mouse (14) and rat (13). In contrast, ST6GalNAc V acts only toward a glycolipid acceptor (15). However, these two enzymes are very similar in terms of the fine substrate structure, i.e. they utilize only GM1b in glycolipid acceptors, resulting in the synthesis of GD1␣. ST6GalNAc VI is almost specific for glycolipid acceptors but is distinct from the other two enzymes in its unique substrate specificity. ST6GalNAc VI could catalyze the transfer of NeuAc by ␣2,6 linkage not only onto GalNAc in GM1b but that in GT1b and GD1a, resulting in the synthesis of GQ1b␣ and GT1a␣, respectively. Therefore, we have identified here for the first time a member of the ST6GalNAc subfamily that can synthesize GQ1b␣ and GT1a␣ as well as GD1␣.
Furthermore, the expression of ST6GalNAc VI was different from that of ST6GalNAc III or V. The ST6GalNAc III gene was expressed mainly in heart, lung, and brain (14). In contrast, ST6GalNAc V was almost specifically expressed in the brain as previously reported by us (15). ST6GalNAc VI was expressed very strongly in colon tissue, at moderate levels in heart, liver, brain, and spleen and weakly in all other tissues examined. Therefore, ST6GalNAc VI seems to be a widely expressed novel ST6GalNAc with a broad substrate specificity for glycolipid structures.
␣-Series gangliosides have been thought to be minor components. GD1␣ was reported as a minor ganglioside in bovine brain tissues (6) and as an accumulated structure in the proximal dendrites and cell bodies of Purkinje cells in murine cerebellum (24). On the other hand, GT1a␣ and GQ1b␣ were found in the rat brain and spinal cord in the nerve terminals of a certain population of cholinergic fibers (29) and in the dorsal and lateral horn of human thoracic cord (25,30). The distribution of GD1␣ and that of GT1a␣ and GQ1b␣ are not necessarily identical. ST6GalNAc VI may synthesize GD1␣ or GT1a␣ and GQ1b␣ depending on the alternative synthesis of asialo-series  4. TLC-immunostaining of the products of the enzyme assay. Glycolipids (5 g) were sialylated with ProtA-ST6GalNAc VI (10 l) for 6 h and purified by C 18 Sep-Pak cartridge, dried, and subjected to TLC with a solvent system of chloroform, methanol, 12 mM MgCl 2 (40:50:10). The complete conversion of each substrate to a less migrating compound was confirmed by TLC followed by visualization with 0.1% premulin reagent. A, TLC-immunostaining with mAb KA-17. Lane 1 contained acidic glycosphingolipids (2.5 g) extracted from bovine brain (BB) consisting of GM1, GD1a, GD1b, and GT1b as major components. Lane 2, standard GD1␣ (2.5 g); lane 4, GM1b sialylated with ST6GalNAc VI. As a control, the same reaction was performed without the enzyme (lane 3). B, TLC-immunostaining with mAb GGR-41. Lane 1, standard GT1a␣ (2.5 g); lane 3, GD1a sialylated with ST6GalNAc VI; lane 5, GT1b sialylated with ST6GalNAc VI. As a control, the same reaction was performed without the enzyme (lanes 2 and 4). Lane 6 was acidic glycosphingolipids (2.5 g) extracted from bovine brain (BB). Ori, origin. gangliosides or ganglio-series gangliosides, respectively. This should be determined by the expression level of GM3 synthase as described previously by our group (20). For the expression of GT1a␣ and GQ1b␣, ST6GalNAc VI should be responsible, since only ST6GalNAc VI can synthesize GT1a␣ and GQ1b␣ to our knowledge. However, for the synthesis of GD1␣, it is difficult to decide which enzyme is responsible in the individual tissues at this moment. There may be several tissue-specific ST6GalNAc FIG. 7. Differential expression of ST6GalNAc V and ST6GalNAc VI genes in mouse brain. Northern blots with 5 g of poly (A) ϩ RNA from mouse cerebrum, cerebellum, and 16-day postcoitum mouse embryo (E16) were probed with mouse ST6GalNAc VI full-length cDNA, ST6GalNAc V cDNA as described (15), and glyceraldehyde-phosphate dehydrogenase (GAPDH). The positions of ribosomal RNAs are indicated at the right.  (FL1-H), respectively. Thin lines represent anti-GQ1b␣ antibody, and solid lines represent controls with the second antibody alone. F, acidic glycosphingolipids were extracted from SK-MEL-37 cells stably transfected with pMIKneo vector or pMIKneo-ST6GalNAc VI and were subjected to TLC-immunostaining as described under "Experimental Procedures." BB, a ganglioside standard of bovine brain gangliosides (5 g); GQ1b␣, 5 g of GQ1b␣ synthesized in vitro from GT1b using ProtA-ST6GalNAc VI; BD/Mock, gangliosides from 125 l of mock-transfected SK-MEL-37 cells; BD/ST6GalNAc VI, gangliosides from 125 l of SK-MEL-37 cells transfected with pMIKneo-ST6GalNAc VI.
FIG. 6. Expression pattern of the ST6GalNAc VI gene in various mouse tissues. Northern blots with 2 g of poly (A) ϩ RNA from various adult mouse tissues were probed with 32 P-labeled mouse ST6GalNAc VI full-length cDNA as described under "Experimental Procedures." The same filters were probed with glyceraldehyde-phosphate dehydrogenase (GAPDH) cDNA after removing the radioactivity. The positions of ribosomal RNAs are indicated at the right. members capable of synthesizing GD1␣. In the expression analysis of the ST6GalNAc V gene, there was a controversial finding that the transcript could not be found in mouse cerebellum, whereas GD1␣ was strongly stained in Purkinje cells by Furuya et al. (24). As shown in Fig. 7, ST6GalNAc VI was definitely expressed in cerebellum, suggesting that this gene, not ST6GalNAc V, is responsible for the GD1␣ expression in mouse cerebellum.
␣-Series gangliosides have been reported as cholinergic nerve-specific molecules (Chol-1) (31,32) or binding molecules to myelin-associated glycoprotein in vitro (33). These studies indicate that ␣-series gangliosides play critical roles in the interaction and communication between neuronal cells and their supportive cells. Furthermore, the immunostaining pattern of the cDNA-transfectant cells indicated that GQ1b␣ is also localized in the cytoplasm and Golgi apparatus, suggesting that GQ1b␣ plays roles not only as a ligand for extracellular molecules but as a regulatory factor of intracellular events (34,35). The availability of the ST6GalNAc VI gene would enable us to clearly demonstrate the roles of ␣-series gangliosides in the neuronal development and functions.
Most studies on the ␣-series of gangliosides have been done with animal tissues or cells. The fact that no human studies have been reported to date suggests that these gangliosides are minor components in human tissues or that no rigorous investigation on the presence of ␣-series gangliosides in human has been tried, if in fact they exist. The use of ST6GalNAc VI should enable us to clarify whether ␣-series gangliosides are playing significant roles in human tissues.