Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids*

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

In this study, using human expressed sequence tags, we have cloned a novel ␣2,3-sialyltransferase, designated ST3Gal VI. ST3Gal VI is a novel Gal␤1,4GlcNAc ␣2,3-sialyltransferase with high specificity for neolactotetraosylceramide and neolactohexaosylceramide as glycolipid substrates. Moreover, ST3Gal VI prefers oligosaccharides containing the terminal Gal␤1, 4GlcNAc structure much more than those containing the * This work was supported by Grant-in-aid for Scientific Research in Priority Areas 10178105, by a Core of Excellence grant from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant-in-aid from Ono Medical Research Foundation. 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) AB022918.
Isolation of ST3Gal VI-Mouse expressed sequence tags (Gen-Bank TM accession nos. W52470, N40607, H06247, AA883549, and H22233) with similarity to mouse ST3Gal V (GM3 synthase) were amplified by the reverse transcription polymerase chain reaction (RT-PCR) method using total RNA prepared from a human melanoma cell line SK-MEL-37 as a template. The sense primer 5Ј-TTGGGAGAAG-GACAACCTTC-3Ј and the antisense primer 5Ј-CCAGGCAGCAACA-GACAGTA-3Ј were used for PCR amplification, which was carried out as follows; 94°C for 1 min, 25 cycles of (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min), and 72°C for 1 min. The RT-PCR-amplified 630-base pair cDNA was cloned into pCR ® 2.1-TOPO vector (Invitrogen, San Diego, CA). The DNA insert was 32 P-labeled with a Megaprime TM DNA labeling system (Amersham, Buckinghamshire, UK) and used to screen the SK-MEL-37 cDNA library. Approximately 4 ϫ 10 5 recombinant MC1061/P3 from a cDNA library prepared from human SK-MEL-37 cells were screened by colony hybridization. Colony lifts were prepared with GeneScreen Plus membrane (NEN Life Science Products). The nucleotide sequence was determined by the dideoxy termination method using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Construction of Expression Vector-A cDNA fragment encoding the open reading frame of ST3Gal VI was prepared by PCR using a 5Ј primer containing a XhoI site, 5Ј-CTCCTCGAGGGTGAGCCAGCCAT-GAGAGGG-3Ј, and a 3Ј primer containing a XbaI site, 5Ј-TCTTCTA-GATCAATCTTGAGTCAAGTTGAT-3Ј and the cloned cDNA fragment as a template. The PCR product was inserted into the XhoI and XbaI sites of pMIKneo vector (kindly provided by Dr. K. Maruyama at Tokyo Medical and Dental University). A truncated form of ST3Gal VI, lacking 34 amino acids from the N-terminus, was prepared by PCR using a 5Ј primer containing an EcoRI site, 5Ј-GAAGAATTCGGAATGAAACG-GAGAAATAAG-3Ј, and a 3Ј primer containing a XhoI site, 5Ј-CTCCTC-GAGTCAATCTTGAGTCAAGTTGAT-3Ј and the cloned cDNA fragment as a template. The product was digested with EcoRI and XhoI and subcloned into these sites of pCD-SA vector (kindly provided by Dr. Tsuji, RIKEN Institute, Wako, Japan).
Cell Culture-Mouse fibroblast L cells and various human cancer cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% fetal calf serum (FCS). Human leukemia cell lines were maintained in RPMI 1640 supplemented with 10% FCS at 37°C in a 5% CO 2 atmosphere.
Preparation of Membrane Fraction-L cells (3 ϫ 10 6 ) were plated in 10-cm dish at least 48 h prior to transfection. Cells were transiently transfected with an expression plasmid (4 g) by DEAE-dextran method (27). After 48 h of culture in DMEM containing 7.5% FCS, the cells were harvested by trypsinization. Cells were pelleted, washed with phosphate-buffered saline (PBS), and lysed in ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride using a nitrogen cavitation apparatus (Parr Instrument Co., Moline, IL) at 400 p. s. i. for 30 min as described by Thampoe et al. (28). 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.
Preparation of Soluble Forms of ST3Gal VI-L cells (10-cm dish) were transfected with pCDSA-hST3Gal VI (4 g) by the DEAE-dextran method and cultured for 16 h in DMEM containing 7.5% FCS. The medium was then replaced with serum-free insulin, transferrin, and selenous acid medium (Becton Dickinson, Bedford, MA) and the cells were cultured for another 32 h. At 48 h after transfection, the culture medium was collected and concentrated 100-fold using Molcut-L ® (Millipore, Tokyo, Japan) and dialyzed against 100 mM sodium cacodylate buffer, pH 6.0.
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 total volume of 50 l for glycolipid acceptors or 20 l for oligosaccharides and asialoglycoproteins. The reaction mixture was incubated at 37°C for 2 h. For glycolipid acceptors, the reaction was terminated by addition of 500 l of water. The products were isolated by C18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by thin layer chromatography (TLC). High performance thin layer chromatography (HPTLC) plates (E. Merck, Darmstadt, Germany) were used. For oligosaccharide substrates, the reaction was terminated by the addition of 20 l of methanol. Oligosaccharide products were separated by TLC with a solvent system of ethanol/pyridine/ n-butanol/acetate/water (100:10:10:3:30). For glycoprotein acceptors, the reaction was terminated by the addition of 20 l of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and the mixtures were directly subjected to SDS-PAGE. The radioactivity on each plate and gel was visualized with a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).
Linkage Analysis by Sialidase Digestion-Five g of neolactotetraosylceramide (nLc4) was sialylated with a soluble form of ST3Gal VI (ST3Gal VI-protA). Five g of GM3 was sialylated with GD3 synthase prepared form the L cells transfected with pMIKneo-ST8Sia I (29) using CMP-[ 14 C]NeuAc (8, 800 dpm/l). The products were purified by C18 Sep-Pak cartridge, dried, and redissolved in 25 l of 50 mM sodium citrate (pH 6.0) and 100 mM NaCl containing 100 g/ml bovine serum albumin. The resulting products were incubated for 2 h at 37°C after the addition of 0.85 unit Salmonella typhimurium LT2 sialidase (New England Biolabs, Beverly, MA). The digestion products were extracted by two volumes of chloroform/methanol (1:1), and the organic phase was collected by partition, dried, and subjected to HPTLC with a solvent system of chloroform/methanol/0.02% CaCl 2 (55:45:10). The plate was exposed to an imaging plate and then analyzed by BAS 2000 image analyzer.
TLC Immunostaining-Twenty g of nLc4 was sialylated with ST3Gal VI using CMP-[ 14 C]NeuAc (4,400 dpm/l) for 6 h, and purified by C18 Sep-Pak cartridge, dried, and subjected to TLC. TLC immunostaining was performed according to the method of Taki et al. (30). After chromatography of the glycolipids, TLC plate was heat-blotted to a polyvinylidene difluoride membrane. The membrane was incubated with monoclonal antibody (mAb) M2590 (7 g/ml) for 1 h, washed, and incubated with biotinylated horse anti-mouse IgM for 1 h. The antibody binding was revealed with ABC-PO (Vector, Burlingame, CA) and HRP-1000 (Konica, Tokyo, Japan) as described previously (31).
Northern Blotting-Total RNA was prepared from human cancer cell lines using TRIZOL ® reagent (Life Technologies, Inc.) according to the manufacturer's instruction. Total RNA (10 g) was separated on 1.2% agarose-formaldehyde gel, then transferred onto a GeneScreen Plus ® membrane. Human Multiple Tissue Northern Blot ® was purchased from CLONTECH. The blots were probed with a gel-purified, [␣-32 P]dCTP-labeled ST3Gal VI cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (26).
Flow Cytometry-Adherent cells were detached in PBS containing 0.5 mM EDTA and 1 mg/ml glucose. After washing with PBS, approximately 5 ϫ 10 5 cells were incubated with mAb 2H5 (CD15s; PharMingen, San Diego, CA) at a dilution of 1:500 (1 g/ml) for 30 min on ice. After washing twice, the cells were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgM ( chain specific) (Zymed Laboratories Inc.) at a 1:200 dilution. After a 30-min incubation on ice, cells were washed twice and subjected to analysis on a FACSCalibur with Cell Quest™ Version 3.1f software (Becton Dickinson). Thresholds for antigen positivity were set at a fluorescence intensity level that excludes 99% of the cells that had been stained without mAb 2H5.

Molecular
Cloning of a cDNA Encoding a Novel ␣2,3-Sialyltransferase-We previously cloned a cDNA encoding mouse ST3Gal V (GM3 synthase) using an expression cloning method. By searching the expressed sequence tag data base, we found sequences (GenBank TM accession nos. W52470, N40607, H06247, AA883549, and H22233) with similarity to mouse ST3Gal V, and obtained the corresponding cDNA fragment by RT-PCR (nucleotide numbers 728 -1375 in Fig. 1A). Approximately 4 ϫ 10 5 colonies of a human melanoma cell line SK-MEL-37 cDNA library were screened using the cDNA fragment as a probe, and nine independent clones (clones 1-9) were obtained. Characterization of the positive clones revealed that clone 2 contained a 1-kilobase pair (kb) insert, clone 3 was 1.2 kb, clone 5 was 1.6 kb, and clone 9 was 1.5 kb in length. From the nucleotide sequence, clone 9 was found to contain a whole open reading frame (Fig. 1A). The nucleotide sequence revealed that the cDNA contains an open reading frame encoding a protein of 331 amino acids with a calculated molecular mass of 38,213 daltons, with six potential N-linked glycosylation sites. The position of the AUG start codon was determined according to the Kozak consensus sequence (32), and the upstream region contained an in-frame stop codon. Hydropathy analysis determined by the Kyte and Doolittle method (33) indicated one prominent hydrophobic segment of 32 residues in length in the amino-terminal region (Gly 3 -Val 34 ), predicting that the protein has type II transmembrane topology characteristic of many other glycosyltransferases cloned to date (Fig. 1B). Comparison of the primary structure of ST3Gal VI protein and the 14 other cloned sialyltransferases indicated that there is significant similarity in two regions, so-called sialylmotifs (34,35). In addition, ST3Gal VI has a TXXXXYPE sequence near the Cterminal end of L-sialylmotif, which is found conserved among the members of ST3Gal subfamily (Fig. 2). These results indicated that this protein belongs to the sialyltransferase gene family and likely the ␣2,3-sialyltransferase subfamily. The predicted protein shows 38%, 34%, and 33% sequence identity to human ST3Gal IV, human ST3Gal III, and mouse ST3Gal V, respectively (Fig. 2). No significant homology in amino acid sequence was observed between the predicted protein and other known sialyltransferases.
Sialyltransferase Activity of the Newly Cloned Enzyme-To analyze the sialyltransferase activity of ST3Gal VI, the expression vector of the cDNA, pMIKneo-ST3Gal VI, was transfected into L cells, and the extracts of the transfected cells were assayed for sialyltransferase activity using CMP-[ 14 C]NeuAc as a donor and glycolipid mixture from bovine blood cells as acceptors. As shown in Fig. 3, the enzyme sialylated asialoglycolipids containing LacCer, nLc4, and nLc6 prepared from acidic glycosphingolipids of bovine red blood cells, and the products co-migrated with sialyl-nLc4 and sialyl-nLc6. In contrast, purified LacCer did not serve as an acceptor for ST3Gal VI. No activity was detected in the extracts from mock-transfected cells. Similar results were obtained using a soluble fusion enzyme ST3Gal VI-protA (data not shown).
Substrate Specificity of ST3Gal VI-To purify the enzyme, a fusion gene consisting of the IgM signal peptide sequence, the protein A IgG binding domain, and the putative active domain of ST3Gal VI (residue number 35-331) was constructed, and transfected into L cells. In this system, the soluble enzyme (ST3Gal VI-protA) would be secreted. Using this soluble form of ST3Gal VI-protA as the enzyme source, we examined the sialyltransferase activity toward various glycolipids. As shown in Sialyltransferase activity of the extracts of the L cells transfected with pMIKneo-ST3Gal VI was measured. Ten g of glycolipid acceptor was incubated with cell extracts containing 20 g of protein in the standard assay condition. BRBC indicated glycolipids containing LacCer, nLc4, and nLc6 prepared from neuraminidase-treated acidic glycosphingolipids extracted from bovine red blood cells. NC indicates that the assay was performed without an acceptor. The samples were subjected to HPTLC with a solvent system of chloroform/methanol/0.02% CaCl 2 (55:45:10). The plate was exposed to an imaging plate and then analyzed by BAS 2000 image analyzer. SA-nLc4, sialyl-neolactotetraosylceramide; SA-nLc6, sialyl-neolactohexaosylceramide.
Next, we examined the sialyltransferase activity toward various glycoproteins. As shown in Fig. 5 and summarized in Table  I, asialofetuin served as an acceptor. However, mucin from bovine submaxillary gland did not. No activity was detected in the extracts from mock-transfected cells.
Linkage Analysis by Sialidase Digestion-To determine the incorporated sialic acid linkage, nLc4 was labeled with CMP-[ 14 C]NeuAc using ST3Gal VI-protA and the product was subjected to digestion with Salmonella typhimurium LT2 sialidase which cleaves only the ␣2,3 linkage. As shown in Fig. 6, [ 14 C] NeuAc-labeled nLc4 was sensitive to digestion, while GD3 synthesized by ST8Sia I (GD3 synthase) was insensitive to the digestion. This result strongly suggested that the product of ST3Gal VI contained the sequence of NeuAc␣2,3Gal-, and that the enzyme is one of the ␤-galactoside ␣2,3-sialyltransferases.
Correlation of the Expression Levels of ST3Gal VI Gene with Sialyl-Le x Expression-In order to investigate whether ST3Gal VI was involved in sialyl-Le x expression, we examined the expression of ST3Gal VI gene along with sialyl-Le x expression levels in several human cancer cell lines. As shown in Fig. 9A, a flow cytometric analysis indicated that these cancer cell lines strongly expressed sialyl-Le x except for MOLT-3. Subsequently, expression of ST3Gal VI in these cells was analyzed by semiquantitative RT-PCR analysis. As shown in Fig. 9B, melanoma cell lines, especially SK-MEL-37 showed a high level of the STGal VI mRNA expression. Among colon cancer cell lines, Lovo and DLD-1 expressed sialyl-Le x on the cell surface, but the expression of ST3Gal VI gene was not observed. Thus, the expression levels of ST3Gal VI did not correlate well with sialyl-Le x expression among cell lines.

DISCUSSION
Since Weinstein et al. isolated a cDNA clone of ␤-galactoside ␣2,6-sialyltransferase in 1987 (37), a number of sialyltransferase genes have been cloned. These cloned sialyltransferases can be classified into four subfamilies based on the linkages they form, i.e. the ST3Gal-, ST6Gal-, ST6GalNAc-, and ST8Siasubfamilies. In a subfamily, some enzymes utilize certain acceptors with high efficiency, but use other acceptors with less efficiency. Therefore, substrate specificities of these enzymes frequently overlap, at least in in vitro analysis. It seems reasonable to think that an acceptor showing the strongest substrate activity in vitro is also the best acceptor in vivo. However, an acceptor that exhibits very weak activity for an enzyme in vitro can be a major acceptor in vivo depending on the expression levels of itself and another enzymes that share the same substrate (38).
To date, four human ␣2,3-sialyltransferase genes have been reported, and human ST3Gal V has recently been identified (39). In this study, we have isolated a novel ST3Gal cDNA that exhibited high homology in L-and S-sialylmotifs with previously cloned ST3Gal genes (Fig. 2). Except for these motifs, this gene did not show any significant homology with other sialyltransferase genes, and even with other ST3Gal genes. Compared with five human ␣2,3-sialyltransferases (ST3Gal) so far cloned, the cloned enzyme exhibit at most 40% homology, indicating that this gene encodes a novel sialyltransferase belonging to the ST3Gal subfamily. This gene is designated ST3Gal VI.
Although mouse cDNA clones of five ST3Gal (I-V) are also available (40), it seems important to restrict the enzyme sources to human when we discuss about the substrate specificity and the expression pattern of ST3Gal VI compared with those of the other ST3Gals. This is because substrate specificities of glycosyltransferases are sometimes quite different between different species. Human ST3Gal I and ST3Gal II utilize Gal␤1,3GalNAc as an acceptor and are thought to be mainly involved in the synthesis of O-glycan and ganglio-series gangliosides, respectively, based on the expression pattern of genes (17,19). ST3Gal III exhibit high activity onto Gal␤1,3GlcNAc on glycoproteins compared with Gal␤1,4GlcNAc and Gal␤1,3GalNAc (20). ST3Gal V utilizes almost exclusively lactosylceramide and forms GM3 (39). The reported substrate specificity of human ST3Gal IV is somewhat confusing. ST3Gal IV isolated from a melanoma library prepared from cells selected for lectin resistance (23) and that from human placenta cloned by PCR approach (22) exhibit quite different preferences of substrates. The former utilizes Gal␤1,4GlcNAc (type II) structure more efficiently than Gal␤1,3GlcNAc (type I), indicating that ST3Gal IV might be involved in the synthesis of sialyl-Le x , a ligand for the selectins (41). However, it seems unlikely that this enzyme synthesizes sialyl-paragloboside (SPG), a precursor of sialyl-Le x on ceramide, since the activity of this enzyme toward glycolipids was very low compared with the activity toward glycoproteins (40). From these facts, it is likely that an unknown ST3Gal gene specific for the synthesis of SPG exists.
ST3Gal VI, as reported in this study, utilizes almost exclusively Gal␤1,4GlcNAc on glycoproteins and glycolipids. Among glycolipid acceptors, ST3Gal VI acts only on nLc4 and nLc6, but not on Lc4, GA1, and LacCer. The efficiency of the sialyltransferase activity toward nLc4 was almost equivalent to those of ST3Gal III and ST3Gal II to type I chain (20) and type III chain (19), respectively. Furthermore, nLc6 exhibits much more incorporation of [ 14 C]NeuAc than nLc4, suggesting that ST3Gal VI preferred polylactosamine type II chains. Consequently, ST3Gal VI is capable of generating NeuAc␣2,3Gal␤1,4GlcNAc structures on glycoproteins and glycolipids including SPG as main products, and is probably involved in the biosynthesis of sialyl-Le x in some tissues.
Results of Northern blotting of ST3Gal VI gene shows predominant expression in placenta, liver, heart, and skeletal muscle. Among human cell lines, melanoma lines exhibited relatively high levels of the gene expression in accord with high expression of sialyl-Le x , indicating that ST3Gal VI might be involved in the synthesis of sialyl-Le x in melanomas, and probably in the synthesis of SPG in placenta (42). However, it seemed unclear whether this gene contributes in the up-regulation of sialyl-Le x synthesis in colon cancer and hematopoietic malignant cell lines. Actual involvement of this ST3Gal VI in the synthesis of sialyl type II structures in vivo remains to be analyzed.
Northern blots showed 1.8-kb major band and 3.0-kb minor band with almost parallel intensities. These bands were rather broad, suggesting the presence of heterogeneity in the size of mRNAs. The fact that cloned cDNAs exhibit various patterns of partial defects or insertions of sequences in the coding region is probably due to alternatively spliced exons (data not shown), explaining the observed heterogeneous mRNAs. Many of those aberrant clones seem non-functional and may have roles in the regulation of the enzyme activity in certain situations.