Molecular Cloning of a Novel alpha 2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids*

Tetsuya OkajimaDagger , Satoshi FukumotoDagger §, Hiroshi MiyazakiDagger , Hideharu Ishida, Makoto Kiso, Keiko FurukawaDagger , Takeshi UranoDagger , and Koichi FurukawaDagger parallel

From the Dagger  Department of Biochemistry, Nagoya University School of Medicine, Tsurumai, Nagoya 466-0065, the § Department of Pediatric Dentistry, Nagasaki University School of Dentistry, Sakamoto, Nagasaki 852-8501, and the  Department of Applied Bio-organic Chemistry, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel member of the human CMP-NeuAc:beta -galactoside alpha 2,3-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 alpha 2,3-sialyltransferase toward Galbeta 1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta 1,4GlcNAc 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Sialyltransferases are a family consisting of more than 14 enzymes that catalyze the transfer of sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc)1 to terminal positions on sugar chains of glycoproteins and glycolipids. Terminal NeuAc residues are key determinants of carbohydrate structures involved in a variety of biological processes and are widely distributed in many cell types (1-3). For example, sialyl-Lewis X (sialyl-Lex) determinants have been reported to be ligands for the three known selectins (E-, P-, and L-selectins), which are cell adhesion molecules involved in the recruitment of leukocytes to lymphoid tissues and the sites of inflammation (4-6). Furthermore, increased expression of sialyl-Lex determinants was suggested to contribute to the metastatic behavior of carcinoma cells (7). Glycosyltransferases involved in the synthesis of sialyl-Lex structures are beta 1,3-N-acetylglucosaminyltransferase (8), beta 1,4-galactosyltransferase (9, 10), alpha 1,3-fucosyltransferases (11-15), and alpha 2,3-sialyltransferases, as described below.

To date, four enzymatically distinct human alpha 2,3-sialyltransferase genes have been cloned and exhibited distinct acceptor substrate specificities. ST3Gal I (16, 17) and II (18, 19) synthesize the sequence NeuAcalpha 2,3Galbeta 1,3GalNAc common to many O-linked oligosaccharides and glycolipids like GM1b, GD1a, and GT1b. ST3Gal III (20, 21) forms a less common sequence, NeuAcalpha 2,3Galbeta 1,3GlcNAc. ST3Gal IV (22, 23) is capable of forming the terminal NeuAcalpha 2,3Galbeta 1,3GalNAc and the NeuAcalpha 2,3Galbeta 1,4GlcNAc sequence found in the carbohydrate moieties of glycoproteins and glycolipids. Recently, we have cloned mouse ST3Gal V (GM3 synthase),2 which exhibits activity almost exclusively toward LacCer. Among five alpha 2,3-sialyltransferases so far isolated, human ST3Gal III and IV are candidates for involvement in the formation of the sialyl-Lex determinant in vivo. However, human ST3Gal III has been shown to utilize Galbeta 1,3GlcNAc much more efficiently than Galbeta 1,4GlcNAc as an acceptor in vitro. The reported substrate specificities of ST3Gal IV have been contradictory for its preference between Galbeta 1,3GalNAc and Galbeta 1,4GlcNAc structure, and for utilization of glycolipids. Therefore, to date, no human alpha 2,3-sialyltransferase that shows high and clear acceptor specificity toward Galbeta 1,4GlcNAc sequence has yet been identified.

In this study, using human expressed sequence tags, we have cloned a novel alpha 2,3-sialyltransferase, designated ST3Gal VI. ST3Gal VI is a novel Galbeta 1,4GlcNAc alpha 2,3-sialyltransferase with high specificity for neolactotetraosylceramide and neolactohexaosylceramide as glycolipid substrates. Moreover, ST3Gal VI prefers oligosaccharides containing the terminal Galbeta 1,4GlcNAc structure much more than those containing the Galbeta 1,3GlcNAc structure, suggesting it is involved in the formation of the sialyl-Lex determinant on glycoproteins and glycolipids. The expression of the gene among normal human tissues and cell lines was also analyzed.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Nomenclature of Cloned Sialyltransferase-- So far five members of human alpha 2,3-sialyltransferase (ST3Gal) have been cloned: ST3Gal I (16), ST3Gal II (18), ST3Gal III (21), ST3Gal IV (22, 23), and ST3Gal V (GenBankTM accession no. AB013302). The alpha 2,3-sialyltransferase cloned in this study is referred to as ST3Gal VI according to Tsuji et al. (24).

Materials-- CMP-NeuAc, LacCer, GM2, GM1, GD1a, GalCer, GT1b, GQ1b, GA1, asialofetuin, and bovine submaxillary asialomucin, were purchased from Sigma. GM3 was purchased from Snow Brand Milk Products Co. (Tokyo, Japan). [alpha -32P]dCTP was from ICN (Costa Mesa, CA). Lex ceramide (Galbeta 1,4(Fucalpha 1,3)GlcNAcbeta 1,3Galbeta 1,4Glcbeta 1Cer) and lactotetraosylceramide (Lc4) were chemically synthesized as described previously (25). Sialyl-neolactotetraosylceramide (sialyl-nLc4) and sialyl-neolactohexaosylceramide (sialyl-nLc6) were prepared from bovine blood cells as described previously (26). The asialo compounds were prepared by digestion with neuraminidase from Vibrio cholerae (Sigma) as described previously (26).

Isolation of ST3Gal VI-- Mouse expressed sequence tags (GenBankTM 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 5'-TTGGGAGAAGGACAACCTTC-3' and the antisense primer 5'-CCAGGCAGCAACAGACAGTA-3' 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 32P-labeled with a MegaprimeTM DNA labeling system (Amersham, Buckinghamshire, UK) and used to screen the SK-MEL-37 cDNA library. Approximately 4 × 105 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 5' primer containing a XhoI site, 5'-CTCCTCGAGGGTGAGCCAGCCATGAGAGGG-3', and a 3' primer containing a XbaI site, 5'-TCTTCTAGATCAATCTTGAGTCAAGTTGAT-3' 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 5' primer containing an EcoRI site, 5'-GAAGAATTCGGAATGAAACGGAGAAATAAG-3', and a 3' primer containing a XhoI site, 5'-CTCCTCGAGTCAATCTTGAGTCAAGTTGAT-3' 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% CO2 atmosphere.

Preparation of Membrane Fraction-- L cells (3 × 106) were plated in 10-cm dish at least 48 h prior to transfection. Cells were transiently transfected with an expression plasmid (4 µg) 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 × 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 µg) 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 MgCl2, 0.3% Triton CF-54, 100 mM sodium cacodylate buffer, pH 6.0, 0.66 mM CMP-NeuAc (Sigma), 4,400 dpm/µl CMP-[14C]NeuAc (Amersham Pharmacia Biotech), the enzyme solution, and substrates in total volume of 50 µl for glycolipid acceptors or 20 µl 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 µl 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 µl 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 µl 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 µg of neolactotetraosylceramide (nLc4) was sialylated with a soluble form of ST3Gal VI (ST3Gal VI-protA). Five µg of GM3 was sialylated with GD3 synthase prepared form the L cells transfected with pMIKneo-ST8Sia I (29) using CMP-[14C]NeuAc (8, 800 dpm/µl). The products were purified by C18 Sep-Pak cartridge, dried, and redissolved in 25 µl of 50 mM sodium citrate (pH 6.0) and 100 mM NaCl containing 100 µg/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% CaCl2 (55:45:10). The plate was exposed to an imaging plate and then analyzed by BAS 2000 image analyzer.

TLC Immunostaining-- Twenty µg of nLc4 was sialylated with ST3Gal VI using CMP-[14C]NeuAc (4,400 dpm/µl) 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 µg/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 µg) 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, [alpha -32P]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 × 105 cells were incubated with mAb 2H5 (CD15s; PharMingen, San Diego, CA) at a dilution of 1:500 (1 µg/ml) for 30 min on ice. After washing twice, the cells were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgM (µ 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 QuestTM 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.

Semiquantitative RT-PCR Analysis-- Total RNA was prepared from cultured cells using TRIZOL® Reagent. Three µg of the RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) using oligo-dT14 primers. One-twentieth volume of the reaction mixture was subjected to PCR (total 50 µl). The reactions were performed using 5' and 3' primers for ST3Gal VI or GAPDH. Primers used were as follows, ST3Gal VI sense primer, 5'-TTGGGAGAAGGACAACCTTC-3' (nucleotides 728-747); ST3Gal VI antisense primer, 5'-CCAGGCAGCAACAGACAGTA-3' (nucleotides 1375-1356); GAPDH sense primer, 5'-CCACCCATGGCAAATTCCA-TGGCA-3', and GAPDH antisense primer, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. The cycling parameters for PCR for ST3Gal VI and GAPDH were 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and the cycle number were 27 for ST3Gal VI and 20 for GAPDH.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Cloning of a cDNA Encoding a Novel alpha 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 (GenBankTM 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 × 105 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 (Gly3-Val34), 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 C-terminal 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 alpha 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.


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Fig. 1.   Nucleotide and deduced amino acid sequences of human ST3Gal VI and hydropathy plot of the protein. A, the deduced amino acid sequence is shown below the nucleotide sequence. The putative transmembrane hydrophobic domain is underlined, and six potential N-linked glycosylation sites are boxed. B, the hydropathy plot was calculated by the method of Kyte and Doolittle (33) with a window of 11 amino acids.


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Fig. 2.   Comparison of sialylmotif L and sialylmotif S of ST3Gal VI with that of 14 previously cloned sialyltransferases. The previously cloned sialyltransferases are the human ST3Gal I (17), human ST3Gal II (19), human ST3Gal III (20), human ST3Gal IV (22, 23), mouse ST3Gal V (GenBankTM accession no. AB013302), human ST6Gal I (43, 44), the chick ST6GalNAc I (45), the mouse ST6GalNAc II (46), the rat ST6GalNAc III (47), the human ST8Sia I (29, 48, 49), human ST8Sia II (50), human ST8Sia III (GenBankTM accession no. AF004668), human ST8Sia IV (51), and human ST8Sia V (GenBankTM accession no. U91641). The sialyltransferase motifs are grouped by the linkages that they form. Shaded letters represent highly conserved amino acids in many sialyltransferases. Shaded and bold letters indicate conserved amino acid residues among the members of ST3Gal subfamily.

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-[14C]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).


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Fig. 3.   Sialyltransferase activity of the newly cloned enzyme. Sialyltransferase activity of the extracts of the L cells transfected with pMIKneo-ST3Gal VI was measured. Ten µg of glycolipid acceptor was incubated with cell extracts containing 20 µg 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% CaCl2 (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.

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 Fig. 4, [14C]NeuAc was incorporated into nLc4 and nLc6 containing Galbeta 1,4GlcNAc sequence at the non-reducing end. ST3Gal VI-protA did not exhibit activity toward Lc4, GA1, and lactosylceramide. Then, we determined the acceptor specificity of the enzyme toward oligosaccharides. As summarized in Table I, ST3Gal VI-protA utilized Galbeta 1,4GlcNAc as the best substrate. Galbeta 1,3GlcNAc showed much less incorporation of [14C]NeuAc. The kinetic analysis using the L cell extracts transfected with pMIKneo-ST3Gal VI showed that the Km value for nLc4 was 0.22 mM.


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Fig. 4.   Thin layer chromatography of sialylated glycosphingolipids. Various glycosphingolipids (0.1 mM) were used as acceptors for ST3Gal VI, and the products were separated on a TLC plate with a solvent system of chloroform/methanol/0.2% CaCl2 (55:45:10). The plate was exposed to a BAS-imaging plate and then analyzed with a BAS 2000 radioimage analyzer. Lex, Lex ceramide; Lc4, lactotetraosylceramide; nLc4, neolactotetraosylceramide; nLc6, neolactohexaosylceramide.

                              
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Table I
Acceptor substrate specificity of ST3 Gal VI
Various acceptor substrates were incubated in the standard assay mixture using ST3Gal VI-protA as an enzyme source. Each substrate was used at a concentration of 0.1 mM for glycolipids and oligosaccharides and 0.2 mg/ml for glycoproteins. Relative rates are calculated as a percentage of the incorporation obtained with nLc6.

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.


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Fig. 5.   Incorporation of sialic acid into asialofetuin by ST3Gal VI. Sialyltransferase activity of ST3Gal VI toward glycoproteins was measured. Forty µg of glycoproteins acceptors was incubated with 8 µg of the cell extracts in the standard assay condition. The samples were boiled in Laemmli sample buffer and subjected to 12.5% SDS-PAGE. The gel was dried and exposed to an imaging plate and then analyzed with a BAS 2000 radioimage analyzer. Additional details were provided under "Experimental Procedures."

Linkage Analysis by Sialidase Digestion-- To determine the incorporated sialic acid linkage, nLc4 was labeled with CMP-[14C]NeuAc using ST3Gal VI-protA and the product was subjected to digestion with Salmonella typhimurium LT2 sialidase which cleaves only the alpha 2,3 linkage. As shown in Fig. 6, [14C] 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 NeuAcalpha 2,3Gal-, and that the enzyme is one of the beta -galactoside alpha 2,3-sialyltransferases.


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Fig. 6.   Linkage analysis of incorporated sialic acids by sialidase digestion. [14C]NeuAc-labeled nLc4 and GD3 were produced from nLc4 and GM3 using ST3Gal VI-protA and GD3 synthase, respectively. The labeled products were then subjected to the treatment with alpha 2,3 sialidase as described under "Experimental Procedures." The resulting glycolipids were separated on a TLC plate with a solvent system of chloroform/methanol/0.2% CaCl2 (55:45:10) and detected with a BAS 2000 radioimage analyzer. SA-nLc4, sialyl-neolactotetraosylceramide.

TLC Immunostaining-- To confirm further that the reaction products were formed by the transfer of sialic acid to the terminal galactose of substrate via alpha 2,3-linkage, TLC immunostaining using mAb M2590 specific to a saccharide arrangement (NeuAcalpha 2,3Galbeta 1,4Glc or NeuAcalpha 2,3Galbeta 1,4GlcNAc) of glycolipids (35) was performed. As shown in Fig. 7, [14C]NeuAc-labeled nLc4 using ST3Gal VI-protA (lane 5) was stained with this antibody (lane 4) as well as GM3 in the standard (lane 1), indicating that this band is sialyl (alpha 2,3)-neolactotetraosylceramide.


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Fig. 7.   TLC immunostaining of sialic acid-incorporated products. TLC-immunostaining was performed as described under "Experimental Procedures." Lane 1, GM3 (1 µg); lane 2, acidic glycosphingolipids extracted from human red blood cells containing GM3 and sialylneolactotetraosylceramide (NeuAcalpha 2,3-nLc4) as major components; lane 4, [14C]NeuAc-labeled sialyl-nLc4 using ST3Gal VI-protA. As a control, the same reaction was performed without CMP-[14C] NeuAc (lane 3). Glycosphingolipids were separated by TLC and blotted to a polyvinylidene difluoride membrane. Immunostaining was done using mAb M2590 to detect GM3 and NeuAcalpha 2,3-nLc4. Lane 5 is an autofluorogram of lane 4 detected with a BAS 2000 radioimage analyzer. SA-nLc4, sialyl-neolactotetraosylceramide; SA-nLc6, sialyl-neolactohexaosylceramide.

Expression of the ST3Gal VI Gene-- To determine the size of ST3Gal VI mRNA and its expression, Northern blot analysis was conducted using the full-length fragment of cDNA as a probe. As shown in Fig. 8, 1.8- and 3.0-kb mRNAs of ST3Gal VI were detected. In adult tissues, the gene expression is abundant in heart, placenta, and liver (Fig. 8A). In human cancer cell lines, strong signals were observed in melanoma lines SK-MEL-37 and SK-MEL-23, while signals were hardly detected in hematopoietic cell lines and an astrocytoma AS or a neuroblastoma line IMR32 (Fig. 8B).


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Fig. 8.   Differential expression of ST3Gal VI in various human tissue and human cell lines. Northern blots with poly(A)+ RNA from various adult human tissues (panel A) and with total RNA (10 µg each) obtained from cultured human cancer cell lines (panel B) were probed with human ST3Gal VI full-length fragment (993 base pairs). The same filter was probed with GAPDH cDNA after removing the radioactivity. The positions of ribosomal RNAs are indicated at the left. The normalized relative intensities of ST3Gal VI signals are represented in C and D, respectively.

Correlation of the Expression Levels of ST3Gal VI Gene with Sialyl-Lex Expression-- In order to investigate whether ST3Gal VI was involved in sialyl-Lex expression, we examined the expression of ST3Gal VI gene along with sialyl-Lex 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-Lex 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-Lex 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-Lex expression among cell lines.


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Fig. 9.   ST3Gal VI gene expression and sialyl-Lex expression on human cancer cell lines. A, flow cytometric analysis of sialyl-Lex on the surface of the cultured human cancer cells. Cells were incubated with anti-sialyl-Lex antibody 2H5, followed by staining with fluorescein isothiocyanate-conjugated anti-mouse IgM as described under "Experimental Procedures." Percentages of positive cells are shown. B, semiquantitative RT-PCR analysis of ST3Gal VI transcripts. Intensities of ST3Gal VI bands in gel normalized by GAPDH bands are presented.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since Weinstein et al. isolated a cDNA clone of beta -galactoside alpha 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 ST8Sia- subfamilies. 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 alpha 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 alpha 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 Galbeta 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 Galbeta 1,3GlcNAc on glycoproteins compared with Galbeta 1,4GlcNAc and Galbeta 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 Galbeta 1,4GlcNAc (type II) structure more efficiently than Galbeta 1,3GlcNAc (type I), indicating that ST3Gal IV might be involved in the synthesis of sialyl-Lex, a ligand for the selectins (41). However, it seems unlikely that this enzyme synthesizes sialyl-paragloboside (SPG), a precursor of sialyl-Lex 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 Galbeta 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 [14C]NeuAc than nLc4, suggesting that ST3Gal VI preferred polylactosamine type II chains. Consequently, ST3Gal VI is capable of generating NeuAcalpha 2,3Galbeta 1,4GlcNAc structures on glycoproteins and glycolipids including SPG as main products, and is probably involved in the biosynthesis of sialyl-Lex 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-Lex, indicating that ST3Gal VI might be involved in the synthesis of sialyl-Lex 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-Lex 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.

    ACKNOWLEDGEMENT

We thank Dr. S. Tsuji for providing an expression vector pCDSA for a protein A fusion enzyme.

    FOOTNOTES

* 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. 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 GenBankTM/EMBL Data Bank with accession number(s) AB022918.

parallel To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466 Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.

2 Fukumoto, S., Miyazaki, H., Urano, T., Furukawa, K., and Furukawa, K. (1999) J. Biol. Chem. 274, in press.

    ABBREVIATIONS

The abbreviations used are: CMP-NeuAc, cytidine 5'-monophospho-N-acetylneuraminic acid; mAb, monoclonal antibody; RT, reverse transcription; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; TLC, thin layer chromatography; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPTLC, high performance thin layer chromatography; kb, kilobase pair(s); SPG, sialyl-paragloboside. The nomenclature of gangliosides is based on that of Svennerholm (54). The abbreviated nomenclature for cloned sialyltransferases follows Ref. 24. The designations of glycosphingolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Nomenclature (55). Lc4, Galbeta 1,3GlcNAcbeta 1,3Galbeta 1,4Glcbeta 1-Cer; nLc4, Galbeta 1,4GlcNAcbeta 1,3Galbeta 1,4Glcbeta 1-Cer; nLc6, Galbeta 1,4GlcNAcbeta 1,3Galbeta 1,4GlcNAcbeta 1,3Gal beta 1,4Glcbeta 1-Cer; Lex, Lewis X (Galbeta 1,4(Fucalpha 1,3)GlcNAcbeta 1,3Galbeta 1,4Glcbeta 1-Cer).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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