Molecular Cloning and Functional Expression of Two Members of Mouse NeuAcalpha 2,3Galbeta 1,3GalNAc GalNAcalpha 2,6-Sialyltransferase Family, ST6GalNAc III and IV*

Young-Choon LeeDagger §, Martina Kaufmann, Shinobu Kitazume-KawaguchiDagger parallel , Mari KonoDagger , Shou TakashimaDagger , Nobuyuki KurosawaDagger **, Hong LiuDagger , Hanspeter Pircher, and Shuichi TsujiDagger Dagger Dagger

From Dagger  Molecular Glycobiology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan and the  Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two cDNA clones encoding NeuAcalpha 2,3Galbeta 1,3GalNAc GalNAcalpha 2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha 2, 3Galbeta 1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta 1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha 2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta 1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions, like cell-cell communication, cell-substrate interaction, adhesion, and protein targeting. The transfer of sialic acids from CMP-Sia1 to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids is catalyzed by a sialyltransferase. Although roles of sialic acids have been proposed in the regulation of many biological phenomena, the purpose of this structural diversity remains largely obscure. To determine the meaning of the diversity of and the regulatory mechanism for the sialylation of glycoconjugates, it is necessary to obtain information on the enzymes themselves and the gene structure of sialyltransferases. Each sialyltransferase exhibits strict specificity for acceptor substrates and linkages (3-6). Although three linkages, Siaalpha 2,6Gal, Siaalpha 2,3Gal, and Siaalpha 2,6GalNAc, are commonly found in glycoproteins (7), and two, Siaalpha 2,3Gal and Siaalpha 2,8Sia, occur frequently in gangliosides (8), each of these linkages has been found in both gangliosides and glycoproteins (8-10).

So far, the cloning of three members of the alpha 2,6-sialyltransferase family (ST6GalNAc I, II and III) has been reported (11-14). The cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12) and mouse (13, 62).2 The overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick ST6GalNAc II is 57.3% to mouse ST6GalNAc II. ST6GalNAc III has been cloned from rat (14), and exhibits very low amino acid sequence identity (8.2-9.8%) to mouse and chick ST6GalNAc I and II.

As far as seen with the expressed recombinant enzymes, the substrate specificity of chick ST6GalNAc I is almost the same as that of the mouse one, and also chick ST6GalNAc II exhibits similar substrate specificity to the mouse enzyme. ST6GalNAc I exhibits the broadest substrate specificity for the following structures: GalNAc-O-Ser/Thr, Galbeta 1,3GalNAc-O-Ser/Thr, and NeuAcalpha 2,3Galbeta 1,3GalNAc-O-Ser/Thr (11). On the other hand, ST6GalNAc II exhibits a narrower substrate specificity, requiring beta -galactosides linked to GalNAc residues, whereas sialic acid residues linked to galactose residues are not essential for its activity, i.e. this enzyme exhibits activity toward Galbeta 1,3GalNAc-O-Ser/Thr and NeuAcalpha 2,3Galbeta 1,3GalNAc-O-Ser/Thr (12, 13). Both genes are expressed in secretory organs, such as the submaxillary and mammary glands, so these enzymes are considered to be involved in the biosynthesis of O-glycans of mucin (11-13). On the other hand, rat ST6GalNAc III exhibits the most restricted substrate specificity, only utilizing the NeuAcalpha 2,3Galbeta 1,3GalNAc sequence as an acceptor (14). This enzyme can transfer sialic acid to both NeuAcalpha 2,3Galbeta 1,3GalNAc-O-Ser/Thr and ganglioside GM1b, suggesting that it cannot discriminate between alpha - and beta -linked GalNAc (14). Incidentally, two types of alpha 2,3-sialyltransferases (ST3Gal I and II) have been cloned that exhibit activity toward Galbeta 1,3GalNAc but which have different substrate preferences for glycoproteins and glycolipids, i.e. ST3Gal I prefers glycoproteins to glycolipids, but II prefers glycolipids (15-18). According to these observations, there may be two scenarios; one is that ST6GalNAc III synthesizes almost all the NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc residues, and the other is that there may be another member of the ST6GalNAc family that has a different substrate preference from that of ST6GalNAc III. To solve this problem, we have extensively performed polymerase chain reaction (PCR) cloning. Comparison of sialyltransferases cloned thus far has revealed highly conserved regions, named sialylmotifs L, S, and VS (11, 19-21), not found in other glycosyltransferases. From the conservation of these sialylmotifs, it was expected that other members of the sialyltransferase gene family have the same motifs. The PCR-based approach with degenerate primers deduced on the conserved sequence in the sialylmotif has resulted in the isolation of several new members of the sialyltransferase gene family (22).

As a result, we have cloned two members of the ST6GalNAc family from mouse, which synthesize NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc residues. A new member, named ST6GalNAc IV, exhibits strong activity toward NeuAcalpha 2,3Galbeta 1,3GalNAc of O-glycans. The other is a homologue of rat ST6GalNAc III. Here, we report the cloning of the cDNAs encoding the two NeuAcalpha 2,3Galbeta 1,3GalNAc GalNAcalpha 2,6-sialyltransferases.

    EXPERIMENTAL PROCEDURES
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Materials-- Fetuin, asialofetuin, bovine submaxillary mucin, alpha 1-acid glycoprotein, CMP-NeuAc, Galbeta 1,3GalNAcalpha 1-benzyl, Galbeta 1,3GalNAc, Galbeta 1,3GlcNAc, Galbeta 1,4GlcNAc, lacto-N-tetraose, benzyl-GalNAc, N-acetyllactosamine and Triton CF-54 were from Sigma. CMP-[14C]NeuAc (11GBq/mmol) was from Amersham Pharmacia Biotech. NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc was from Seikagaku Co. NANase I, Newcastle disease virus sialidase, and sialidase from Vibrio cholerae were from Oxford Glycosystems and Roche Molecular Biochemicals, respectively. Synthetic primers were obtained from Espec Oligo Service (Japan). The restriction endonucleases were from Takara (Japan) and Toyobo (Japan). Galbeta 1,3GalNAc was sialylated using the secreted form of ST3Gal I expressed in COS-7 cells (16). NeuAcalpha 2,3Galbeta 1,3GalNAc was purified by preparative TLC (ethanol/1-butanol/pyridine/water/acetate = 100/10/10/30/3) and subsequent DEAE-Sephadex A-25 anionic exchange chromatography. NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol was prepared by the reduction of NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc (23).

PCR Cloning with Degenerate Oligonucleotides-- A mouse ST6GalNAc III cDNA fragment was prepared by PCR amplification. The primers used were rat ST6GalNAc III cDNA, 5'-ATCCATAGCGCCATGGCCTGCATC-3' (nucleotides -12 to 12), and 5'-TCACGGTCAGGAAGCACAGCATCA-3' (complementary to the rat ST6GalNAc III coding strand; nucleotides 916-939). The amplified 940-bp cDNA was subcloned into the EcoRV site of a pBluescript SK(+) vector (Stratagene). A mouse brain cDNA library was constructed and screened using the PCR-amplified mouse ST6GalNAc III cDNA as described previously (11), and full-length mouse ST6GalNAc III cDNA was isolated by rapid amplification of 5'-cDNA ends (RACE)-PCR.

To isolate the new sialyltransferases, PCR was performed as described previously (11) with two primers (5'-primer ST-107, 5'-TGGGCCTTGG(Ino)2(A/C)AGGTGTGCTGTTG-3', and 3'-primer ST-205, 5'-AGGCGAATGGTAGTTTTTG(A/T)GCCCACATC-3') deduced from the conserved region in mouse ST3Gal I and II. The standard molecular cloning techniques described by Ausubel et al. were used (24).

PCR RACE-- Amplification of the 5'-end of mouse ST6GalNAc III cDNA was performed as described previously (13). cDNA was synthesized by reverse transcription (Superscript II, Life Technologies, Inc.) of 5 µg of mouse brain poly(A)+ RNA and NB41A3 poly(A)+ RNA using primer RT-181, 5'-TTAGGCTGTCCAAAGCAGTTAGG-3' (complementary to the ST6GalNAc III coding strand, nucleotides 181-204), and was A-tailed. Two consecutive PCRs were performed with two nested sets of primers, NotI-d(T)18 (Amersham Pharmacia Biotech) and RT-181, and then following primers, 5'-AACTGGAAGAATTCGCGGCCGCAGGAA-3', and RT-91, 5' -CTTGAGGATGCAGGCCATGGCGCTATG-3' (complementary to the ST6GalNAc III coding strand, nucleotides -9 to 18). The cDNA was amplified through 35 cycles of a step program (94 °C, 30 s; 55 °C, 30 s; 72 °C, 40 s). The amplified products were subcloned and sequenced.

Preparation of Soluble ST6GalNAc Proteins Fused with Protein A-- A truncated form of ST6GalNAc III and IV, lacking the first 28 and 36 amino acids of the open reading frame, respectively, was prepared by PCR amplification using 5'-primers containing an EcoRI site and 3'-primers containing a XhoI site, i.e. 5'-primer, 5'-CGCCTTGAATTCAATGATGCGACTTTC-3' (ST6GalNAc III Sec; nucleotides 76-99), and 3'-primer, CCACAGCTCGAGTGTCATGGTCGG-3', (ST6GalNAc III Tail; complementary to the coding strand, nucleotides 930-953) for ST6GalNAc III, and 5'-primer, 5'-CGCGAATTCTGCCTGGACCGCCACCTCCC-3' (R1-Sec; nucleotides 109-128), and 3'-primer 5'-GCGCTCGAGAACTACTTGGCCCTCCAGG-3' (R1-Tail; complementary to the coding strand, nucleotides 893-911) for ST6GalNAc IV. The resulting amplified 878- and 803-bp fragments were subcloned into the EcoRV sites of pBluescript SK(+). The inserted fragments were cut out by digestion with EcoRI and XhoI, and then inserted into the EcoRI and XhoI sites of expression vector pcDSA (25).

The insert junctions were confirmed by restriction enzyme and DNA sequencing. The resulting plasmids consisted of the IgM signal peptide sequence, the protein A IgG binding domain, and a truncated form of ST6GalNAc III (pCDB8ST) and IV(pCDR1ST), respectively. Each expression plasmid (pCDB8ST and pCDR1ST: 20 µg) was transiently transfected into COS-7 cells on a 150-mm plate using LipofectAMINETM reagent (Life Technologies, Inc.). Each protein A-fused ST6GalNAc III and IV expressed in the medium was absorbed to an IgG-Sepharose gel (Amersham Pharmacia Biotech; 50 µl of resin/50 ml of culture medium; Ref. 25) and used as the enzyme source.

We also constructed an expression vector containing the whole coding region of ST6GalNAc III, in which a 1337-bp fragment from the NH2 terminus was inserted into the pcDL-SRalpha vector, named pcDL-SRalpha B8ST for kinetic analysis. The vector was transiently transfected into COS-7 cells as described above. After 5 h of transfection, the culture medium was changed to Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 48 h, the COS-7 cells were collected and the membrane-bound proteins were extracted by sonication in 20 mM MES buffer (pH 6.4) containing 0.3% Triton CF-54. After centrifugation of the cell lysate at 10,000 × g for 15 min, the resultant supernatant was used as the enzyme source. In this case, equivalent amounts of protein from COS cells stably transfected with pcDL-SRalpha were assayed in parallel as control experiment, and then the values obtained were subtracted from those obtained with cells expressing the full-length enzyme.

Sialyltransferase Assays and Linkage Analysis-- Sialyltransferase assays were performed as described previously (13). In brief, enzyme activity was measured in 50 mM MES buffer (pH 6.0), 1 mM MgCl2, 1 mM CaCl2, 0.5% Triton CF-54, 100 µM CMP-[14C]NeuAc (10.2 KBq), an acceptor substrate, and an enzyme preparation, in a total volume of 10 µl. As acceptor substrates, 10 µg of proteins, 5 µg of glycolipids, or 10 µg of oligosaccharides were used. The enzyme reaction was performed at 37 °C for 2 h.

For linkage analysis of sialic acids, [14C]NeuAc-incorporated fetuin was synthesized with ST3Gal I (16), ST6GalNAc I (12), ST8Sia II (26), ST6GalNAc III, and IV. The sialylated fetuin was treated with a linkage-specific sialidase, NANase I (specific for alpha 2,3-linked sialic acids), NANase III (specific for alpha 2,3-, alpha 2,6-. and alpha 2,8-linked sialic acids), or Newcastle disease virus sialidase (specific for alpha 2,3- and alpha 2,8-linked sialic acids)(27). After the sialidase treatment, the desialylated glycoprotein was subjected to SDS-polyacrylamide gel electrophoresis (gradient gel, 5-20%).

To obtain oligosaccharide portion of 14C-sialylrated fetuin, the sialylation of fetuin was carried out essentially as described, but on a 10-fold larger scale. To maximize the product yield, the incubation period was extended to 24 h. The incubation mixture was then treated with 0.1 N NaOH, 1 M NaBH4 at 37 °C for 48 h, and neutralized by the gradual addition of acetic acid in an ice bath. A sample was then desalted by Sephadex G-25 chromatography (1.3 × 25 cm). The 14C-sialylated oligosaccharide alditol and reference oligosaccharide NeuAcalpha 2, 3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol were treated with various kinds of sialidases. A radioactive sample containing at least 10,000 cpm or a sialylated sample containing at least 5 µg of sialic acid was spotted onto a TLC plate (Merck, Darmstadt, Germany) and then developed with ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3 (21) or 1-propanol/aqueous ammonia/water = 6/1/2.5 (28). The chromatogram was visualized with a BAS2000 radio image analyzer for 14C-sialylated sample (Fuji Film) or the resorcinol method for nonradioactive sample (29).

Analysis of ST6GalNAc III and IV Gene Expression-- The level of the ST6GalNAc III transcript was determined by competitive PCR (30). For the construction of a competitor DNA, PCR was performed with the ST6GalNAc III gene-specific primers, 5'-ATGGATACATAAATGTGAGGACC-3' (nucleotides 185-207) and 5'-GTGGATACTGTAGCAGGCATCCA-3' (complementary to the ST6GalNAc III coding strand; nucleotides 677-699), with ST6GalNAc III cDNA as the template. The amplified fragment (515 bp) was subcloned into pKF18k (Takara, Japan), and then subjected to site-directed mutagenesis using a mutagenic primer, 5'-TGAGGAAGATCTCGGCTACATG-3' (nucleotides 345-366; BglII linker is bold and italic) and a Mutan-Super Express Km kit (Takara, Japan). From the mutagenized plasmid, a 204-bp BglII fragment was deleted and the mutagenized plasmid was self-ligated, giving rise to a plasmid harboring a 315-bp competitor DNA fragment.

Poly(A)+ RNAs from various mouse tissues were reverse-transcribed to cDNAs using oligo(dT) (Amersham Pharmacia Biotech) as a primer with Superscript II (Life Technologies, Inc.). These single-stranded cDNAs were mixed with 0.35 pg of the competitor DNA and 40 pmol each of the above ST6GalNAc III gene-specific primers, and then competitive PCR was performed.

To determine the level of ST6GalNAc IV, Northern blot analysis (24) was performed using 5 µg of poly(A)+ RNAs from various mouse tissues. The 0.8-kb fragment of the NH2-terminal truncated form of ST6GalNAc IV (nucleotides 109-911) was used as the probe.

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Identification and Sequence of ST6GalNAc III and New Sialyltransferase cDNA Clones from Mice-- PCR with primers based on the rat ST6GalNAc III cDNA sequence gave a 0.9-kb cDNA. This fragment was used as the hybridization probe to screen a mouse brain cDNA library. Some independent clones containing a single open reading frame encoding a protein of 305 amino acids, showing 94.4% identity with rat ST6GalNAc III, were obtained (Fig. 1A). According to the following results, these clones encode a mouse homologue of ST6GalNAc III.


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Fig. 1.   Nucleotide and deduced amino acid sequences of mouse ST6GalNAc III and IV. The nucleotide and amino acid sequences of mouse ST6GalNAc III (A) and IV (B) are numbered from the presumed start codon and initiation methionine, respectively. The double underlined amino acids correspond to a putative transmembrane domain. Sialylmotifs L and S are boxed by solid and dashed lines, respectively. The positions of the PCR primers are indicated by arrows.

Next, in order to obtain clones of the new members of the sialyltransferase family, PCR with two degenerate oligonucleotides (ST-107 and ST-205), which were designed based on the 5'- and 3'-sequences of sialylmotif L, respectively, was performed with mouse brain cDNA as a template. A fragment of the expected size of approximately 150 bp was obtained. Among the PCR recombinants, one clone, designated as pCRR1, has a unique amino acid sequence distinct from that of the known sialylmotifs. The identity of the sialylmotif L of pCRR1 with those of ST6GalNAc I, II and III is 44.4%, 46.6%, and 64.4%, respectively.

A mouse brain cDNA library was screened with the cDNA insert of pCRR1 to isolate the complete coding sequence of the gene. The screening of about 106 independent clones yielded several overlapping clones, which were isolated and sequenced. The nucleotide sequence of one cDNA clone included an open reading frame of 906 bp, coding for 302 amino acids with a molecular mass of 34.2 kDa, starting with a methionine codon at nucleotide 1 with a conventional initiation sequence (31). The nucleotide and deduced amino acid sequences of the new sialyltransferase family member are shown in Fig. 1B. This protein has a type II transmembrane topology, containing a 23-amino acid NH2-terminal hydrophobic sequence bordered by charged residues, as has been found for all glycosyltransferases cloned to date. Comparison of this primary sequence with other amino acid sequences in DNA and protein data banks revealed similarity in some regions to all cloned sialyltransferases. One region (named sialylmotif L, residues 75-119) in the center of the protein, consisting of a 45-amino acid stretch, shows 42.2-64.4% sequence identity, whereas another, in the COOH-terminal portion (named sialylmotif S, residues 211-235), exhibits 20.0-60.0% identity. The overall amino acid sequence identity of this protein is 11.9% to mouse ST6GalNAc I (62),2 10.3% to mouse ST6GalNAc II (13), and 43.0% to mouse ST6GalNAc III, respectively (Table I). These results suggest that the cloned gene belongs to the sialyltransferase gene family. In fact, the following results revealed that it is a member of the ST6GalNAc family, so it was named ST6GalNAc IV.

                              
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Table I
Amino acid-identity (%) of members of the ST6GalNAc family

Both the Cloned DNAs Encode GalNAc alpha 2,6-Sialyltransferase-- To facilitate functional analysis of the enzyme, it was desirable to produce a soluble and condensable form of enzyme that could be secreted from the cells. First of all, sequences corresponding to the putative stem and active domains of ST6GalNAc III and IV were respectively fused to the immunoglobulin signal peptide sequence followed by the IgG binding domain of protein A (pCDB8ST and pCDR1ST). After transfecting each of the constructs into COS-7 cells, the enzyme secreted into the medium was condensed with IgG-Sepharose and used for further experiments. As shown in Table II, among the glycolipids examined in this study, only GM1b, i.e. not asialoGM1, served as an acceptor substrate for ST6GalNAc III and IV. ST6GalNAc III showed higher activity toward GM1b, of which the product comigrated with authentic GD1alpha in two different solvent systems (data not shown) than ST6GalNAc IV. ST6GalNAc III and IV also exhibited activity toward fetuin, but very low activity toward asialofetuin (Table II). These results suggested that the new sialyltransferase (ST6GalNAc IV) requires the NeuAcalpha 2,3Galbeta 1,3GalNAc residue in fetuin and GM1b just like ST6GalNAc III (Fig. 2). On the other hand, GD1a, which has the NeuAcalpha 2,3Galbeta 1,3GalNAc sequence and an additional NeuAc residue at the internal galactose, did not serve as an acceptor substrate (Fig. 2). It should be noted that the oligosaccharide, NeuAcalpha 2,3Galbeta 1,3GalNAc was a good acceptor substrate for ST6GalNAc IV (Table III), while such an oligosaccharide was a poor acceptor substrate for ST6GalNAc III. When the relative enzyme activities of ST6GalNAc III and IV toward fetuin were, respectively, set to 100%, the activity to oligosaccharide NeuAcalpha 2,3Galbeta 1,3GalNAc was 290% in case of ST6GalNAc IV, while only 0.5% of the activity was detected in case of ST6GalNAc III. ST6GalNAc IV enzyme activities toward nonsialylated Galbeta 1,3GalNAc and disialylated NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc were almost negligible (Table III).

                              
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Table II
Acceptor specificities of mouse ST6GalNAc III and ST6GalNAc IV
R represents the remainder of the N-linked oligosaccharide chains. NT, not tested.


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Fig. 2.   Thin layer chromatography of sialylated glycosphingolipids. A series of glycosphingolipids as acceptor substrates (GM3 in lane 1, asialoGM1 in lane 2, GM1b in lane 3, GD1a in lane 4, and paragloboside in lane 5), CMP-[14C]NeuAc as donor substrate, and ST6GalNAc IV were used for sialyltransferase assays as described under "Experimental Procedures." The glycosphingolipid fractions were extracted from the reaction mixtures and separated on TLC plates in chloroform/methanol/0.2% CaCl2 = 55/45/10, and visualized using a BAS2000 radioimage analyzer (A) or with orcinol-H2SO4 (B). Note that the product of the transferase acting on GM1b is GD1alpha , though the orcinol reagent does not show a spot at the GD1alpha position in the GM1b lane in B.

                              
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Table III
Relative activities of mouse ST6GalNAc IV toward oligosaccharides

The truncated form of ST6GalNAc III exhibited enzyme activity that was too low (18 fmol/h/ml of medium) to determine kinetic parameters. Therefore, the full-length ST6GalNAc III was also subcloned into expression vector pCDL-SRalpha to yield pcDL-SRalpha B8ST. The enzyme fraction (3.6 pmol/h/µl of cell lysate) was then obtained as described under "Experimental Procedures." The COS cell lysates intrinsically exhibit strong ST3Gal I and II, and significant ST6Gal I activities; thus, this enzyme fraction is not suitable for analyzing substrate specificity but for estimating Km and relative Vmax/Km values for acceptor substrates. The apparent Km value for GM1b was 200 µM, which was lower than those for fetuin (8,000 µM) and NeuAcalpha 2,3Galbeta 1,3GalNAc-benzyl (670 µM). The relative Vmax/Km value for GM1b was 1.0, which was higher than those for fetuin (0.31) and NeuAcalpha 2,3Galbeta 1,3GalNAc-benzyl (0.019). These results suggested that the expressed ST6GalNAc III prefers glycolipids to glycoproteins.

Linkage Specificity of the Two Sialyltransferases-- For linkage analysis, [14C]NeuAc-incorporated fetuin, which was synthesized with ST3Gal I (16), ST6GalNAc I (12), ST8Sia II (26), and ST6GalNAc III and IV, respectively, and each sialylated fetuin was treated with linkage-specific sialidases, i.e., NANase I (specific for alpha 2,3-linked sialic acids), NANase III (specific for alpha 2,3-, alpha 2,6-, and alpha 2,8-linked sialic acids), and Newcastle disease virus sialidase (specific for alpha 2,3- and alpha 2,8-linked sialic acids)(27) (Fig. 3). The resulting patterns of the ST6GalNAc III and IV enzymes were virtually identical to that of ST6GalNAc I. The [14C]NeuAc residue of fetuin sialylated by both the enzymes was removed by the NANase III treatment but not by the NANase I or Newcastle disease virus sialidase treatment (Fig. 4). These results indicate that the incorporated sialic acids each contain an alpha 2,6-linkage, and the expressed enzymes are the mouse homologue of rat ST6GalNAc III, and a new member of the ST6GalNAc family, ST6GalNAc IV, respectively.


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Fig. 3.   Linkage analysis of incorporated sialic acids. [14C]Sialylfetuin sialylated with ST6GalNAc IV (A) and III (B), ST3Gal I (C), ST6GalNAc I (D), or ST8Sia II (E) was treated with each sialidase. The resulting product was subjected to SDS-polyacrylamide gel electrophoresis (gradient gel, 5-20%) and radioactivity was detected with a BAS2000 radioimage analyzer (Fuji Film). -, without sialidase treatment; I, NANase I treatment; III, NANase III treatment; NDV, treatment with sialidase of Newcastle disease virus.


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Fig. 4.   Thin layer chromatography of the oligosaccharide alditols derived from fetuin 14C-sialylated by ST6GalNAc IV. The TLC plates were developed in ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3 (A) or 1-propanol/aqueous ammonia/water = 6/1/2.5 (B). The oligosaccharide alditols derived on 0.1 N NaOH, 1 M NaBH4 treatment of 14C-sialylated fetuin (lanes 1-4) and NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol, as a reference (lanes 5-8), were, respectively, treated in the absence of sialidase (lanes 1 and 5) or the presence of alpha 2,3-linkage-specific NANase I (lanes 2 and 6), alpha 2,3- and alpha 2,8-specific Newcastle disease virus sialidase (lanes 3 and 7), or V. cholerae sialidase (lanes 4 and 8). Each sample was then chromatographed on a TLC plate and visualized with a BAS2000 radioimage analyzer (Fuji Film) for 14C-sialylated fetuin products (lanes 1-4) or with resorcinol reagent for reference oligosaccharide products (lanes 5-8). Oligo 1, NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol; Oligo 2, Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol.

To confirm the linkage specificity of ST6GalNAc III and IV, the following experiments were performed. Although we report here the results of ST6GalNAc IV, the results for ST6GalNAc III is the same as those for IV. The 14C-sialylated oligosaccharide alditol was prepared by beta -elimination of the sialylated fetuin with ST6GalNAc IV. A desalted sample was then spotted onto a TLC plate and developed with ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3. All of the radioactive product migrated as a low molecular compound, i.e. no radioactivity remained at the origin, suggesting that 14C-sialylation occurred exclusively on O-linked glycan chains of fetuin (data not shown). Furthermore, more than 70% of the radioactivity comigrated with the reference oligosaccharide, NeuAcalpha 2, 3Galbeta 1,3(NeuAcalpha 2,6)-GalNAc-ol (data not shown). This radioactive material was isolated by preparative TLC and used for linkage analysis (Fig. 4). The reference oligosaccharide, NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol, was also used for comparison. The 14C-sialylated oligosaccharide alditols were detected by BAS2000 radioimage analyzer (Fig. 4, lanes 1-4). In case of reference oligosaccharide, resorcinol reagent was used for detection (Fig. 4, lanes 5-8). On NANase I or Newcastle disease virus digestion, NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol (oligo 1, lane 5) was converted to Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol (oligo 2, lanes 6 and 7). With these sialidase treatments, the radioactive band that co-migrated with oligo 1 (lane 1) migrated to the same position as oligo 2 (lanes 2 and 3). It should be noted that the digestion with NANase I and Newcastle disease virus sialidase was partial for some reason. When NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol (oligo 1) was treated with V. cholerae sialidase (alpha 2,3, alpha 2,6, and alpha 2,8 linkage-specific sialidase), it was converted to Galbeta 1,3GalNAc-ol, which was not detectable with the resorcinol method (lane 8). With this treatment, the radioactive band comigrated with neither oligo 1 nor oligo 2, but with NeuAc, indicating that the linkage type of the [14C]NeuAc residue in the oligosaccharide alditol is alpha 2,6 (lane 4). The reason that [14C]NeuAc band in lane 4 (Fig. 4A) was remarkably sharp could be because the prepared 14C-sialylated oligosaccharide alditol contained more salts, which sometimes affect the migration pattern on TLC plate. Taken together, the results strongly suggest that the 14C-sialylated oligosaccharide alditol derived from fetuin is NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc-ol. Thus, the cloned enzyme is a new member of the ST6GalNAc-family, named ST6GalNAc IV.

Expression of the ST6GalNAc III and IV Genes in Mouse Tissues-- To examine the expression of the mouse ST6GalNAc III gene in various tissues, Northern blot hybridization was performed using 5 µg of poly(A)+ RNA from various adult mouse tissues, which gave only faint signals and could not be used to examine the specific gene expression of the ST6GalNAc III gene. Therefore, we performed competitive reverse transcription-PCR to determine in which tissues the ST6GalNAc III gene is expressed. In adult tissues, significant levels of expression that could not be detected on Northern blot analysis but could be detected on reverse transcription-PCR were observed in brain, lung, and heart, followed by lower levels of expression in kidney, mammary gland, spleen, thymus, and testis (Fig. 5A). We also estimated the amount of the ST6GalNAc III transcript in brain during development (Fig. 5B). The amount of the ST6GalNAc III transcript was highest at E12, although whole embryos at E7, E11, and E12 were used to isolate poly(A)+ RNA. The amount of the ST6GalNAc III transcript in brain was lower at E16 than at embryonal stages (E11 and E12), and then kept almost similar levels during mouse development, suggesting that the mouse ST6GalNAc III gene may be highly transcribed in tissues other than brain at embryonic stages.


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Fig. 5.   Estimation of the amounts of the mouse ST6GalNAc III and IV transcripts. A, competitive PCR was performed using single-stranded cDNAs reverse-transcribed from poly(A)+RNA derived from various tissues (ICR mouse) and 0.35 pg of the competitor DNA. In parallel, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was amplified to estimate the amounts of the cDNAs used. Br, brain; SG, salivary gland; Th, thymus; He, heart; Lu, lung; Li, liver; Sp, spleen; Ki, kidney; In, small intestine; Co, colon; Te, testis; MG, mammary gland. B, competitive PCR was performed using single-stranded cDNAs reverse-transcribed from poly(A)+ RNA derived at different stages of development (embryo (Emb) and brain (Br)) and 0.35 pg of the competitor DNA. In parallel, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was amplified to estimate the amounts of the cDNAs used. C, Northern blot analysis was performed using poly(A)+ RNA (5 µg) derived from 8-week ICR mouse tissues. Br, brain; SG, salivary gland; Th, thymus; Lu, lung; He, heart; Li, liver; Ki, kidney; Sp, spleen; In, small intestine; Co, colon; Te, testis; MG, mammary gland. D, Northern blot analysis was performed using poly(A)+ RNA (5 µg) prepared from 12-day postcoital ICR mouse embryos (E12), and 1-day (P1), 3-week (3w), and 8-week (8w) ICR mouse brains and livers. The hybridization probe was prepared from the NH2-terminal truncated fragment (nucleotides 109-111) of ST6GalNAc IV.

The mRNA size and distribution of the ST6GalNAc IV gene were determined by Northern blot analysis (Fig. 5, C and D). Three transcripts (1.9, 2.2, and 3.6 kb) were observed in ICR mouse tissues (8-week-old mice). Strong signals were observed in brain and colon, and moderate ones in lung, heart, thymus, and spleen (Fig. 5C). The expression in brain was developmentally regulated. Analysis of RNAs from embryonal stage (E12), and 1-day, 3-week, and 8-week brain revealed three RNA species of 1.9, 2.2, and 3.6 kb. The 1.9- and 2.2-kb mRNA were abundantly expressed.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have described the isolation and characterization of cDNAs for encoding third and fourth types of mouse GalNAc alpha 2,6-sialyltransferase (ST6GalNAc III and IV). The mouse ST6GalNAc III shared very high sequence similarity (94.4%) at the amino acid level with the rat one (14). The mouse ST6GalNAc IV cloned in this study turned out to be encoded for a novel type of sialyltransferase. The cDNAs were isolated by PCR cloning method based on the highly conserved regions from previously cloned sialyltransferases (11, 19-21). The cDNA for ST6GalNAc IV was also isolated independently by mRNA differential display method by comparing the gene expressions between native and activated CD8+ cells.3

Based on the following observations, we concluded that the cDNAs we isolated were indeed for ST6GalNAc III and IV, which transfer CMP-NeuAc with an alpha 2,6-linkage to a GalNAc residue on NeuAcalpha 2,3Galbeta 1,3GalNAc of glycoproteins and glycolipids. First, fetuin, which contains the O-glycosidically linked NeuAcalpha 2,3Galbeta 1,3GalNAc sequence (32), was shown to serve as a good acceptor for both enzymes. However, both asialofetuin (contains the Galbeta 1,3GalNAc sequence) and asialo-bovine submaxillary mucin (5% of the total carbohydrate chains contain Galbeta 1,3GalNAc sequences) (33) served as much poorer acceptors compared with fetuin. Second, the study of the sensitivity of 14C-sialylated oligosaccharide alditol to various sialidase including NANase I, III, V. cholerae sialidase, and Newcastle disease virus sialidase revealed that the sialylated product was NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc and the linkage type of the [14C]NeuAc was alpha 2,6. The 14C-sialylated oligosaccharide alditol was derived from the fetuin sialylated by both enzymes with CMP-[14C]NeuAc. Furthermore, GM1b could be served as an acceptor for both enzymes, and the product was GD1alpha .

Similar to other glycosyltransferases, ST6GalNAc IV has a type II membrane protein topology, a short NH2-terminal cytoplasmic tail, a hydrophobic signal-anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain (6). The location of the transmembrane domain was determined by hydropathy plot according to the Kyte and Doolittle method (34). The transmembrane domain was 23 amino acids long from position 14 to 36. We also noticed that ST6GalNAc IV was the smallest protein (302 amino acid) among all cloned sialyltransferases. This was mainly because of the very short stem region of ST6GalNAc IV. The size of stem regions among members of the ST6GalNAc family varies to a great degree. ST6GalNAc IV had only 38 amino acid residues between the transmembrane region and sialylmotif L, while ST6GalNAc I, II, and III have 261, 123, and 53 amino acid residues (13, 62),2 respectively. These differences may have important implications for the in vivo functions of individual enzymes, although this remains to be clarified.

The four members belonging to ST6GalNAc family can be classified into two subfamilies according to the sequence similarity and substrate specificity differences (Fig. 6 and Tables I and III). ST6GalNAc I and II belong to one subfamily, and III and IV belong to the other. A dendrogram constructed by the method of Higgins and Sharp (35) suggested that one subfamily (III and IV) is separated from other sialyltransferase families, suggesting a great difference in domain structure (Fig. 6). The dendrogram also showed that the other ST6GalNAc subfamily (I and II) was closely related to the group of ST3Gal families. As reported previously (13, 15-17, 36-43),4 mouse ST3Gal family contains four cysteine residues, which are conserved in both chick and mouse ST6GalNAc I and II. This structure is the so-called Kurosawa motif, Cys-Xaa75-82-Cys-Xaa1-2-Cys-Ala-Xaa-Val-Xaa150-160-Cys (Xaa denotes any amino acid residue). However, mouse and rat ST6GalNAc III, and mouse ST6GalNAc IV do not contain this motif, nor do any members of the ST8Sia family (21, 25, 26, 37, 44-54)5 or ST6Gal I (55-59).6 This indicates that one ST6GalNAc subfamily (I and II) and the ST3Gal family, but not another one (III and IV), share common domain structures.


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Fig. 6.   Dendrogram of the cloned mouse sialyltransferases.[enspace]After analyzing the deduced amino acid sequences of cloned mouse sialyltransferases according to Higgins and Sharp (35), a dendrogram was constructed. Note that one subfamily of the ST6GalNAc family (III and IV) is separated from other sialyltransferase families, and that the other ST6GalNAc subfamily (I and II) is near the group of ST3Gal families. Parentheses indicate the accession number of GenBankTM/EBI data base.

The mouse ST6GalNAc III and IV almost have the same substrate specificities, but they differ in substrate preference (Tables II-IV). ST6GalNAc III preferred glycolipid as substrate over glycoprotein. On the other hand, ST6GalNAc IV preferred glycoprotein as substrate over glycolipid. For example, the activity of mouse ST6GalNAc III toward GM1b was stronger than that toward fetuin, which is similar to rat ST6GalNAc III (14). However, mouse ST6GalNAc IV exhibited stronger activity toward fetuin than GM1b. It has been reported that GD1alpha is expressed highly in embryonic rat brain, but the expression level decreases dramatically in adult brain (60). Additionally, GD1alpha has been assumed to be a molecular component of a variety of important biological processes including metastasis of highly virulent lymphomas and motor learning as elaborated by Purkinje cells (60, 61). The spatial and temporal expression of the rat ST6GalNAc III gene correlates well with the expression of GD1alpha . In the case of mouse ST6GalNAc III, its gene expression in brain and other tissues also seems to correlate with that of GD1alpha , although the amount of the transcript was relatively low in adult tissues. Based on these observations, ST6GalNAc III could be a strong candidate for GD1alpha synthase among members of the ST6GalNAc family.

                              
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Table IV
Comparison of the substrate specificities of members of the ST6GalNAc family
A, B, and C, significant enzyme activity could be detected. The activity strength order is A > B > C. ---, enzyme activity could not be detected or was negligible.

It is known that trisaccharide, NeuAcalpha 2,3Galbeta 1,3GalNAc, cannot be an acceptor substrate for mouse ST6GalNAc I, II. However, it did serve as a substrate for ST6GalNAc III, although it was not a good one. Similar results were reported for rat ST6GalNAc III (14). On the other hand, mouse ST6GalNAc IV showed strong enzyme activity toward NeuAcalpha 2, 3Galbeta 1,3GalNAc. It is also interesting that the peptide portion of an acceptor substrate is not necessary for the ST6GalNAc IV activity. The expression levels of ST6GalNAc IV in various adult mouse tissues were very different from those of III. The mRNA expression of ST6GalNAc IV could be easily detected by Northern blotting using 5 µg of poly(A)+RNA in mouse brain, thymus, lung, heart, spleen, and colon, but the RNA expression was too low to be detected in the case of ST6GalNAc III. Because NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2,6)GalNAc structures are found in almost all tissues, ST6GalNAc IV may be the main candidate for synthesizing the NeuAcalpha 2,3Galbeta 1,3(NeuAcalpha 2, 6)GalNAc residue, which is usually found in the O-linked glycan chains of glycoproteins.

At present, we still cannot answer the question whether or not there are separate sialyltransferases that are responsible mainly for glycolipid or glycoprotein synthesis. This is a important point for understanding the nature of biosynthesis for glycolipids and glycoproteins. Finding the second type of NeuAcalpha 2,3Galbeta 1,3GalNAc GalNAc alpha 2,6-sialyltransferase may help to solve this problem. The controversial point is how to distinguish the functions of ST6GalNAc III and IV in vivo. Work related to this is currently in progress. Mouse ST3Gal I and II, which both use Galbeta 1,3GalNAc-residue as substrate (16, 17), also have different preferences for glycolipids and glycoproteins. The existence of these groups, or subfamilies, is probably important for fine control of the expression of sialylglycoconjugates, resulting in stage- and tissue-specific variety. The study concernnig the four members of the ST6GalNAc family will help us to understand the sialylglycoconjugates' biological functions during the course of development.

    ACKNOWLEDGEMENTS

We thank Dr. James C. Paulson for the valuable discussion and for giving us the cDNA sequence of rat ST6GalNAc III prior to publication, and Dr. Yoshitaka Nagai, Director of the Glycobiology Research Group, and Dr. Tomoya Ogawa, Coordinator of the Group, Frontier Research Program of the Institute of Physical and Chemical Research (RIKEN), for their support in this work.

    FOOTNOTES

* This work was supported by Grants-in-aid 10152263 and 10178104 for Scientific Research on Priority Areas and Grant-in-aid C 09680639 for Scientific Research from the Ministry of Education of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y11342 (mouse ST6GalNAc III) and Y15779, Y15780, and AJ007310 (mouse ST6GalNAc IV).

§ Present address: Div. of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University, Saha-ku, Pusan 604-714, Korea.

parallel Special postdoctoral researcher, Institute of Physical and Chemical Research (RIKEN).

** Present address: Dept. of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Dagger Dagger To whom correspondence should be addressed. Tel.: 81-48-467-9615; Fax: 81-48-462-4692; E-mail: stsuji{at}postman.riken.go.jp.

2 The accession number of the cDNA sequence of mouse ST6GalNAc I is Y11274.

3 M. Kaufmann, C. Blaser, S. Takashima, S. Tsuji, and H. Pircher (1999) Int. Immunol., in press.

4 In addition to the references, the following data also support it: EMBL accession nos. X96667, AF059321, AF035250, and Y15003.

5 In addition to the references, the following data also support it: EMBL accession nos. U73176, L38677, U82762, AF004668, AF003092, AF008194, and U90215.

6 In addition to the references, the following data also support it: EMBL accession nos. A17362, 23699, and Y15111.

    ABBREVIATIONS

The abbreviations used are: Sia, sialic acid; NANase, N-acetylneuraninidase; ST6GalNAc I, GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3); ST6GalNAc II, Galbeta 1,3GalNAc GalNAc alpha 2,6-sialyltransferase; ST6GalNAc III, the first type of Siaalpha 2,3Galbeta 1,3GalNAc GalNAcalpha 2,6-sialyltransferase (EC 2.4.99.7); ST6GalNAc IV, the second type of Siaalpha 2,3Galbeta 1,3GalNAc GalNAcalpha 2,6-sialyltransferase (EC 2.4.99.7); NeuAc, N-acetylneuraminic acid; CMP-NeuAc, cytidine 5'-monophospho-N-acetylneuraminic acid; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); E, embryonic day; RACE, rapid amplification of cDNA ends; MES, 4-morpholineethanesulfonic acid. The nomenclature for gangliosides follows the system of Svennerholm (1). The abbreviated nomenclature for cloned sialyltransferases follows the system of Tsuji et al. (2).

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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