©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Sia2,3Gal1,4GlcNAc 2,8-Sialyltransferase from Mouse Brain (*)

Yukiko Yoshida , Naoya Kojima , Nobuyuki Kurosawa , Toshiro Hamamoto , Shuichi Tsuji (§)

From the (1)Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cDNA encoding a new 2,8-sialyltransferase (ST8Sia III), which exhibits activity toward the Sia2,3Gal1,4GlcNAc sequences of N-linked oligosaccharides, was cloned from mouse brain by means of the polymerase chain reaction-based approach. The predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4% identity with those of so far cloned mouse 2,8-sialyltransferases, i.e. G synthase (ST8Sia I) and STX (ST8Sia II), respectively. Transfection of the protein A-fused ST8Sia III gene into COS-7 cells led to 2,8-sialyltransferase activity toward sialylated glycoproteins and 2,3-sialylated glycosphingolipids, such as 2,3-sialylparagloboside and G. However, the kinetic properties of ST8Sia III revealed that it is much more specific to N-linked oligosaccharides of glycoproteins than glycosphingolipids. The expression pattern of the ST8Sia III gene was clearly different from those of other 2,8-sialyltransferase genes. The expression of the ST8Sia III gene was tissue and stage specific. The ST8Sia III gene was expressed only in brain and testis, and it appeared first in 20 postcoitum embryonal brain and then decreased. Therefore, the new 2,8-sialyltransferase is closely involved in brain development.


INTRODUCTION

Sialic acids are ubiquitous in the oligosaccharide side chains of glycoconjugates of a wide variety of animals(1) . Sia2,8Sia sequences are widely observed in various gangliosides, such as G, G, and b- and c-series gangliosides,()and are found more specifically in glycoproteins of mammals(2) . It has been reported that Sia2,8Sia sequences are associated with only two proteins, the neural cell adhesion molecule (N-CAM)()(3-5) and the subunit of the voltage-gated sodium channels in rat brain(6) . Recently, we cloned an 2,8-sialyltransferase, namely G synthase (ST8Sia I)(7) , and showed that a developmentally regulated sialyltransferase (STX, ST8Sia II) exhibited N-glycan 2,8-sialyltransferase activity(8) . Many sialyltransferase genes have been recently cloned(7, 8, 9, 10, 11, 12, 13, 14, 15) , but only two of them were confirmed to be genes encoding 2,8-sialyltransferases. However, the substrate specificities of these two 2,8-sialyltransferases cannot fully explain how all the known Sia2,8Sia sequences in glycolipids and glycoproteins of mammals are synthesized. For example, G synthase was reported only to synthesize G, i.e. not G, G, or other b-series gangliosides(7) . Even if the cloned N-glycan 2,8-sialyltransferase (STX) is involved in the polysialic acid synthesis in the case of N-CAM, it must not be the sole enzyme synthesizing polysialic acids of N-CAM because STX itself does not exhibit polymerization activity as to sialic acids(8) . In addition, STX was shown to be expressed within limited neural developmental stages(8, 16, 17) , while polysialylated N-CAM was reported to exist at almost all stages(6, 18) . The expression of polysialic acids is developmentally regulated, the embryonic forms, having a high sialic acid content, undergoing postnatal conversion to the adult forms with a low sialic acid content(6, 19, 20) . This evidence strongly suggests that there must be other types of 2,8-sialyltransferase.

Comparison of so far cloned sialyltransferases has revealed highly conserved regions (7, 16, 21) named sialyl motifs L and S, which are not found in other glycosyltransferases such as fucosyltransferases, galactosyltransferases, or N-acetylglucosaminyltransferases. From the conservation of the sialyl motifs, it was expected that other members of the sialyltransferase gene family might have the same motifs. A PCR-based approach involving degenerate primers based on the conserved sequences of the sialyl motifs has resulted in the isolation of several new members of the sialyltransferase gene family(8, 13, 14, 15, 16) . Here, we report the cloning of a cDNA encoding a new type of 2,8-sialyltransferase using a PCR-based approach.


EXPERIMENTAL PROCEDURES

Materials

Unless otherwise indicated, the materials used in this study were essentially the same as in previous studies(7, 13, 14, 15) . Lactosylceramide, G, G, G, G, and G were purchased from Sigma; G and paragloboside were from IATRON (Tokyo). 2,3- and 2,6-sialylparaglobosides (SPGs) were kind gifts from Dr. Iwamori (Tokyo University). Glycoproteins (fetuin, asialofetuin, 1-acid glycoprotein, ovomucoid, transferrin, and bovine submaxillary mucin) were from Sigma. Asialo-1-acid glycoprotein and asialo-ovomucoid were prepared by mild acid hydrolysis of glycoproteins (0.02 N HCl, 80 °C, 1 h). Protein A-Sepharose was from Pharmacia Biotech Inc.

PCR Cloning with Degenerate Oligonucleotides

PCR was performed using degenerate primers (5`-primer OP-L, T(G/A)(A/C)AGA(A/C)(A/T)TG(C/T)GC(G/C)(G/A)T(G/C)GTGGG(A/C)AA; 3`-primer OP-S, CA(C/A)(A/T)G(A/G)GAAGGGCCAGAAGCCATA) deduced from conserved regions in STX (rat brain) (16) and G synthase (human melanoma cells)(7) . Total RNA from 3-day-old mouse brain was used as a template to synthesize cDNA. The cycling parameters were 94 °C for 40 s, 37 °C for 40 s, and 72 °C for 1 min for the first 5 cycles, followed by 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 1 min for 30 cycles. The 0.5-kb PCR products were blunt-ended, kinated, and then subcloned into the SmaI site of pUC119. The subclones were characterized by sequencing.

Approximately 10 plaques of a 3-day-old mouse brain cDNA library (14) were screened with the 0.5-kb PCR fragments. Standard molecular cloning techniques, described by Maniatis and co-workers (22), were used.

Construction and Purification of ST8Sia III Fused with Protein A

A truncated form of ST8Sia III, lacking the first 39 amino acids of the open reading frame, was prepared by PCR amplification with 5`- and 3`-primers containing an XhoI site, respectively (5`-CATCTTCTCGAGTCCCAAGTACGCCAGCCCG-3` and 5`-TTCCATCTCGAGTTCTTAGGCACAGTGTGACAG-3`). The amplified and digested 1028-bp XhoI fragment was inserted into the XhoI site of a pcDSA vector(8) . The single insertion in the correct orientation was finally analyzed by restriction enzyme and DNA sequencing, and the resulting plasmid was designated as pcDSA-O3, which consisted of an IgM signal peptide sequence, a protein A IgG binding domain, and the truncated form of ST8Sia III. COS-7 cells were transiently transfected with 10 µg of pcDSA-ST8Sia III using the DEAE-dextran procedure and cultured as previously described(8) . After 48 h of transfection, the culture medium was collected, and the protein A-mouse STX expressed in the medium was adsorbed to IgG-Sepharose (15 µl of resin/10 ml of culture medium) at 4 °C for 16 h. The resin was collected by centrifugation, washed three times with phosphate-buffered saline, suspended in 50 µl (final volume) of Dulbecco's modified Eagle medium without fetal bovine serum, and used as the soluble enzyme.

Sialyltransferase Assays and Product Characterization

The enzyme activity was measured in the presence of 0.1 M sodium cacodylate buffer (pH 6.0), 10 mM MgCl, 2 mM CaCl, 0.5% Triton CF-54, 100 µM CMP-[C]NeuAc (0.25 µCi), 10 µg of acceptor substrate, and 2 µl of enzyme preparation in a total volume of 10 µl, as described in the previous paper(7) . After 4 h of incubation at 37 °C, the reaction was terminated by the addition of SDS-PAGE loading buffer (10 µl), and the incubation mixtures were directly subjected to SDS-PAGE (for glycoprotein acceptors). For glycolipid acceptors, the incubation mixtures were applied on a C-18 column (Sep-Pak Vac, 100 mg; Waters, Milford, MA), which had been washed with water. The glycolipids were eluted from the column with methanol, dried, and then subjected to chromatography on an HPTLC plate (Merck, Germany) with a solvent system of chloroform, methanol, and 0.02% CaCl (55:45:10), as previously described(7) . Acceptor substrates were visualized by staining with Coomassie Brilliant Blue (for glycoproteins) or by the orcinol/HSO method (for glycolipids). The radioactive materials in glycoproteins or glycolipids were visualized with a BAS2000 radio image analyzer (Fuji Film, Japan), and the radioactivity incorporated into acceptor glycoproteins was counted.

For linkage analysis of sialic acids, fetuin sialylated with the enzyme was precipitated with 70% ethanol, washed three times with 70% ethanol, dissolved in water, and then digested with a linkage-specific recombinant sialidase, NANase I (specific for 2,3-linked sialic acids, 0.1 units/ml), NANase II (specific for 2,3- and 2,6-linked sialic acids, 0.5 units/ml), or NANase III (specific for 2,3-, 2,6- and 2,8-linked sialic acids, 0.35 units/ml) (FACE, Glyko, Inc., Navato, CA) at 37 °C for 8 h.

For preparation of desialylated or de-N-glycosylated fetuin, fetuin (100 µg) was digested with NANAase I (0.1 units/ml), NANAase II (0.5 units/ml), or NANAase III (0.35 units/ml) in a total volume of 20 µl for 24 h at 37 °C or with N-glycanase (1.5 units; Genzyme, Cambridge, MA) in a total volume of 20 µl at 37 °C for 36 h. After inactivation of the enzyme by boiling for 1 min, the resulting desialylated or de-N-glycosylated glycoproteins were used as acceptors.

Northern Blot Analysis

5 µg of total RNA was fractionated on a denaturing formaldehyde-agarose gel (1%) and then transferred onto a nylon membrane (Nytran, Schleicher & Schuell). The full-length ST8Sia III cDNA (1205 bp) was amplified by PCR using synthetic oligonucleotide primers (5`-AGGCTCGAGCTCTCAATGGACCGATT-3` and 5`-TTCCATCTCGAGTTCTTAGGCACAGTGTGACAG-3`) from 3-day-old mouse brain cDNA. The full-length mouse G synthase and mouse STX (8) fragments were prepared by PCR amplification, subcloned, and then sequenced. These fragments were radiolabeled and used as probes.


RESULTS

Cloning and Nucleotide Sequencing of a New Sialyltransferase cDNA

Last year, the gene encoding G synthase (ST8Sia I) was cloned from human (7, 23, 24) and mouse.()Recently, we identified the enzymatic activity of mouse STX (ST8Sia II) as that of an N-glycan 2,8-sialyltransferase(8) . To obtain the new -2,8-sialyltransferase, we conducted PCR cloning experiments involving two degenerate oligonucleotide primers based on two highly conserved regions, sialyl motifs L and S, of human ST8Sia I (7) and rat ST8Sia II(16) . The PCR product corresponding to a 0.5-kb fragment was subcloned and sequenced. Among several clones, one, pCRO3, encoded a peptide exhibiting 35.6 and 41.9% identity to the 160-amino acid region of mouse ST8Sia I and mouse ST8Sia II,()respectively.

To isolate the complete coding sequence of the gene containing the 0.5-kb fragment, we screened a mouse brain cDNA library using the pCRO3 probe. Sequence analysis of the largest clone (1.7 kb, CRO3) revealed a continuous 380-amino acid open reading frame, including 74 bp of a 5`- and 465 bp of a 3`-non-coding region (Fig. 1).


Figure 1: Nucleotide and deduced amino acid sequences of mouse ST8Sia III. The nucleotide and amino acid sequences are numbered from the presumed start codon and initiation methionine, respectively. The doubleunderlined amino acids correspond to a putative transmembrane domain. The asterisks indicate potential N-glycosylation sites (Asn-X-Ser/Thr). Sialyl motifs L and S are boxed by solid and dashedlines, respectively. The positions of the PCR primers are indicated by arrows.



The predicted amino acid sequence encoding a protein with a type II transmembrane topology, as found for so far cloned sialyltransferases, consisted of a NH-terminal cytoplasmic tail, a transmembrane domain, a proline-rich stem region, and a large COOH-terminal active domain.

Cloned Gene Encoded a New 2,8-Sialyltransferase

To facilitate functional analysis of the new sialyltransferase, expression plasmid pcDSA-O3 was constructed and transfected into COS-7 cells, and the protein A-fused protein adsorbed to IgG-Sepharose in the medium was used as the enzyme source. In view of the sequence similarity of the sialyl motifs to ST8Sia I and ST8Sia II, we examined the sialyltransferase activity toward several gangliosides and glycoproteins as acceptor substrates. When fetuin was used as an acceptor, we detected strong sialyltransferase activity, as seen in the case of mouse ST8Sia II. No activity toward fetuin was observed in the culture medium from cells transfected with the vector alone. As shown in Fig. 2A, sialylated glycoproteins such as 1-acid glycoprotein, fetuin, ovomucoid, and transferrin served as acceptors for the new sialyltransferase. However, the new sialyltransferase did not exhibit activity toward asialo-glycoproteins at all.


Figure 2: Incorporation of sialic acids into various glycoproteins and glycolipids by the cloned sialyltransferase. A, various glycoproteins were incubated with the cloned sialyltransferase secreted by COS-7 cells transfected with pcDSA-O3 as a protein A-fused soluble enzyme, as described under ``Experimental Procedures,'' and then the reaction mixtures were analyzed by SDS-PAGE. Lane1, 1-acid glycoprotein; lane2, asialo-1-acid glycoprotein; lane3, fetuin; lane4, asialofetuin; lane5, ovomucoid; lane6, asialo-ovomucoid; lane7, human transferrin; lane8, bovine transferrin; lane9, bovine submaxillary mucin (BSM); lane10, no acceptor substrate glycoprotein. Lanes11 and 12 show the incorporation of sialic acids into fetuin by the culture medium of COS-7 cells transfected with the vector alone and pcDSA-O3, respectively. It should be noted that aisalo-glycoproteins did not serve as acceptors at all. B, the various glycolipids indicated in the panel were incubated with the cloned sialyltransferase, and the resulting glycolipids were analyzed by HPTLC with a solvent system of CHCl, CHOH, 0.2% CaCl (55:45:10). It should be noted that 2,3-SPG, but not 2,6-SPG, served as an acceptor for the new sialyltransferase.



C-Labeled sialic acid incorporation from CMP-[C]NeuAc was also observed when G was used as an acceptor substrate (Fig. 2B), as seen in the case of G synthase (ST8Sia I). This sialyltransferase exhibits low activity toward G. 2,3-SPG (Sia2,3Gal1,4GlcNAc1,3Gal1,4Glc1,1Cer) served as the best acceptor substrate among the glycolipids examined for this sialyltransferase. On the other hand, 2,6-SPG did not serve as an acceptor at all. Other gangliosides, such as G, G, G, G, and G as well as neutral glycosphingolipids, did not serve as acceptor substrates for this sialyltransferase (). There was no sialyltransferase activity toward gangliosides, including 2,3-SPG as well as neutral glycosphingolipids, in the medium obtained from COS-7 cells transfected with the vector without the insert.

The product synthesized from G by the sialyltransfer-ase comigrated with authentic G (NeuAc2,8NeuAc2,3Gal1,4Glc1,1Cer) on HPTLC with two different solvent systems. In addition, a C-sialylated ganglioside was eluted from DEAE-Sephadex at the position of disialylated gangliosides (data not shown). The linkages of the incorporated sialic acids were also confirmed by digestion of C-sialylated fetuin with linkage-specific sialidases. The incorporated C-labeled sialic acids were completely resistant to treatment with 2,3-specific or 2,6- and 2,3-specific sialidase but almost completely disappeared on treatment with 2,3-, 2,6-, and 2,8-specific sialidase (Fig. 3). Although it has been shown that NeuAc2,9NeuAc and NeuAc2,7NeuAc linkages are present in glycosphingolipids and glycoproteins, and we did not determine the linkages by chemical analyses, e.g. methylation analysis, the linkage analyses performed here indicated that the sialic acids incorporated by the new sialyltransferase were most probably linked to terminal sialic acids through 2,8 linkages and the synthesized Sia2,8Sia sequences. Therefore, the cloned gene encoded a new 2,8-sialyltransferase (ST8Sia III).


Figure 3: Linkage analysis of incorporated sialic acids. Fetuin was C-sialylated with the cloned ST8Sia III, and then the C-sialylated glycoprotein (1000 cpm, 10 µg) was digested with 2,3-specific sialidase (NANase I), 2,3- and 2,6-specific sialidase (NANase II), or 2,3-, 2,6-, and 2,8-specific sialidase (NANase III). C-Sialylated fetuin was also digested with N-glycanase (1.5 units) at 37 °C for 36 h. The resulting glycoproteins were subjected to SDS-PAGE and visualized with a BAS2000 image analyzer (A), and then the residual radioactivity at the position of enzyme-treated fetuin was quantified (B). C, I, II, III, and N indicate treatment with no enzyme, NANase I, NANase II, NANase III, and N-glycanase, respectively.



In view of the fact that the new 2,8-sialyltransferase exhibits activity toward 2,3-SPG and G but not toward 2,6-SPG, it may exhibit activity only toward the Sia2,3Gal sequence. This possibility was confirmed by measuring the activity toward desialylated fetuin. The activity of the new 2,8-sialyltransferase toward desialylated fetuin on treatment with 2,3-specific sialidase, as well as that on treatment with 2,3- and 2,6-specific or 2,3-, 2,6-, and 2,8-specific sialidase, was completely abolished (Fig. 4). Under the same digestion conditions, 2,3-SPG was desialylated by 2,3-specific sialidase, but 2,6-SPG was completely resistant to treatment with 2,3-specific sialidase.


Figure 4: Effects of treatment with sialidase and N-glycanase of fetuin on the activity of ST8Sia III. Fetuin was digested with NANase I, II, and III, respectively, and the resulting desialylated glycoproteins were incubated with protein A-fused soluble mouse ST8Sia III and subjected to SDS-PAGE; then, the radioactivity incorporated into the desialylated glycoproteins was visualized and quantified with a BAS2000 radio image analyzer. C, I, II, and III indicate treatment with no enzyme, NANase I, NANase II, and NANase III, respectively. Glycoproteins were first digested with N-glycanase. Then, the resulting de-N-glycosylated glycoproteins were incubated with mouse ST8Sia III and CMP-[C]NeuAc, and the incorporated sialic acids were visualized and counted. C and N indicate treatment with no enzyme and N-glycanase, respectively.



To determine whether the sialic acids are incorporated into N-linked oligosaccharides or O-linked oligosaccharides of fetuin, C-sialylated fetuin was digested with N-glycanase. The sialic acids incorporated into fetuin were completely released from the protein (Fig. 3). In addition, N-glycanase-treated fetuin did not serve as an acceptor (Fig. 4). Since G, G, G, and O-linked oligosaccharides in fetuin, which contain Sia2,3Gal1,3GalNAc sequences, did not serve as acceptors for this sialyltransferase, and 2,3-SPG was a good acceptor for it, it is specific for the Sia2,3Gal1,4GlcNAc sequences of N-linked oligosaccharides of glycoproteins as well as glycolipids.

Comparison of Acceptor Substrate Specificities between the New 2,8-Sialyltransferase and So Far Cloned 2,8-Sialyltransferases

The acceptor substrate specificity of the new 2,8-sialyltransferase (ST8Sia III) was compared with those of so far cloned 2,8-sialyltransferases, i.e. G synthase (ST8Sia I) and STX (ST8Sia II), as shown in . STX exhibited sialyltransfer activity only toward sialylated glycoproteins such as 1-acid glycoprotein or fetuin, with no activity being detected toward glycolipids including G and 2,3-SPG, while G synthase exhibited activity only toward G, i.e. not toward sialylated glycoproteins. Comparison of the substrate specificities of these two 2,8-sialyltransferases showed that of the new sialyltransferase was rather broad. Both sialylated glycoproteins and glycolipids served as acceptors for this sialyltransferase. Although the substrate specificity for glycoproteins of ST8Sia III was similar to that of ST8Sia II, fetuin acts as a better acceptor (10-fold) than 1-acid glycoprotein in the case of ST8Sia III, whereas the incorporation of sialic acids into fetuin was almost the same as that into -acid glycoprotein in the case of ST8Sia II. Thus, the structure of oligosaccharides on glycoproteins acting as acceptors for ST8Sia III is probably different from that in the case of ST8Sia II. On the other hand, the substrate specificity of ST8Sia III toward glycolipids was rather similar to that of ST8Sia I (G synthase) because both sialyltransferases were able to synthesize G from G. Surprisingly, ST8Sia III, but not ST8Sia I, could synthesize G from G. The apparent K values of ST8Sia III for 2,3-SPG, G, and G were 68, 588, and 3300 µM, respectively (). The V/K values shown in strongly indicate that 2,3-SPG is a much more suitable acceptor for ST8Sia III than G or G. In addition, the V/K values for fetuin indicate that ST8Sia III is much more specific for complex-type N-linked oligosaccharides, which contain Sia2,3Gal1,4GlcNAc sequences.

Tissue-specific and Developmentally Regulated Expression of the New 2,8-Sialyltransferase Gene

To evaluate the expression pattern and message size of the new sialyltransferase gene, we isolated total RNA from several mouse tissues (brain, heart, liver, lung, kidney, spleen, salivary gland, thymus, testis, and placenta) and analyzed them by Northern blotting. Three RNA species, 6.7, 2.2, and 1.7 kb, were expressed in brain (Fig. 5, A and B). Strong expression of a 3.7-kb transcript was observed in testis but not in brain (Fig. 5A). The distribution of these transcripts was similar to that in the case of STX (ST8Sia II).


Figure 5: Northern blot analyses of the ST8Sia III gene. A, total RNAs (5 µg each) were prepared from various adult mouse tissues. The hybridization probe was the 1205-bp XhoI fragment of ST8Sia III cDNA. B, total RNAs (5 µg each) were prepared from 14 and 20 postcoitum mouse embryo (E-14 and E-20) and 3-day-old, 2-week-old, and 8-week-old mouse brains. The full-length cDNAs for ST8Sia III, mouse ST8Sia II (STX), and mouse STSia I (G synthase) were used as probes, respectively.



We previously reported that expression of the STX (ST8Sia II) gene was detected in fetal and newborn mouse brain(8) . To compare the transcription pattern of the ST8Sia III gene with those of other 2,8-sialyltransferase genes during mouse brain development, total RNA prepared from the brains of 14 and 20 postcoitum fetal and 3-day-old, 2-week-old, and 8-week-old mice was analyzed by Northern blot hybridization (Fig. 5B). The transcripts of ST8Sia III appeared first in 20 postcoitum fetal brain and decreased thereafter during development. On the other hand, a 6.0-kb transcript of ST8Sia II was first detected in 14 postcoitum fetal brain, and then its level increased to reach a peak in 20 postcoitum fetal brain. Then, the ST8Sia II message decreased to an almost undetectable level by 2 weeks after birth. An approximately 9-kb transcript of ST8Sia I was also expressed in the brain throughout development, its level being highest in 20 postcoitum fetal brain. These results suggest that the three enzyme genes are expressed differently during brain development.


DISCUSSION

We cloned a new cDNA encoding an 2,8-sialyltransferase (ST8Sia III) from a mouse brain cDNA library by a PCR-based approach based on the sialyl motif sequences of so far cloned 2,8-sialyltransferases, i.e. G synthase (ST8Sia I) and STX (ST8Sia II). The expression of the ST8Sia III gene is regulated in a tissue- and developmental stage-specific manner, like STX, i.e. it is expressed in brain but not in other tissues except testis and strongly so in the embryonal stage. ST8Sia III synthesizes the Sia2,8Sia sequence through 2,8-sialyltransfer to Sia2,3Gal1,4GlcNAc-R, since 2,3-SPG, not 2,6-SPG, served as the best acceptor substrate for this enzyme among the several glycolipids examined. Comparison of the acceptor substrate specificities and kinetic properties revealed that ST8Sia III is specific to complex-type N-linked oligosaccharides of glycoproteins, which contain Sia2,3Gal1,4GlcNAc sequences, even though ST8Sia III exhibits activity toward glycosphingolipids such as 2,3-SPG. Although G and G serve as acceptors for ST8Sia III in vitro, they must not be actual acceptors in vivo because transfection of the ST8Sia III gene into G- or G-expressing cells did not lead to expression of G or G (data not shown). However, it should be noted that ST8Sia III exhibits the ability to synthesize Sia2,8Sia2,8Sia sequences from Sia2,8Sia sequences.

It has been shown that poly-2,8-sialosyl sialyltransferase activity is restricted to an early stage of development(25) . From the results of chemical analysis (26) and overexpression of Gal1,4GlcNAc2,6-sialyltransferase during Xenopus embryogenesis(27) , it was suggested that polysialic acids are attached to the Sia2,3-Gal residues of N-linked oligosaccharides. The gene expression pattern and substrate specificity of ST8Sia III suggested that it is very closely involved in the initial step of sialic acid polymerization, i.e. the biosynthesis of Sia2,8Sia2,3Gal of N-glycans.

We previously reported that ST8Sia II (STX), which is highly regulated during development of the brain, also only exhibits activity toward N-linked oligosaccharides of glycoproteins, and it was suggested to be involved in polysialic acid chain biosynthesis(8) . The reason why two different types of 2,8-sialyltransferase with similar substrate specificities toward N-linked oligosaccharides exist in mouse brain is not clear at present. One possibility is that the glycoproteins, which act as acceptor substrates for ST8Sia II (STX) and ST8Sia III, are different. Indeed, at least two brain glycoproteins, i.e. N-CAM and the subunit of voltage-gated sodium channels, are known to be polysialylated(3, 4, 5, 6) . Another possibility is that the two enzymes may exhibit activity toward almost the same acceptor glycoproteins in vivo but are controlled through different regulation systems. The gene expression of ST8Sia II (STX) and that of ST8Sia III during brain development is distinct from each other. The ST8Sia II gene appeared in fetal brain and then completely disappeared, at least in 2-week-old mouse brain, while the ST8Sia III gene was still expressed in 2-week-old mouse brain. It has been reported that the expression of polysialic acids of N-CAM is developmentally regulated, the embryonic form, having a high sialic acid content, undergoing postnatal conversion to the adult form with a low sialic acid content(6, 19, 20) . ST8Sia II and ST8Sia III may be responsible for the polysialic acid chain biosynthesis of the embryonic and postnatal forms of N-CAM, respectively. Identification of the actual substrates for the two sialyltransferases is required. However, the restricted and developmentally regulated expression of two different 2,8-sialyltransferases for glycoproteins in the brain led us to the conclusion that the synthesis of Sia2,8Sia sequences of glycoproteins is closely related to brain and neural development, like the differential expression of Sia2,8Sia-containing gangliosides.

  
Table: Comparison of the acceptor substrate specificities of the three cloned 2,8-sialyltransferases


  
Table: Kinetic properties of ST8Sia III



FOOTNOTES

*
This work was supported by the following Grants-in-aid for Scientific Research on Priority Areas 0625213 and 05274103 (to S. T.) and for the Encouragement of Young Scientists 06770042 (to N. K.), from the Ministry of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X80502.

§
To whom correspondence should be addressed. Tel.: 81-48-462-1111 (ext. 6521); Fax: 81-48-462-4692.

The nomenclature for gangliosides follows the system of Svennerholm (28).

The abbreviations used are: N-CAM, neural cell adhesion molecule; ST8Sia I, G synthase; ST8Sia II, STX reported as a developmentally regulated member of the sialyltransferase family by Livingston and Paulson (16); ST8Sia III, a new member of the 2,8-sialyltransferase family reported in this paper; PCR, polymerase chain reaction; HPTLC, high performance thin layer chromatography; 2,3-SPG, 2,3-sialylparagloboside (IVNeuAc nLcOseCer); 2,6-SPG, 2,6-sialylparagloboside (IVNeuAc nLcOseCer); kb, kilobase(s); bp, base pairs; PAGE, polyacrylamide gel electrophoresis.

Y. Yoshida, N. Kojima, T. Hamamoto, and S. Tsuji, GenBank/EMBL Data Bank accession no. X84235.

Y. Yoshida, N. Kojima, N. Kurosawa, Y.-C. Lee, and S. Tsuji, GenBank/EMBL Data Bank accession no. X83562.


ACKNOWLEDGEMENTS

We are grateful to Dr. Masao Iwamori (Tokyo University) for providing the 2,3- and 2,6-sialylparaglobosides.


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