Expression Cloning of Mouse cDNA of CMP-NeuAc:Lactosylceramide alpha 2,3-Sialyltransferase, an Enzyme That Initiates the Synthesis of Gangliosides*

Satoshi FukumotoDagger §, Hiroshi MiyazakiDagger , George Goto§, Takeshi UranoDagger , Keiko FurukawaDagger , and Koichi FurukawaDagger parallel

From the Dagger  Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Nagoya 466-0065 and the § Department of Pediatric Dentistry, Nagasaki University School of Dentistry, 1-7-1, Sakamoto, Nagasaki 852-8102, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression cloning of a cDNA for the alpha 2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha 2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha 2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gangliosides are acidic, sialic acid-containing glycosphingolipids and are ubiquitously expressed in all tissues and cells (1, 2). Their carbohydrate structures are strictly regulated, depending on the development of tissues, differentiation, and malignant transformation of cells (3, 4). Ganglioside synthesis is catalyzed by glycosyltransferases, and the combination of expression levels of various glycosyltransferase genes determines characteristic patterns of ganglioside expression (5-8). Among the gangliosides, GM3 is the simplest, having only three sugars, and is widely distributed in all over the body of vertebrates (1). Because GM3 is a precursor for all other ganglioside series gangliosides and hematosides (7), its biosynthesis strongly affects the levels of the more complex gangliosides.

Recent progress in the molecular cloning of glycosyl-transferase genes responsible for the synthesis of gangliosides has enabled us and other investigators to analyze the regulatory mechanisms for the ganglioside expression and biological functions of them by manipulation of those genes to modify the ganglioside profiles in cultured cells and in experimental animals (9). In our laboratory, we have isolated genes encoding ganglioside GM2/GD21 synthase (10), GD3 synthase gene (11), and GM1/GD1b/GA1 synthase (12) and have analyzed expression of these genes in various tissues and malignant cells (8, 13, 14). We have also established gene knock-out mice lines in which all complex ganglioside series gangliosides are genetically eliminated in the whole body (15). The resulting phenotypic changes in those mutant mice pose new questions about the substitutive roles of simpler ganglioside structures (e.g. GM3 and GD3) for the eliminated complex gangliosides.

In the present study, we isolated a mouse cDNA clone that determines expression of ganglioside GM3 and have analyzed its expression pattern in various tissues. This gene product corresponds to GM3-specific sialyltransferase (CMP-NeuAc:lactosylceramide alpha 2,3-sialyltransferase, alpha 2,3S-T) (EC 2.4.99.-). Because of the critical role that GM3 synthase plays in the biosynthesis of all gangliosides, this probe will be extremely useful for the analysis of the biological functions of acidic glycosphingolipids.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue Cultures and Cells-- Cell lines were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented by 10% fetal bovine serum. L cell and B78 (a subline of mouse B16 melanoma) were kindly provided by Dr. A. P. Albino at Sloan-Kettering Cancer Center in New York. L cell is a mouse fibroblast line with no alpha 2,3-sialyltransferase activity (for lactosylceramide acceptor) (16) and minimal GM3 expression. B78 expresses GM3 ganglioside almost exclusively (10).

Plasmids and cDNA Library-- A cDNA library of mouse fibrosarcoma CMS5j was prepared using poly(A)+ RNA and the pCDM8 expression vector (Invitrogen, San Diego, CA). This library contained 5.0 × 106 independent colonies. The strain of bacterial host used was Escherichia coli MC1061/P3.

Expression Cloning of Mouse GM3 Synthase cDNA-- Plasmids of the cDNA library were amplified, purified, and then transfected into L cells together with plasmid pdl3027 (polyoma T gene kindly provided by C. Basilico (17)) using DEAE-dextran (Amersham Pharmacia Biotech) as described previously (10). Subconfluent L cells, 2 × 106 in 10-cm dishes (Corning, Corning, NY), were co-transfected with 8 µg each of cDNA library plasmid and pdl3027. After 48-60 h, the transfected cells were detached from plates and incubated with a mouse monoclonal antibody M2590 (mouse IgM) (18) (Nihon Biotest Research Institute, Tokyo, Japan) at 20 µg/ml on ice for 45 min. After washing, cells were plated on dishes coated with goat anti-mouse IgM (Cappel, Durham, NC) as described previously (10). Plasmid DNA was rescued from the panned cells by preparing Hirt extracts and transformed into MC1061/P3. Expanded plasmids were transfected again, and the same procedure was repeated five times. Using isolated colony extracts and microscale DEAE-dextran transfection and immunofluorescence assay, we isolated a cDNA clone that determines the expression of ganglioside GM3 in L cells.

DNA Sequencing-- The sequence was determined by dideoxynucleotide termination sequencing using the PRISM dye terminator cycle sequencing kit and model 373 DNA sequencer (Applied Biosystems, Foster City, CA). The cDNA insert of the cloned pM3T-7 was cleaved by HindIII and NotI and then inserted into HindIII/NotI sites of phagemid BlueScript- vector. Deletion mutants of this clone were prepared with a Kilo-Sequence deletion kit (Takara, Kyoto).

Transfection of the Cloned cDNA-- Isolated cDNA clone pM3T-7 (in pCDM8) was transfected into L cells by the DEAE-dextran method, and the transfectant cells were used for the analyses of ganglioside expression and of the enzyme activity after 60 h. Plasmid pM3T-7/MIKneo was constructed by inserting XhoI/NotI fragment of pM3T-7 into XhoI/NotI sites of pMIKneo expression vector (kindly provided by Dr. K. Maruyama at Tokyo Medical Dental School).

Enzyme Assay-- The enzyme activity of alpha 2,3S-T was measured as described previously (19). Briefly, to prepare membrane fractions, samples were lysed using a nitrogen cavitation apparatus. Nuclei were removed by low speed centrifugation, and the supernatant was centrifuged at 105,000 × g for 1 h at 4 °C. To analyze enzyme activity of alpha 2,3S-T, the reaction mixture contained the following in a volume of 50 µl: 100 mM sodium cacodylate-HCl (pH 6.0), 10 mM MgCl2, 0.3% Triton CF-54 (Sigma), 325 µM lactosylceramide (Lac-Cer) (Sigma), 400 mM CMP-NeuAc (Sigma), CMP-[14C]NeuAc (105 dpm) (NEN Life Science Products), and membrane containing 30 µg of protein. After incubation for 3 h at 37 °C in a shaking water bath, the products were isolated by a C18 Sep-Pak cartridge (Warters, Milford, MA) and analyzed by TLC and fluorography as described (16). The protein concentration in the membrane preparation was determined by Lowry's methods (20). Glycosphingolipids and asialo-fetuin were purchased from Sigma.

RNA Extraction and Northern Blotting-- Total RNA was extracted from various tissues of mice with acid phenol methods as described previously (21). 20 µg each of total RNA was applied and separated on 1.0% agarose/formaldehyde gel. After blotting onto nylon membrane (GeneScreen Plus, Dupont), they were hybridized with [32P]dCTP-labeled cDNA probe of pM3T-7. The expression levels of the gene among tissues were estimated by using a Bio-Imaging Analyzer BAS2000 (Fuji Film, Tokyo).

Extraction of Gangliosides and TLC Analysis-- Gangliosides were extracted according to Furukawa et al. (22). Briefly, glycolipids were extracted from about 400 µl of packed cells of transfectants and control cells transfected with a vector alone using chloroform/methanol (2:1, 1:1, and 1:2) sequentially. After desalting, gangliosides were isolated by DEAE-Sephadex A-50 (Amersham Pharmacia Biotech) ion exchange chromatography. TLC was performed on a high performance TLC plates (Merck) using the solvent system chloroform:methanol:2.5 N NH4OH (60:35:8) and sprayed by resorcinol for detection of bands. For standard gangliosides, bovine brain ganglioside mixture (Wako Chemical Inc., Tokyo) and GM3 (Snow-brand, Tokyo) were used.

Homology Search-- Nucleotide and amino acid sequence homology search was carried out using the internet program BLAST (National Center for Biotechnology Information). Amino acid sequence and hydropathy analyses were performed with a software GENETYX-MAC version 8.0 (Software Development, Tokyo, Japan).

Flow Cytometry-- Cell surface expression of gangliosides was analyzed by flow cytometry (Becton Dickinson, Mountain View, CA) using anti-GM3 monoclonal antibody M2590. The cells were incubated with monoclonal antibodies for 45 min on ice and stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgM (Cappel). Intensity of staining was measured in arbitrary units as the log of fluorescent intensity and displayed on a 4 decade scale. Control cells for flow cytometry were prepared by using second antibody alone.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of alpha 2,3S-T cDNA Clones-- L cells were transfected with a cDNA library prepared from CMS5j and pdl3027 containing the polyoma T gene. After five cycles of transfection, panning, and Hirt extraction, a cDNA clone pM3T-7 was obtained.

Synthesis of GM3 in the Transfectant Cells of the Cloned cDNA-- pM3T-7 in pCDM8 or pMIKneo vector was transiently introduced into L cells, which lack GM3 expression and GM3 synthase activity (16), to confirm in flow cytometry whether the clone determines the expression of GM3 on the cell surface. As shown in Fig. 1, L cells transfected with pM3T-7 expressed definite amount of GM3, whereas those transfected with the vector alone did not. Glycosphingolipids extracted from the transfectant cells were analyzed to confirm GM3 synthesis. As shown in Fig. 2, the transfectant cells with pM3T-7/MIKneo showed a definite GM3 band in TLC, although the parent cell (data not shown) and the transfectant cells with pMIKneo alone showed a very faint band, suggesting that the parent L cell expresses a minimal level of the enzyme.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Transient expression of GM3 in L cells. A, GM3 synthesis reaction. B, flow cytometry of cDNA transfectant cells. Cloned cDNA pM3T-7 in pCDM8 (right panel) or pCDM8 alone (left panel) was transfected by DEAE-dextran into L cells. Expression of GM3 was analyzed after 60 h using GM3-specific monoclonal antibody M2590 in flow cytometry as described under "Materials and Methods." The x axis is fluorescence intensity, and the y axis is relative cell number. Thin lines are with monoclonal antibody, and thick lines are controls.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Synthesis of GM3 in the L cells transfected with alpha 2,3S-T cDNA. L cells were transiently transfected with pM3T-7/MIKneo or pM3T-7/CDM8 (data not shown), and glycosphingolipids were extracted after 60 h as described under "Materials and Methods." A, the acidic fractions were isolated and separated in TLC. St, ganglioside standard of bovine brain gangliosides (totally 10 µg) plus purified GM3 (0.3 µg). Lane L cell/pMIKneo is gangliosides from L cells transfected with pMIKneo alone. Lane L cell/alpha 2,3S-T/pMIKneo is gangliosides from L cells transfected with 1 µg/ml of pM3T-7/MIKneo. The solvent used was chloroform, methanol, 0.2% CaCl2 (60:35:8). Resorcinol spray was performed for the detection of bands. B, comparison of GM3 quantity measured by the intensities of standard GM3 bands (data not shown) and bands in A using a densitometer.

Sequence of the Insert of pM3T-7-- Fig. 3 shows the entire sequence of the insert of pM3T-7 determined by sequence analysis of the original pM3T-7 in pCDM8 or deletion constructs of phagemid BlueScript- containing XhoI/NotI fragment. Total size of this insert was 1710 base pairs comprising a 12-base pair 5'-untranslated region, a continuous open reading frame of 1077 base pairs, and 621 base pairs of 3'-untranslated region. The initiation codon at the beginning of the open reading frame is embedded within a sequence similar to the Kozak consensus initiation sequence (23, 24). This open reading frame predicts a 359-amino acid protein with a molecular mass of 41,244 daltons. Search of currently available protein and nucleic acid data bases identified a number of genes with significant sequence homology to this cDNA. A majority of those genes were sialyltransferases, and highly homologous regions were detected at the sequences of sialylmotif L, sialyl motif S, and a few other regions. Among sialyl-transferase cDNAs, ST3Gal III (ST3N) (Galbeta 1,3/1,4GlcNAc alpha 2,3S-T) generally showed the highest homology.2 The second group is ST3Gal III (Galbeta 1,3GalNAc/Galbeta 1,4GlcNAc alpha 2,3S-T). The next groups are ST3Gal IA (ST3O) (Galbeta 1,3GalNAc alpha 2,3S-T) and ST3Gal II (ST3GalA2) (Galbeta 1,3GalNAcalpha 2,3S-T). These latter two groups showed approximately similar levels of homology. The next groups are ST6 groups and ST8Sia groups, which showed the lowest homology among these sialyltransferases. Inspection and hydropathy of the predicted protein sequence suggested that this enzyme molecule has a similar structural organization to those of reported glycosyltransferase genes (Fig. 3). A single hydrophobic segment with 17 amino acids was present at near the amino terminus. This putative signal anchor sequence would place 13 residues within the cytosolic compartment and 329 amino acids within the Golgi lumen as a catalytic domain. The predicted amino acid sequence indicated the presence of three N-glycosylation sites at amino acids 180-182, 224-226, and 334-336 (Fig. 3).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 3.   Nucleotide sequences of cloned alpha 2,3S-T pM3T-7. Top, deduced amino acid sequences for the single open reading frame. The putative transmembrane region with 17 amino acids was double underlined. Candidates of N-glycosylation site were marked by a single underline. Bottom, hydropathy analysis of the coding region based on the deduced amino acids according to Hopp and Woods (40).

Products of the alpha 2,3S-T cDNA Can Catalyze Only GM3 Synthesis-- We then analyzed the enzyme activity of GM3 synthase in membrane extracts from those transfectants. As shown in Fig. 4A, the transfectant cells with pM3T-7/MIKneo (alpha 2,3S-T/pMIKneo) showed very high GM3 synthase activity when lactosylceramide was used as an acceptor (1394 units). On the other hand, transfectant cells with pMIKneo alone were completely negative. Thus, this clone was able to determine the expression of GM3 and alpha 2,3S-T activity when introduced into a mouse cell line, strongly suggesting that this cDNA encodes the GM3 synthase. Using the membrane extracts, the enzyme activity was examined toward various acceptor compounds such as galactosylceramide, glucosylceramide, asialo-GM2 (GA2), asialo-GM1 (GA1), GM3, GM1, GD1b, GD1a, GT1b, etc., as shown in Fig. 4B. Asialo-fetuin was also examined. None of the acceptors examined showed significant levels of [14C]NeuAc incorporation except lactosylceramide, indicating that this sialyltransferase is almost specific for the synthesis of GM3 as reported by Iber et al. (25). Only GA1 and GA2 showed minimal levels of incorporation. Asialo-fetuin also showed no incorporation of [14C]NeuAc (data not shown).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   alpha 2,3S-T activity in the extracts from transient transfectant cells of the cloned cDNA. A, enzyme activity with lactosylceramide as acceptor was measured as described under "Materials and Methods." Membrane fractions were prepared from B78 and L cells transfected with pMIKneo alone or pM3T-7/MIKneo (alpha 2,3S-T/pMIKneo). As shown in the inset, GM3 band was observed in the TLC of the products. B, comparison of enzyme activity for various acceptor structures (325 µM each was used) as indicated in the figure.

Northern Blot Analysis-- Expression levels of the alpha 2,3S-T gene in various tissues of mice were examined by Northern blots using total RNA. Among various tissues examined, brain showed the highest level, and then testis, heart and liver also showed relatively high levels, whereas almost all tissues showed some levels of the gene expression (Fig. 5). Mouse melanoma B78 also expressed a high level of transcript, consistent with the serological data indicating high levels of GM3 expression.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 5.   Northern blots of alpha 2,3S-T (GM3 synthase) gene. A, 20 µg each of total RNA from mouse tissues was separated on agarose gel and then blotted onto nylon membrane. Hybridization with 32P-labeled probe derived from pM3T-7 was performed as described under "Materials and Methods." B, relative expression levels of mRNA of alpha 2,3S-T gene among mouse tissues were measured by Bio-Imaging Analyzer (Fuji, Tokyo) and presented as a percentage of the value of B78.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have identified a cDNA that determines the expression of GM3 and induces alpha 2,3S-T activity in transfected cells. Furthermore, the deduced amino acid sequence indicated the presence of the sialyl motifs, which has been recognized among all mammalian and chicken sialyltransferases cloned so far (26), suggesting that this cDNA represents the GM3 synthase gene. Examination of the sequence showed features in common with other alpha 2,3-sialyltransferases. As shown in Fig. 6, the derived GM3 synthase sequence contained a number of characteristic regions commonly detected only in the sequences of ST3Gal alpha 2,3-sialyltransferases (see enclosed boxes in sialyl motif L and S). These regions may be involved in the recognition of acceptor structures. The conserved YPE sequence was also noted by Sasaki in a recent review (27). In addition, the ST8Sia group showed a common sequence (NPS) in the carboxyl terminus region of the sialyl motif L. This sequence contrasted with the sequence YPE, which was commonly detected among ST3Gal group, supporting the idea that this region might be involved in the acceptor recognition.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Sequence alignment of sialyl motif regions of representative sialyltransferases. Amino acid sequences of sialyl motif L and S of nine sialyltransferases were aligned. Top, sialyl motif L. Bottom, sialyl motif S. Identical amino acid(s) in the majority of sialyltransferases are indicated by bold letters, and identical amino acids only among ST3Gal(s) were enclosed by boxes. Derivations are as follows: ST3Gal(GM3), reported in this paper; ST3Gal I, ST3GalA1 (41); ST3Gal II, ST3Gal A.2 (42); ST3Gal III, mST3Gal III (43); ST3Gal IV, mST3Gal IV (43); ST6GalNAc II, Galbeta 1,3GalNAc-specific GalNAcalpha 2,6S-T (44); ST8SiaI, GD3 synthase (11); ST8SiaII, polysialic acid synthase (45); ST8SiaIV, N-glycan alpha 2,8S-T (46).

The properties of the GM3 synthase predicted from the amino acid sequence (Fig. 3, A and B) and demonstrated by the analysis with extracts from cDNA-transfected cells were not so different from those of previously purified GM3 synthases. For example, Km for lactosylceramide and CMP-NeuAc of the GM3 synthase expressed in the transfected cells of pM3T-7 were 270 µM and 313 µM, respectively (data not shown). These values were of similar magnitude to the results of Preuss et al. (28), i.e. 80 µM (Km for lactosylceramide) and 210 µM (Km for CMP-NeuAc), respectively. High specificity for lactosyl-ceramide was also similar, although a few acceptors besides lactosylceramide also showed low levels of activity in their results. On the other hand, the predicted molecular mass of the enzyme was 41,244 Da, whereas that in their paper was 76 kDa in SDS-polyacrylamide gel electrophoresis. This discrepancy might be partially due to the glycosylation of the enzyme protein. Actually, the predicted amino acid sequence contained three possible sites of N-glycosylation (Fig. 3A). Otherwise, differences of the tissues and species used as sources of mRNA and enzymes need to be considered.

Among gangliosides, GM3 has the simplest carbohydrate structure and is located at the starting point of the synthesis of all ganglioside series gangliosides. Almost all mammalian tissues and cells contain GM3 (1), suggesting its important biological role. Moreover, a number of studies have shown that GM3 functions in the regulation of platelet-derived growth factor receptor (29) and epidermal growth factor receptor (30-32) in cell adhesion (33-35), in the regulation of keratinocyte proliferation (36), and in the promotion of the differentiation of HL60 leukemia cells (37). Recently, Yamamura et al. (38) reported that GM3 co-localized with various signaling molecules such as c-Src and a low molecular GTP-binding protein Rho on the cell surface of B16 forming a glycolipid-enriched microdomain. Therefore, GM3 itself might play important roles at diverse situations for the regulation of cell proliferation and differentiation. Following the isolation of the GM3 synthase gene, the effects of GM3 endogenously generated in the cells can be studied to confirm the possible functions described above. Moreover, human cDNA of GM3 synthase has recently been cloned by Ishii et al. (39), enabling us to analyze the regulatory mechanisms of leukocyte differentiation by gangliosides.

Besides the importance of GM3 itself, alpha 2,3S-T is also important, because it can regulate the amount of all gangliosides in particular tissues and cells. For example, although a majority of human melanoma cells characteristically express ganglioside GD3, they also express very high level of GM3. The high GD2 expression in neuroblastoma cells is also partly based on the high expression of GM3. Because GM3 expression is ubiquitous, its importance is often not appreciated. However, the quantity of GM3 is quite different among different normal tissues and various tumor cells. GM3 seems to control the expression levels of more specific and complex gangliosides by adjusting the amounts of precursor structures. Therefore, the mechanisms for the regulation of the gene expression of alpha 2,3S-T are of great importance and interest.

Mammalian brain tissues usually express low levels of GM3 compared with the level of complex gangliosides. However, the actual synthesis of GM3 should be much higher than expected from the amount of GM3, because all complex ganglioside series gangliosides are synthesized through GM3. Results of Northern blotting clearly indicated that GM3 synthesis takes place most actively in the brain among the normal tissues tested (Fig. 5). During the study of substrate specificity of the cloned alpha 2,3S-T, we found that GD1b specifically inhibited the synthesis of GM3 with endogenous acceptor lactosylceramide in membrane preparations (data not shown). This fact indicates that later members of the biosynthetic pathway (GD1b in this case) may regulate alpha 2,3S-T activity by a feedback mechanism in the brain tissue. The regulatory mechanisms of GM3 expression at the levels of gene expression and post-translation are presently being investigated.

    ACKNOWLEDGEMENTS

We thank Dr. Y. Yoshikai in the Department of Bioprotection at Nagoya University for providing the opportunity to use the flow cytometry machine and Dr. K. O. Lloyd at Memorial Sloan-Kettering Cancer Center for carefully reading the manuscript.

    FOOTNOTES

* This work was supported by Grants-in-Aid for Scientific Research of Priority Areas 05274103 and Core of Excellence from the Ministry of Education, Science, Sports, and Culture 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) AB013302.

These authors contributed equally to this work.

parallel To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069.

2 We propose here to assign the name of ST3Gal V to GM3 synthase.

    ABBREVIATIONS

The abbreviations used are: GM2, GalNAcbeta 1right-arrow4 (NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glc Cer; GD2, GalNAcbeta 1right-arrow4 (NeuAcalpha 2right-arrow8 NeuAcalpha 2right-arrow 3)Galbeta 1right-arrow4Glc-Cer; GD3, NeuAcalpha 2right-arrow8 NeuAcalpha 2right-arrow3 Galbeta 1right-arrow4 Glc-Cer; GM3, NeuAcalpha 2right-arrow3Galbeta 1right-arrow4Glc-Cer; Lac-Cer (CDH), Galbeta 1right-arrow4Glc-Cer; GA2, GalNAcbeta 1right-arrow4Galbeta 1right-arrow4Glc-Cer; GM1, Galbeta 1right-arrow3GalNAcbeta 1right-arrow4 (NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glc-Cer; GA1, Galbeta 1right-arrow3GalNAcbeta 1right-arrow4Galbeta 1right-arrow4 Glc-Cer; GD1a, NeuAcalpha 2right-arrow3Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Gal beta 1right-arrow4Glc-Cer; GD1b, Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow8NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glc-Cer; GT1b, Neu Acalpha 2right-arrow3Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(Neu Acalpha 2right-arrow8NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glc-Cer (ganglioside nomenclature is based on that of Svennerholm (47)); alpha 2, 3S-T, alpha 2,3-sialyltransferase (the nomenclature of sialyltransferases is based on that of Tsuji et al. (48)).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Wiegandt, H. (ed) (1985) Glycolipids, pp. 199-260, Elsevier Science Publishing Co., Inc., New York
  2. Kaufmann, B., Basu, S., and Roseman, S. (1967) in Inborn Disorders of Sphingolipid Metabolism (Anderson, S. M., and Volk, B. W., eds), pp. 193-213, Pergamon, New York
  3. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764[CrossRef][Medline] [Order article via Infotrieve]
  4. Furukawa, K., and Lloyd, K. O. (1990) in Human Melanoma: From Basic Science to Clinical Application (Ferrone, S., ed), pp. 15-30, Springer, Heidelberg, Germany
  5. Pohlentz, G., Klein, D., Schwarzmann, G., Schmitz, D., and Sandhoff, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7044-7048[Abstract]
  6. Iber, H., Kaufmann, R., Pohlentz, G., Schwarzmann, G., and Sandhoff, K. (1989) FEBS Lett. 248, 18-22[CrossRef][Medline] [Order article via Infotrieve]
  7. Iber, H., and Sandhoff, K. (1989) FEBS Lett. 254, 124-128[CrossRef][Medline] [Order article via Infotrieve]
  8. Yamashiro, S., Ruan, S., Furukawa, K., Tai, T., Lloyd, K. O., Shiku, H., and Furukawa, K. (1993) Cancer Res. 53, 5305-5400
  9. Lloyd, K. O., and Furukawa, K. (1998) Glycoconj. J. 15, 627-636[CrossRef][Medline] [Order article via Infotrieve]
  10. Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, K. O., Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089[Abstract/Free Full Text]
  11. Haraguchi, M., Yamashiro, S., Yamamoto, A., Furukawa, K., Takamiya, K., Lloyd, K. O., Shiku, H., and Furukawa, K (1994) Proc. Natl. Acad. Sci. U. S. A 91, 10455-10459[Abstract/Free Full Text]
  12. Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., Furukawa, K., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799[Abstract/Free Full Text]
  13. Yamashiro, S., Okada, M., Haraguchi, M., Furukawa, K., Lloyd, K. O., Shiku, H., and Furukawa, K. (1995) Glycoconj. J 12, 894-900[Medline] [Order article via Infotrieve]
  14. Yamamoto, A., Haraguchi, M., Yamashiro, S., Fukumoto, S., Furukawa, K., Takamiya, K., Atsuta, M., Shiku, H., and Furukawa, K. (1996) J. Neurochem. 66, 26-34[Medline] [Order article via Infotrieve]
  15. Takamiya, K., Yamamoto, A., Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., Sakae, M., Kishikawa, M., Shiku, H., Furukawa, K., and Aizawa, S. (1996) Proc. Natl. Acad Sci. U. S. A. 93, 10662-10667[Abstract/Free Full Text]
  16. Yamashiro, S., Haraguchi, M., Furukawa, K., Takamiya, K., Yamamoto, A., Nagata, Y., Lloyd, K. O., Shiku, H., and Furukawa, K. (1995) J. Biol. Chem. 270, 6149-6155[Abstract/Free Full Text]
  17. Dailey, L., and Basilico, C. (1985) J. Virol. 54, 739-749[Medline] [Order article via Infotrieve]
  18. Wakabayashi, S., Saito, T., Shinohara, N., Okamoto, S., Tomioka, H., and Taniguchi, M. (1984) J. Invest. Dermatol 83, 128-133[Abstract]
  19. Ruan, S., and Lloyd, K. O. (1992) Cancer Res. 52, 5725-5731[Abstract]
  20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  21. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  22. Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K., Lundblad, A., Mattes, M. J., and Lloyd, K. O. (1985) Biochemistry 24, 7820-7826[Medline] [Order article via Infotrieve]
  23. Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve]
  24. Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract]
  25. Iber, H., vanEchten, G., and Sandhoff, K. (1991) Eur. J. Biochem 195, 115-120[Abstract]
  26. Paulson, J. C., and Colley, K. J. (1989) J. Biol. Chem 264, 17615-17618[Free Full Text]
  27. Sasaki, K. (1996) Trends Glycosci. Glycotechnol. 8, 195-215
  28. Preuss, U., Gu, X., Gu, T., and Yu, R. K. (1993) J. Biol. Chem 268, 26273-26278[Abstract/Free Full Text]
  29. Bremer, E. G., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825[Abstract/Free Full Text]
  30. Bremer, E. G., Schlessinger, J., and Hakomori, S. (1986) J. Biol. Chem 261, 2434-2440[Abstract/Free Full Text]
  31. Weis, F. M., and Davis, R. J. (1990) J. Biol. Chem. 265, 12059-12066[Abstract/Free Full Text]
  32. Song, W., Welti, R., Hafner-Strauss, S., and Rintoul, D. A. (1993) Biochemistry 32, 8602-8607[Medline] [Order article via Infotrieve]
  33. Kojima, N., and Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558[Abstract/Free Full Text]
  34. Kojima, N., Shiota, M., Sadahira, Y., Handa, K., and Hakomori, S. (1992) J. Biol. Chem. 267, 17264-17270[Abstract/Free Full Text]
  35. Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222[Abstract/Free Full Text]
  36. Paller, A. S., Amsmeier, S. L., Alvarez-Franco, M., and Bremer, E. G. (1993) J. Invest. Dermatol. 100, 841-845[Abstract]
  37. Nojiri, H., Takaku, F., Terui, Y., Miura, Y., and Saito, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 782-786[Abstract]
  38. Yamamura, S., Handa, K., and Hakomori, S. (1997) Biochem. Biophys. Res. Commun 236, 218-222[CrossRef][Medline] [Order article via Infotrieve]
  39. Ishii, A., Ohta, M., Watanabe, Y., Sakoe, K., Nakamura, M., Inokuchi, J., Sanai, Y., and Saito, M. (1998) J. Biol. Chem 273, 31652-31655[Abstract/Free Full Text]
  40. Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad Sci. U. S. A. 78, 3824-3828[Abstract]
  41. Lee, Y.-C., Kurosawa, N., Hamamoto, T., Nakaoka, T., and Tsuji, S. (1993) Eur. J. Biochem. 216, 377-385[Abstract]
  42. Lee, Y.-C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T., and Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033[Abstract/Free Full Text]
  43. Kono, M., Ohyama, Y., Lee, Y.-C., Hamamoto, T., Kojima, and Tsuji, S. (1996) Glycobiology 7, 469-479[Abstract]
  44. Kurosawa, N., Inoue, M., Yoshida, Y., and Tsuji, S (1996) J. Biol. Chem. 271, 15109-15116[Abstract/Free Full Text]
  45. Yoshida, Y., Kurosawa, N., Kanematsu, T., Kojima, N., and Tsuji, S. (1996) J. Biol. Chem. 271, 30167-30173[Abstract/Free Full Text]
  46. Yoshida, Y., Kojima, N., and Tsuji, S. (1995) J. Biochem. (Tokyo) 118, 658-664[Abstract]
  47. Svennerholm, L. (1963) J. Neurochem. 10, 613-623[Medline] [Order article via Infotrieve]
  48. Tsuji, S., Datta, A. K., and Paulson, J. C. (1996) Glycobiology 6, v-xiv[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.