Molecular Cloning of a Novel
2,3-Sialyltransferase (ST3Gal VI)
That Sialylates Type II Lactosamine Structures on Glycoproteins and
Glycolipids*
Tetsuya
Okajima
,
Satoshi
Fukumoto
§,
Hiroshi
Miyazaki
,
Hideharu
Ishida¶,
Makoto
Kiso¶,
Keiko
Furukawa
,
Takeshi
Urano
, and
Koichi
Furukawa
From the
Department of Biochemistry, Nagoya
University School of Medicine, Tsurumai, Nagoya 466-0065, the
§ Department of Pediatric Dentistry, Nagasaki University
School of Dentistry, Sakamoto, Nagasaki 852-8501, and the
¶ Department of Applied Bio-organic Chemistry, Faculty of
Agriculture, Gifu University, Gifu 501-1193, Japan
 |
ABSTRACT |
A novel member of the human
CMP-NeuAc:
-galactoside
2,3-sialyltransferase (ST) subfamily,
designated ST3Gal VI, was identified based on BLAST analysis of
expressed sequence tags, and a cDNA clone was isolated from a human
melanoma line library. The sequence of ST3Gal VI encoded a type II
membrane protein with 2 amino acids of cytoplasmic domain, 32 amino
acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III,
ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts
from L cells transfected with ST3Gal VI cDNA in a expression vector
and a fusion protein with protein A showed an enzyme activity of
2,3-sialyltransferase toward Gal
1,4GlcNAc structure on
glycoproteins and glycolipids. In contrast to ST3Gal III and
ST3Gal IV, this enzyme exhibited restricted substrate specificity,
i.e. it utilized Gal
1,4GlcNAc on glycoproteins, and
neolactotetraosylceramide and neolactohexaosylceramide, but not
lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is
involved in the synthesis of sialyl-paragloboside, a precursor of
sialyl-Lewis X determinant.
 |
INTRODUCTION |
Sialyltransferases are a family consisting of more than 14 enzymes
that catalyze the transfer of sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid
(CMP-NeuAc)1 to terminal
positions on sugar chains of glycoproteins and glycolipids. Terminal
NeuAc residues are key determinants of carbohydrate structures involved
in a variety of biological processes and are widely distributed in
many cell types (1-3). For example, sialyl-Lewis X
(sialyl-Lex) determinants have been reported to be ligands
for the three known selectins (E-, P-, and L-selectins), which are cell
adhesion molecules involved in the recruitment of leukocytes to
lymphoid tissues and the sites of inflammation (4-6). Furthermore,
increased expression of sialyl-Lex determinants was
suggested to contribute to the metastatic behavior of carcinoma cells
(7). Glycosyltransferases involved in the synthesis of
sialyl-Lex structures are
1,3-N-acetylglucosaminyltransferase (8),
1,4-galactosyltransferase (9, 10),
1,3-fucosyltransferases
(11-15), and
2,3-sialyltransferases, as described below.
To date, four enzymatically distinct human
2,3-sialyltransferase
genes have been cloned and exhibited distinct acceptor substrate specificities. ST3Gal I (16, 17) and II (18, 19) synthesize the
sequence NeuAc
2,3Gal
1,3GalNAc common to many O-linked
oligosaccharides and glycolipids like GM1b, GD1a, and GT1b. ST3Gal III
(20, 21) forms a less common sequence, NeuAc
2,3Gal
1,3GlcNAc.
ST3Gal IV (22, 23) is capable of forming the terminal
NeuAc
2,3Gal
1,3GalNAc and the NeuAc
2,3Gal
1,4GlcNAc sequence
found in the carbohydrate moieties of glycoproteins and glycolipids.
Recently, we have cloned mouse ST3Gal V (GM3
synthase),2 which exhibits
activity almost exclusively toward LacCer. Among five
2,3-sialyltransferases so far isolated, human ST3Gal III and IV are
candidates for involvement in the formation of the sialyl-Lex determinant in vivo. However, human
ST3Gal III has been shown to utilize Gal
1,3GlcNAc much more
efficiently than Gal
1,4GlcNAc as an acceptor in vitro.
The reported substrate specificities of ST3Gal IV have been
contradictory for its preference between Gal
1,3GalNAc and
Gal
1,4GlcNAc structure, and for utilization of glycolipids.
Therefore, to date, no human
2,3-sialyltransferase that shows high
and clear acceptor specificity toward Gal
1,4GlcNAc sequence has yet
been identified.
In this study, using human expressed sequence tags, we have cloned a
novel
2,3-sialyltransferase, designated ST3Gal VI. ST3Gal VI is a
novel Gal
1,4GlcNAc
2,3-sialyltransferase with high specificity for neolactotetraosylceramide and neolactohexaosylceramide as glycolipid substrates. Moreover, ST3Gal VI prefers oligosaccharides containing the terminal Gal
1,4GlcNAc structure much more than those containing the Gal
1,3GlcNAc structure, suggesting it is involved in the formation of the sialyl-Lex determinant on
glycoproteins and glycolipids. The expression of the gene among normal
human tissues and cell lines was also analyzed.
 |
EXPERIMENTAL PROCEDURES |
Nomenclature of Cloned Sialyltransferase--
So far five
members of human
2,3-sialyltransferase (ST3Gal) have been cloned:
ST3Gal I (16), ST3Gal II (18), ST3Gal III (21), ST3Gal IV (22, 23), and
ST3Gal V (GenBankTM accession no. AB013302). The
2,3-sialyltransferase cloned in this study is referred to as ST3Gal
VI according to Tsuji et al. (24).
Materials--
CMP-NeuAc, LacCer, GM2, GM1, GD1a, GalCer, GT1b,
GQ1b, GA1, asialofetuin, and bovine submaxillary asialomucin, were
purchased from Sigma. GM3 was purchased from Snow Brand Milk Products
Co. (Tokyo, Japan). [
-32P]dCTP was from ICN (Costa
Mesa, CA). Lex ceramide
(Gal
1,4(Fuc
1,3)GlcNAc
1,3Gal
1,4Glc
1Cer) and
lactotetraosylceramide (Lc4) were chemically synthesized as described
previously (25). Sialyl-neolactotetraosylceramide (sialyl-nLc4) and
sialyl-neolactohexaosylceramide (sialyl-nLc6) were prepared from bovine
blood cells as described previously (26). The asialo compounds were
prepared by digestion with neuraminidase from Vibrio
cholerae (Sigma) as described previously (26).
Isolation of ST3Gal VI--
Mouse expressed sequence tags
(GenBankTM accession nos. W52470, N40607, H06247, AA883549,
and H22233) with similarity to mouse ST3Gal V (GM3 synthase) were
amplified by the reverse transcription polymerase chain reaction
(RT-PCR) method using total RNA prepared from a human melanoma cell
line SK-MEL-37 as a template. The sense primer
5'-TTGGGAGAAGGACAACCTTC-3' and the antisense primer
5'-CCAGGCAGCAACAGACAGTA-3' were used for PCR amplification, which was
carried out as follows; 94 °C for 1 min, 25 cycles of (94 °C for
1 min, 55 °C for 1 min, and 72 °C for 1 min), and 72 °C for 1 min. The RT-PCR-amplified 630-base pair cDNA was cloned into
pCR®2.1-TOPO vector (Invitrogen, San Diego, CA). The DNA
insert was 32P-labeled with a MegaprimeTM DNA
labeling system (Amersham, Buckinghamshire, UK) and used to screen the
SK-MEL-37 cDNA library. Approximately 4 × 105
recombinant MC1061/P3 from a cDNA library prepared from human SK-MEL-37 cells were screened by colony hybridization. Colony lifts
were prepared with GeneScreen Plus membrane (NEN Life Science Products). The nucleotide sequence was determined by the dideoxy termination method using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Construction of Expression Vector--
A cDNA fragment
encoding the open reading frame of ST3Gal VI was prepared by PCR using
a 5' primer containing a XhoI site, 5'-CTCCTCGAGGGTGAGCCAGCCATGAGAGGG-3', and a 3' primer containing a
XbaI site, 5'-TCTTCTAGATCAATCTTGAGTCAAGTTGAT-3' and the
cloned cDNA fragment as a template. The PCR product was inserted
into the XhoI and XbaI sites of pMIKneo vector
(kindly provided by Dr. K. Maruyama at Tokyo Medical and Dental
University). A truncated form of ST3Gal VI, lacking 34 amino acids from
the N-terminus, was prepared by PCR using a 5' primer
containing an EcoRI site, 5'-GAAGAATTCGGAATGAAACGGAGAAATAAG-3', and a 3' primer containing a
XhoI site, 5'-CTCCTCGAGTCAATCTTGAGTCAAGTTGAT-3' and the
cloned cDNA fragment as a template. The product was digested with
EcoRI and XhoI and subcloned into these sites of
pCD-SA vector (kindly provided by Dr. Tsuji, RIKEN Institute, Wako, Japan).
Cell Culture--
Mouse fibroblast L cells and various human
cancer cell lines were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 7.5% fetal calf serum (FCS). Human leukemia
cell lines were maintained in RPMI 1640 supplemented with 10% FCS at
37 °C in a 5% CO2 atmosphere.
Preparation of Membrane Fraction--
L cells (3 × 106) were plated in 10-cm dish at least 48 h prior to
transfection. Cells were transiently transfected with an expression
plasmid (4 µg) by DEAE-dextran method (27). After 48 h of
culture in DMEM containing 7.5% FCS, the cells were harvested by
trypsinization. Cells were pelleted, washed with phosphate-buffered saline (PBS), and lysed in ice-cold PBS containing 1 mM
phenylmethylsulfonyl fluoride using a nitrogen cavitation apparatus
(Parr Instrument Co., Moline, IL) at 400 p. s. i. for 30 min as
described by Thampoe et al. (28). Nuclei were removed by low
speed centrifugation, and supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. The pellet was resuspended in
ice-cold 100 mM sodium cacodylate buffer, pH 6.0.
Preparation of Soluble Forms of ST3Gal VI--
L cells (10-cm
dish) were transfected with pCDSA-hST3Gal VI (4 µg) by the
DEAE-dextran method and cultured for 16 h in DMEM containing 7.5%
FCS. The medium was then replaced with serum-free insulin, transferrin,
and selenous acid medium (Becton Dickinson, Bedford, MA) and the cells
were cultured for another 32 h. At 48 h after transfection,
the culture medium was collected and concentrated 100-fold using
Molcut-L® (Millipore, Tokyo, Japan) and dialyzed against
100 mM sodium cacodylate buffer, pH 6.0.
Sialyltransferase Assay--
The sialyltransferase assay was
performed in a mixture containing 10 mM MgCl2,
0.3% Triton CF-54, 100 mM sodium cacodylate buffer, pH
6.0, 0.66 mM CMP-NeuAc (Sigma), 4,400 dpm/µl
CMP-[14C]NeuAc (Amersham Pharmacia Biotech), the enzyme
solution, and substrates in total volume of 50 µl for glycolipid
acceptors or 20 µl for oligosaccharides and asialoglycoproteins. The
reaction mixture was incubated at 37 °C for 2 h. For glycolipid
acceptors, the reaction was terminated by addition of 500 µl of
water. The products were isolated by C18 Sep-Pak cartridge (Waters,
Milford, MA) and analyzed by thin layer chromatography (TLC). High
performance thin layer chromatography (HPTLC) plates (E. Merck,
Darmstadt, Germany) were used. For oligosaccharide substrates, the
reaction was terminated by the addition of 20 µl of methanol.
Oligosaccharide products were separated by TLC with a solvent system of
ethanol/pyridine/n-butanol/acetate/water (100:10:10:3:30).
For glycoprotein acceptors, the reaction was terminated by the addition
of 20 µl of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading
buffer and the mixtures were directly subjected to SDS-PAGE. The
radioactivity on each plate and gel was visualized with a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).
Linkage Analysis by Sialidase Digestion--
Five µg of
neolactotetraosylceramide (nLc4) was sialylated with a soluble form of
ST3Gal VI (ST3Gal VI-protA). Five µg of GM3 was sialylated with GD3
synthase prepared form the L cells transfected with pMIKneo-ST8Sia I
(29) using CMP-[14C]NeuAc (8, 800 dpm/µl). The products
were purified by C18 Sep-Pak cartridge, dried, and redissolved in 25 µl of 50 mM sodium citrate (pH 6.0) and 100 mM NaCl containing 100 µg/ml bovine serum albumin. The
resulting products were incubated for 2 h at 37 °C after the addition of 0.85 unit Salmonella typhimurium LT2 sialidase
(New England Biolabs, Beverly, MA). The digestion products were
extracted by two volumes of chloroform/methanol (1:1), and the organic
phase was collected by partition, dried, and subjected to HPTLC with a
solvent system of chloroform/methanol/0.02% CaCl2
(55:45:10). The plate was exposed to an imaging plate and then analyzed
by BAS 2000 image analyzer.
TLC Immunostaining--
Twenty µg of nLc4 was sialylated with
ST3Gal VI using CMP-[14C]NeuAc (4,400 dpm/µl) for
6 h, and purified by C18 Sep-Pak cartridge, dried, and subjected
to TLC. TLC immunostaining was performed according to the method of
Taki et al. (30). After chromatography of the glycolipids,
TLC plate was heat-blotted to a polyvinylidene difluoride membrane. The
membrane was incubated with monoclonal antibody (mAb) M2590 (7 µg/ml)
for 1 h, washed, and incubated with biotinylated horse anti-mouse
IgM for 1 h. The antibody binding was revealed with ABC-PO
(Vector, Burlingame, CA) and HRP-1000 (Konica, Tokyo, Japan) as
described previously (31).
Northern Blotting--
Total RNA was prepared from human cancer
cell lines using TRIZOL® reagent (Life Technologies, Inc.)
according to the manufacturer's instruction. Total RNA (10 µg) was
separated on 1.2% agarose-formaldehyde gel, then transferred onto a
GeneScreen Plus® membrane. Human Multiple Tissue Northern
Blot® was purchased from CLONTECH. The
blots were probed with a gel-purified, [
-32P]dCTP-labeled ST3Gal VI cDNA or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described
previously (26).
Flow Cytometry--
Adherent cells were detached in PBS
containing 0.5 mM EDTA and 1 mg/ml glucose. After washing
with PBS, approximately 5 × 105 cells were incubated
with mAb 2H5 (CD15s; PharMingen, San Diego, CA) at a dilution of 1:500
(1 µg/ml) for 30 min on ice. After washing twice, the cells were
stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgM
(µ chain specific) (Zymed Laboratories Inc.) at a
1:200 dilution. After a 30-min incubation on ice, cells were washed
twice and subjected to analysis on a FACSCalibur with Cell QuestTM
Version 3.1f software (Becton Dickinson). Thresholds for antigen
positivity were set at a fluorescence intensity level that excludes
99% of the cells that had been stained without mAb 2H5.
Semiquantitative RT-PCR Analysis--
Total RNA was prepared
from cultured cells using TRIZOL® Reagent. Three µg of
the RNA was reverse transcribed by Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc.) using oligo-dT14
primers. One-twentieth volume of the reaction mixture was subjected to
PCR (total 50 µl). The reactions were performed using 5' and 3'
primers for ST3Gal VI or GAPDH. Primers used were as follows, ST3Gal VI
sense primer, 5'-TTGGGAGAAGGACAACCTTC-3' (nucleotides 728-747); ST3Gal
VI antisense primer, 5'-CCAGGCAGCAACAGACAGTA-3' (nucleotides
1375-1356); GAPDH sense primer, 5'-CCACCCATGGCAAATTCCA-TGGCA-3', and
GAPDH antisense primer, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. The cycling
parameters for PCR for ST3Gal VI and GAPDH were 94 °C for 30 s,
55 °C for 30 s, and 72 °C for 1 min, and the cycle number were 27 for ST3Gal VI and 20 for GAPDH.
 |
RESULTS |
Molecular Cloning of a cDNA Encoding a Novel
2,3-Sialyltransferase--
We previously cloned a cDNA encoding
mouse ST3Gal V (GM3 synthase) using an expression cloning method. By
searching the expressed sequence tag data base, we found sequences
(GenBankTM accession nos. W52470, N40607, H06247, AA883549, and H22233) with similarity to mouse ST3Gal V, and obtained the
corresponding cDNA fragment by RT-PCR (nucleotide numbers 728-1375
in Fig. 1A). Approximately
4 × 105 colonies of a human melanoma cell line
SK-MEL-37 cDNA library were screened using the cDNA fragment as
a probe, and nine independent clones (clones 1-9) were obtained.
Characterization of the positive clones revealed that clone 2 contained
a 1-kilobase pair (kb) insert, clone 3 was 1.2 kb, clone 5 was 1.6 kb,
and clone 9 was 1.5 kb in length. From the nucleotide sequence, clone 9 was found to contain a whole open reading frame (Fig. 1A).
The nucleotide sequence revealed that the cDNA contains an open
reading frame encoding a protein of 331 amino acids with a calculated
molecular mass of 38,213 daltons, with six potential
N-linked glycosylation sites. The position of the AUG start
codon was determined according to the Kozak consensus sequence (32),
and the upstream region contained an in-frame stop codon. Hydropathy
analysis determined by the Kyte and Doolittle method (33) indicated one
prominent hydrophobic segment of 32 residues in length in the
amino-terminal region (Gly3-Val34), predicting
that the protein has type II transmembrane topology characteristic of
many other glycosyltransferases cloned to date (Fig. 1B).
Comparison of the primary structure of ST3Gal VI protein and the 14 other cloned sialyltransferases indicated that there is significant
similarity in two regions, so-called sialylmotifs (34, 35). In
addition, ST3Gal VI has a TXXXXYPE sequence near the
C-terminal end of L-sialylmotif, which is found conserved among the members of ST3Gal subfamily (Fig.
2). These results indicated that this
protein belongs to the sialyltransferase gene family and likely the
2,3-sialyltransferase subfamily. The predicted protein shows 38%,
34%, and 33% sequence identity to human ST3Gal IV, human ST3Gal III,
and mouse ST3Gal V, respectively (Fig. 2). No significant homology in
amino acid sequence was observed between the predicted protein and
other known sialyltransferases.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 1.
Nucleotide and deduced amino acid sequences
of human ST3Gal VI and hydropathy plot of the protein.
A, the deduced amino acid sequence is shown below
the nucleotide sequence. The putative transmembrane hydrophobic domain
is underlined, and six potential N-linked
glycosylation sites are boxed. B, the hydropathy
plot was calculated by the method of Kyte and Doolittle (33) with a
window of 11 amino acids.
|
|

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of sialylmotif L and sialylmotif S
of ST3Gal VI with that of 14 previously cloned sialyltransferases.
The previously cloned sialyltransferases are the human ST3Gal I (17),
human ST3Gal II (19), human ST3Gal III (20), human ST3Gal IV (22, 23),
mouse ST3Gal V (GenBankTM accession no. AB013302), human
ST6Gal I (43, 44), the chick ST6GalNAc I (45), the mouse ST6GalNAc II
(46), the rat ST6GalNAc III (47), the human ST8Sia I (29, 48, 49),
human ST8Sia II (50), human ST8Sia III (GenBankTM accession
no. AF004668), human ST8Sia IV (51), and human ST8Sia V
(GenBankTM accession no. U91641). The sialyltransferase
motifs are grouped by the linkages that they form. Shaded
letters represent highly conserved amino acids in many
sialyltransferases. Shaded and bold
letters indicate conserved amino acid residues among the
members of ST3Gal subfamily.
|
|
Sialyltransferase Activity of the Newly Cloned Enzyme--
To
analyze the sialyltransferase activity of ST3Gal VI, the expression
vector of the cDNA, pMIKneo-ST3Gal VI, was transfected into L
cells, and the extracts of the transfected cells were assayed for
sialyltransferase activity using CMP-[14C]NeuAc as a
donor and glycolipid mixture from bovine blood cells as acceptors. As
shown in Fig. 3, the enzyme sialylated
asialoglycolipids containing LacCer, nLc4, and nLc6 prepared from
acidic glycosphingolipids of bovine red blood cells, and the products
co-migrated with sialyl-nLc4 and sialyl-nLc6. In contrast, purified
LacCer did not serve as an acceptor for ST3Gal VI. No activity was
detected in the extracts from mock-transfected cells. Similar results
were obtained using a soluble fusion enzyme ST3Gal VI-protA (data not
shown).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 3.
Sialyltransferase activity of the newly
cloned enzyme. Sialyltransferase activity of the extracts of the L
cells transfected with pMIKneo-ST3Gal VI was measured. Ten µg of
glycolipid acceptor was incubated with cell extracts containing 20 µg
of protein in the standard assay condition. BRBC indicated
glycolipids containing LacCer, nLc4, and nLc6 prepared from
neuraminidase-treated acidic glycosphingolipids extracted from bovine
red blood cells. NC indicates that the assay was performed
without an acceptor. The samples were subjected to HPTLC with a solvent
system of chloroform/methanol/0.02% CaCl2 (55:45:10). The
plate was exposed to an imaging plate and then analyzed by BAS 2000 image analyzer. SA-nLc4, sialyl-neolactotetraosylceramide;
SA-nLc6, sialyl-neolactohexaosylceramide.
|
|
Substrate Specificity of ST3Gal VI--
To purify the enzyme, a
fusion gene consisting of the IgM signal peptide sequence, the protein
A IgG binding domain, and the putative active domain of ST3Gal VI
(residue number 35-331) was constructed, and transfected into L cells.
In this system, the soluble enzyme (ST3Gal VI-protA) would be secreted.
Using this soluble form of ST3Gal VI-protA as the enzyme source, we
examined the sialyltransferase activity toward various glycolipids. As shown in Fig. 4, [14C]NeuAc
was incorporated into nLc4 and nLc6 containing Gal
1,4GlcNAc sequence
at the non-reducing end. ST3Gal VI-protA did not exhibit activity
toward Lc4, GA1, and lactosylceramide. Then, we determined the acceptor
specificity of the enzyme toward oligosaccharides. As summarized in
Table I, ST3Gal VI-protA utilized
Gal
1,4GlcNAc as the best substrate. Gal
1,3GlcNAc showed much less
incorporation of [14C]NeuAc. The kinetic analysis using
the L cell extracts transfected with pMIKneo-ST3Gal VI showed that the
Km value for nLc4 was 0.22 mM.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 4.
Thin layer chromatography of sialylated
glycosphingolipids. Various glycosphingolipids (0.1 mM) were used as acceptors for ST3Gal VI, and the products
were separated on a TLC plate with a solvent system of
chloroform/methanol/0.2% CaCl2 (55:45:10). The plate was
exposed to a BAS-imaging plate and then analyzed with a BAS 2000 radioimage analyzer. Lex, Lex
ceramide; Lc4, lactotetraosylceramide; nLc4,
neolactotetraosylceramide; nLc6,
neolactohexaosylceramide.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Acceptor substrate specificity of ST3 Gal VI
Various acceptor substrates were incubated in the standard assay
mixture using ST3Gal VI-protA as an enzyme source. Each substrate was
used at a concentration of 0.1 mM for glycolipids and
oligosaccharides and 0.2 mg/ml for glycoproteins. Relative rates are
calculated as a percentage of the incorporation obtained with nLc6.
|
|
Next, we examined the sialyltransferase activity toward various
glycoproteins. As shown in Fig. 5 and
summarized in Table I, asialofetuin served as an acceptor. However,
mucin from bovine submaxillary gland did not. No activity was detected
in the extracts from mock-transfected cells.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Incorporation of sialic acid into
asialofetuin by ST3Gal VI. Sialyltransferase activity of ST3Gal VI
toward glycoproteins was measured. Forty µg of glycoproteins
acceptors was incubated with 8 µg of the cell extracts in the
standard assay condition. The samples were boiled in Laemmli sample
buffer and subjected to 12.5% SDS-PAGE. The gel was dried and exposed
to an imaging plate and then analyzed with a BAS 2000 radioimage
analyzer. Additional details were provided under "Experimental
Procedures."
|
|
Linkage Analysis by Sialidase Digestion--
To determine the
incorporated sialic acid linkage, nLc4 was labeled with
CMP-[14C]NeuAc using ST3Gal VI-protA and the product was
subjected to digestion with Salmonella typhimurium LT2
sialidase which cleaves only the
2,3 linkage. As shown in Fig.
6, [14C] NeuAc-labeled nLc4
was sensitive to digestion, while GD3 synthesized by ST8Sia I (GD3
synthase) was insensitive to the digestion. This result strongly
suggested that the product of ST3Gal VI contained the sequence of
NeuAc
2,3Gal-, and that the enzyme is one of the
-galactoside
2,3-sialyltransferases.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Linkage analysis of incorporated sialic acids
by sialidase digestion. [14C]NeuAc-labeled nLc4 and
GD3 were produced from nLc4 and GM3 using ST3Gal VI-protA and GD3
synthase, respectively. The labeled products were then subjected to the
treatment with 2,3 sialidase as described under "Experimental
Procedures." The resulting glycolipids were separated on a TLC plate
with a solvent system of chloroform/methanol/0.2% CaCl2
(55:45:10) and detected with a BAS 2000 radioimage analyzer.
SA-nLc4, sialyl-neolactotetraosylceramide.
|
|
TLC Immunostaining--
To confirm further that the reaction
products were formed by the transfer of sialic acid to the terminal
galactose of substrate via
2,3-linkage, TLC immunostaining using mAb
M2590 specific to a saccharide arrangement
(NeuAc
2,3Gal
1,4Glc or NeuAc
2,3Gal
1,4GlcNAc) of glycolipids
(35) was performed. As shown in Fig. 7,
[14C]NeuAc-labeled nLc4 using ST3Gal VI-protA (lane
5) was stained with this antibody (lane 4) as well as
GM3 in the standard (lane 1), indicating that this band is
sialyl (
2,3)-neolactotetraosylceramide.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 7.
TLC immunostaining of sialic
acid-incorporated products. TLC-immunostaining was performed as
described under "Experimental Procedures." Lane 1, GM3
(1 µg); lane 2, acidic glycosphingolipids extracted from
human red blood cells containing GM3 and
sialylneolactotetraosylceramide (NeuAc 2,3-nLc4) as major components;
lane 4, [14C]NeuAc-labeled sialyl-nLc4 using
ST3Gal VI-protA. As a control, the same reaction was performed without
CMP-[14C] NeuAc (lane 3). Glycosphingolipids
were separated by TLC and blotted to a polyvinylidene difluoride
membrane. Immunostaining was done using mAb M2590 to detect GM3 and
NeuAc 2,3-nLc4. Lane 5 is an autofluorogram of
lane 4 detected with a BAS 2000 radioimage
analyzer. SA-nLc4, sialyl-neolactotetraosylceramide;
SA-nLc6, sialyl-neolactohexaosylceramide.
|
|
Expression of the ST3Gal VI Gene--
To determine the size of
ST3Gal VI mRNA and its expression, Northern blot analysis was
conducted using the full-length fragment of cDNA as a probe. As
shown in Fig. 8, 1.8- and 3.0-kb
mRNAs of ST3Gal VI were detected. In adult tissues, the gene
expression is abundant in heart, placenta, and liver (Fig.
8A). In human cancer cell lines, strong signals were
observed in melanoma lines SK-MEL-37 and SK-MEL-23, while signals were
hardly detected in hematopoietic cell lines and an astrocytoma AS or a
neuroblastoma line IMR32 (Fig. 8B).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Differential expression of ST3Gal VI in
various human tissue and human cell lines. Northern blots with
poly(A)+ RNA from various adult human tissues (panel
A) and with total RNA (10 µg each) obtained from cultured human
cancer cell lines (panel B) were probed with human ST3Gal VI
full-length fragment (993 base pairs). The same filter was probed with
GAPDH cDNA after removing the radioactivity. The positions of
ribosomal RNAs are indicated at the left. The normalized
relative intensities of ST3Gal VI signals are represented in
C and D, respectively.
|
|
Correlation of the Expression Levels of ST3Gal VI Gene with
Sialyl-Lex Expression--
In order to investigate whether
ST3Gal VI was involved in sialyl-Lex expression, we
examined the expression of ST3Gal VI gene along with
sialyl-Lex expression levels in several human cancer cell
lines. As shown in Fig. 9A, a
flow cytometric analysis indicated that these cancer cell lines
strongly expressed sialyl-Lex except for MOLT-3.
Subsequently, expression of ST3Gal VI in these cells was analyzed by
semiquantitative RT-PCR analysis. As shown in Fig. 9B,
melanoma cell lines, especially SK-MEL-37 showed a high level of the
STGal VI mRNA expression. Among colon cancer cell lines, Lovo and
DLD-1 expressed sialyl-Lex on the cell surface, but the
expression of ST3Gal VI gene was not observed. Thus, the expression
levels of ST3Gal VI did not correlate well with sialyl-Lex
expression among cell lines.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
ST3Gal VI gene expression and
sialyl-Lex expression on human cancer cell lines.
A, flow cytometric analysis of sialyl-Lex on the
surface of the cultured human cancer cells. Cells were incubated with
anti-sialyl-Lex antibody 2H5, followed by staining with
fluorescein isothiocyanate-conjugated anti-mouse IgM as described under
"Experimental Procedures." Percentages of positive cells are shown.
B, semiquantitative RT-PCR analysis of ST3Gal VI
transcripts. Intensities of ST3Gal VI bands in gel normalized by GAPDH
bands are presented.
|
|
 |
DISCUSSION |
Since Weinstein et al. isolated a cDNA clone of
-galactoside
2,6-sialyltransferase in 1987 (37), a number of
sialyltransferase genes have been cloned. These cloned
sialyltransferases can be classified into four subfamilies based on the
linkages they form, i.e. the ST3Gal-, ST6Gal-, ST6GalNAc-,
and ST8Sia- subfamilies. In a subfamily, some enzymes utilize certain
acceptors with high efficiency, but use other acceptors with less
efficiency. Therefore, substrate specificities of these enzymes
frequently overlap, at least in in vitro analysis. It seems
reasonable to think that an acceptor showing the strongest substrate
activity in vitro is also the best acceptor in
vivo. However, an acceptor that exhibits very weak activity for an
enzyme in vitro can be a major acceptor in vivo
depending on the expression levels of itself and another enzymes that
share the same substrate (38).
To date, four human
2,3-sialyltransferase genes have been reported,
and human ST3Gal V has recently been identified (39). In this study, we
have isolated a novel ST3Gal cDNA that exhibited high homology in
L- and S-sialylmotifs with previously cloned ST3Gal genes (Fig. 2).
Except for these motifs, this gene did not show any significant
homology with other sialyltransferase genes, and even with other ST3Gal
genes. Compared with five human
2,3-sialyltransferases (ST3Gal) so
far cloned, the cloned enzyme exhibit at most 40% homology,
indicating that this gene encodes a novel sialyltransferase
belonging to the ST3Gal subfamily. This gene is designated ST3Gal VI.
Although mouse cDNA clones of five ST3Gal (I-V) are also available
(40), it seems important to restrict the enzyme sources to human when
we discuss about the substrate specificity and the expression pattern
of ST3Gal VI compared with those of the other ST3Gals. This is because
substrate specificities of glycosyltransferases are sometimes quite
different between different species. Human ST3Gal I and ST3Gal II
utilize Gal
1,3GalNAc as an acceptor and are thought to be mainly
involved in the synthesis of O-glycan and ganglio-series
gangliosides, respectively, based on the expression pattern of genes
(17, 19). ST3Gal III exhibit high activity onto Gal
1,3GlcNAc on
glycoproteins compared with Gal
1,4GlcNAc and Gal
1,3GalNAc (20).
ST3Gal V utilizes almost exclusively lactosylceramide and forms GM3
(39). The reported substrate specificity of human ST3Gal IV is somewhat
confusing. ST3Gal IV isolated from a melanoma library prepared from
cells selected for lectin resistance (23) and that from human placenta
cloned by PCR approach (22) exhibit quite different preferences of substrates. The former utilizes Gal
1,4GlcNAc (type II) structure more efficiently than Gal
1,3GlcNAc (type I), indicating that ST3Gal
IV might be involved in the synthesis of sialyl-Lex, a
ligand for the selectins (41). However, it seems unlikely that this
enzyme synthesizes sialyl-paragloboside (SPG), a precursor of
sialyl-Lex on ceramide, since the activity of this enzyme
toward glycolipids was very low compared with the activity toward
glycoproteins (40). From these facts, it is likely that an unknown
ST3Gal gene specific for the synthesis of SPG exists.
ST3Gal VI, as reported in this study, utilizes almost exclusively
Gal
1,4GlcNAc on glycoproteins and glycolipids. Among glycolipid acceptors, ST3Gal VI acts only on nLc4 and nLc6, but not on Lc4, GA1,
and LacCer. The efficiency of the sialyltransferase activity toward
nLc4 was almost equivalent to those of ST3Gal III and ST3Gal II to type
I chain (20) and type III chain (19), respectively. Furthermore, nLc6
exhibits much more incorporation of [14C]NeuAc than nLc4,
suggesting that ST3Gal VI preferred polylactosamine type II chains.
Consequently, ST3Gal VI is capable of generating NeuAc
2,3Gal
1,4GlcNAc structures on glycoproteins and glycolipids including SPG as main products, and is probably involved in the biosynthesis of sialyl-Lex in some tissues.
Results of Northern blotting of ST3Gal VI gene shows predominant
expression in placenta, liver, heart, and skeletal muscle. Among human
cell lines, melanoma lines exhibited relatively high levels of the gene
expression in accord with high expression of sialyl-Lex,
indicating that ST3Gal VI might be involved in the synthesis of
sialyl-Lex in melanomas, and probably in the synthesis of
SPG in placenta (42). However, it seemed unclear whether this gene
contributes in the up-regulation of sialyl-Lex synthesis in
colon cancer and hematopoietic malignant cell lines. Actual involvement
of this ST3Gal VI in the synthesis of sialyl type II structures
in vivo remains to be analyzed.
Northern blots showed 1.8-kb major band and 3.0-kb minor band with
almost parallel intensities. These bands were rather broad, suggesting
the presence of heterogeneity in the size of mRNAs. The fact that
cloned cDNAs exhibit various patterns of partial defects or
insertions of sequences in the coding region is probably due to
alternatively spliced exons (data not shown), explaining the observed
heterogeneous mRNAs. Many of those aberrant clones seem
non-functional and may have roles in the regulation of the enzyme
activity in certain situations.
 |
ACKNOWLEDGEMENT |
We thank Dr. S. Tsuji for providing an
expression vector pCDSA for a protein A fusion enzyme.
 |
FOOTNOTES |
*
This work was supported by Grant-in-aid for Scientific
Research in Priority Areas 10178105, by a Core of Excellence grant from
the Ministry of Education, Science, Sports and Culture of Japan, and by
a grant-in-aid from Ono Medical Research Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB022918.
To whom correspondence should be addressed: Dept. of
Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai,
Showa-ku, Nagoya 466 Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069;
E-mail: koichi{at}med.nagoya-u.ac.jp.
2
Fukumoto, S., Miyazaki, H., Urano, T., Furukawa,
K., and Furukawa, K. (1999) J. Biol. Chem. 274, in press.
 |
ABBREVIATIONS |
The abbreviations used are:
CMP-NeuAc, cytidine
5'-monophospho-N-acetylneuraminic acid;
mAb, monoclonal
antibody;
RT, reverse transcription;
PCR, polymerase chain reaction;
DMEM, Dulbecco's modified Eagle's medium;
FCS, fetal calf serum;
PBS, phosphate-buffered saline;
TLC, thin layer chromatography;
PAGE, polyacrylamide gel electrophoresis;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HPTLC, high performance thin
layer chromatography;
kb, kilobase pair(s);
SPG, sialyl-paragloboside.
The nomenclature of gangliosides is based on that of Svennerholm (54).
The abbreviated nomenclature for cloned sialyltransferases follows Ref.
24. The designations of glycosphingolipids are abbreviated according to
the recommendations of the IUPAC-IUB Commission on Nomenclature (55).
Lc4, Gal
1,3GlcNAc
1,3Gal
1,4Glc
1-Cer;
nLc4, Gal
1,4GlcNAc
1,3Gal
1,4Glc
1-Cer;
nLc6, Gal
1,4GlcNAc
1,3Gal
1,4GlcNAc
1,3Gal
1,4Glc
1-Cer;
Lex, Lewis X
(Gal
1,4(Fuc
1,3)GlcNAc
1,3Gal
1,4Glc
1-Cer).
 |
REFERENCES |
-
Hakomori, S.
(1981)
Annu. Rev. Biochem.
50,
733-764[CrossRef][Medline]
[Order article via Infotrieve]
-
Paulson, J. C.
(1989)
Trends Biochem. Sci.
14,
272-276[CrossRef][Medline]
[Order article via Infotrieve]
-
Varki, A.
(1992)
Curr. Opin. Cell Biol.
4,
257-266[Medline]
[Order article via Infotrieve]
-
Valki, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7390-7397[Abstract]
-
McEver, R. P.
(1994)
Curr. Opin. Immunol.
6,
75-84[CrossRef][Medline]
[Order article via Infotrieve]
-
Springer, T. A.
(1994)
Cell
76,
301-314[Medline]
[Order article via Infotrieve]
-
Hakomori, S.
(1991)
Curr. Opin. Immunol.
3,
646-653[Medline]
[Order article via Infotrieve]
-
Kawashima, H.,
Yamamoto, K.,
Osawa, T.,
and Irimura, T.
(1993)
J. Biol. Chem.
268,
27118-27126[Abstract/Free Full Text]
-
Narimatsu, H.,
Sinha, S.,
Brew, K.,
Okayama, H.,
and Qasba, P. K.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
4720-4724[Abstract]
-
Schwientek, T.,
Almeida, R.,
Levery, S. B.,
Holmes, E. H.,
Bennett, E.,
and Clausen, H
(1998)
J. Biol. Chem.
273,
29331-29340[Abstract/Free Full Text]
-
Kukowska-Latallo, J. F.,
Larsen, R. D.,
Nair, R. P.,
and Lowe, J. B.
(1990)
Genes Dev.
4,
1288-1303[Abstract]
-
Weston, B. W.,
Smith, P. L.,
Kelly, R. J.,
and Lowe, J. B.
(1992)
J. Biol. Chem.
267,
24575-24584[Abstract/Free Full Text]
-
Weston, B. W.,
Nair, R. P.,
Larsen, R. D.,
and Lowe, J. B.
(1992)
J. Biol. Chem.
267,
4152-4160[Abstract/Free Full Text]
-
Goelz, S. E.,
Hession, C.,
Goff, D.,
Griffiths, B.,
Tizard, R.,
Newman, B.,
Chi-Rosso, G.,
and Lobb, R.
(1990)
Cell
63,
1349-1356[Medline]
[Order article via Infotrieve]
-
Sasaki, K.,
Kurata, K.,
Funayama, K.,
Nagata, M.,
Watanabe, E.,
Ohta, S.,
Hanai, N.,
and Nishi, T.
(1994)
J. Biol. Chem.
269,
14730-14737[Abstract/Free Full Text]
-
Gillespie, W.,
Kelm, S.,
and Paulson, J. C.
(1992)
J. Biol. Chem.
267,
21004-21010[Abstract/Free Full Text]
-
Kitagawa, H.,
and Paulson, J. C.
(1994)
J. Biol. Chem.
269,
17872-17878[Abstract/Free Full Text]
-
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]
-
Kim, Y. J.,
Kim, K. S.,
Kim, S. H.,
Kim, C. H.,
Ko, J. H.,
Choe, I. S.,
Tsuji, S.,
and Lee, Y. C.
(1996)
Biochem. Biophys. Res. Commun.
228,
324-327[CrossRef][Medline]
[Order article via Infotrieve]
-
Kitagawa, H.,
and Paulson, J. C.
(1993)
Biochem. Biophys. Res. Commun.
194,
375-382[CrossRef][Medline]
[Order article via Infotrieve]
-
Wen, D. X.,
Livingston, B. D.,
Medzihradszky, K. F.,
Kelm, S.,
Burlingame, A. L.,
and Paulson, J. C.
(1992)
J. Biol. Chem.
267,
21011-21019[Abstract/Free Full Text]
-
Kitagawa, H.,
and Paulson, J. C.
(1994)
J. Biol. Chem.
269,
1394-1401[Abstract/Free Full Text]
-
Sasaki, K.,
Watanabe, E.,
Kawashima, K.,
Sekine, S.,
Dohi, T.,
Oshima, M.,
Hanai, N.,
Nishi, T.,
and Hasegawa, M.
(1993)
J. Biol. Chem.
268,
22782-22787[Abstract/Free Full Text]
-
Tsuji, S.,
Datta, A. K.,
and Paulson, J. C.
(1996)
Glycobiology
6,
v-vii[Medline]
[Order article via Infotrieve]
-
Mitsuoka, C.,
Sawada-Kasugai, M.,
Ando-Furui, K.,
Izawa, M.,
Nakanishi, H.,
Nakamura, S.,
Ishida, H.,
Kiso, M.,
and Kannagi, R.
(1998)
J. Biol. Chem.
273,
11225-11233[Abstract/Free Full Text]
-
Furukawa, K.,
Chait, B. T.,
and Lloyd, K. O.
(1988)
J. Biol. Chem.
263,
14939-14947[Abstract/Free Full Text]
-
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]
-
Thampoe, I. J.,
Furukawa, K.,
Vellve, E.,
and Lloyd, K. O.
(1989)
Cancer Res.
49,
6258-6264[Abstract]
-
Haraguchi, M.,
Yamashiro, S.,
Yamamoto, A.,
Furukawa, K.,
Takamiya, K.,
Lloyd, K.,
Shiku, H.,
and Furukawa, K.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10455-10459[Abstract/Free Full Text]
-
Taki, T.,
Handa, S.,
and Ishikawa, D.
(1994)
Anal. Biochem.
221,
312-316[CrossRef][Medline]
[Order article via Infotrieve]
-
Miyazaki, H.,
Fukumoto, S.,
Okada, M.,
Hasegawa, T.,
Furukawa, K.,
and Furukawa, K.
(1997)
J. Biol. Chem.
272,
24794-24799[Abstract/Free Full Text]
-
Kozak, M.
(1986)
Cell
44,
283-292[Medline]
[Order article via Infotrieve]
-
Kyte, J.,
and Doolittle, R. F.
(1982)
J. Mol. Biol.
157,
105-132[Medline]
[Order article via Infotrieve]
-
Datta, A. K.,
and Paulson, J. C.
(1995)
J. Biol. Chem.
270,
1497-1500[Abstract/Free Full Text]
-
Datta, A. K.,
Sinha, A.,
and Paulson, J. C.
(1998)
J. Biol. Chem.
273,
9608-9614[Abstract/Free Full Text]
-
Hirabayashi, Y.,
Hamaoka, A.,
Matsumoto, M.,
Matsubara, T.,
Tagawa, M.,
Wakabayashi, S.,
and Taniguchi, M.
(1985)
J. Biol. Chem.
260,
13328-13333[Abstract/Free Full Text]
-
Weinstein, J.,
de Souza-e-Silva, U.,
and Paulson, J. C.
(1982)
J. Biol. Chem.
257,
13835-13844[Abstract/Free Full Text]
-
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]
-
Ishii, A.,
Ohta, M.,
Watanabe, Y.,
Matsuda, K.,
Ishiyama, K.,
Sakoe, K.,
Nakamura, M.,
Inokuchi, J.,
Sanai, Y.,
and Saito, M.
(1998)
J. Biol. Chem.
273,
31652-31655[Abstract/Free Full Text]
-
Kono, M.,
Ohyama, Y.,
Lee, Y. C.,
Hamamoto, T.,
Kojima, N.,
and Tsuji, S.
(1997)
Glycobiology
7,
469-479[Abstract]
-
Miyamoto, D.,
Takashima, S.,
Suzuki, T.,
Nishi, T.,
Sasaki, K.,
Morishita, Y.,
and Suzuki, Y.
(1995)
Biochem. Biophys. Res. Commun.
217,
852-858[CrossRef][Medline]
[Order article via Infotrieve]
-
Taki, T.,
Matsuo, K.,
Yamamoto, K.,
Matsubara, T.,
Hayashi, A.,
Abe, T.,
and Matsumoto, M.
(1988)
Lipids
23,
192-198[Medline]
[Order article via Infotrieve]
-
Grundmann, U.,
Nerlich, C.,
Rein, T.,
and Zettlmeissl, G.
(1990)
Nucleic Acids Res.
18,
667[Medline]
[Order article via Infotrieve]
-
Stamenkovic, I.,
Asheim, H. C.,
Deggerdal, A.,
Blomhoff, H. K.,
Smeland, E. B.,
and Funderud, S. J.
(1990)
J. Exp. Med.
172,
641-643[Abstract]
-
Kurosawa, N.,
Hamamoto, T.,
Lee, Y. C.,
Nakaoka, T.,
Kojima, N.,
and Tsuji, S.
(1994)
J. Biol. Chem.
269,
1402-1409[Abstract/Free Full Text]
-
Kurosawa, N.,
Inoue, M.,
Yoshida, Y.,
and Tsuji, S.
(1996)
J. Biol. Chem.
271,
15109-15116[Abstract/Free Full Text]
-
Sjoberg, E. R.,
Kitagawa, H.,
Glushka, J.,
van Halbeek, H.,
and Paulson, J. C.
(1996)
J. Biol. Chem.
271,
7450-7459[Abstract/Free Full Text]
-
Sasaki, K.,
Kurata, K.,
Kojima, N.,
Kurosawa, N.,
Ohta, S.,
Hanai, N.,
Tsuji, S.,
and Nishi, T.
(1994)
J. Biol. Chem.
269,
15950-15956[Abstract/Free Full Text]
-
Nara, K.,
Watanabe, Y.,
Maruyama, K.,
Kasahara, K.,
Nagai, Y.,
and Sanai, Y.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7952-7956[Abstract]
-
Scheidegger, E. P.,
Sternberg, L. R.,
Roth, J.,
and Lowe, J. B.
(1995)
J. Biol. Chem.
270,
22685-22688[Abstract/Free Full Text]
-
Nakayama, J.,
Fukuda, M. N.,
Fredette, B.,
Ranscht, B.,
and Fukuda, M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7031-7035[Abstract]
-
Spiro, R. G.,
and Bhoyroo, V. D.
(1974)
J. Biol. Chem.
249,
5704-5717[Abstract/Free Full Text]
-
Tsuji, T.,
and Osawa, T.
(1986)
Carbohydr. Res.
151,
391-402[CrossRef][Medline]
[Order article via Infotrieve]
-
Svennerholm, L.
(1963)
J. Neurochem.
10,
613-623[Medline]
[Order article via Infotrieve]
-
IUPAC-IUB Commission on Nomenclature.
(1977)
Lipids
12,
455-68[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.