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1,3-galactosyltransferase That
Determines the Expression of
GD1b/GM1/GA1*
(Received for publication, May 8, 1997, and in revised form, July 8, 1997)
§¶,¶
,§,,
From the ABSTRACT Using an anti-GD1b monoclonal
antibody, expression cloning of a cDNA for the
INTRODUCTION Gangliosides are amphipathic glycolipid molecules containing
sialic acid and present abundantly in the nervous system of vertebrates (1, 2). A number of studies on gangliosides have been performed showing
that they play important roles in the development, maintenance, and
repair of the nervous system (3-6). Furthermore, results of many of
these studies suggested biological functions of gangliosides as
neurotrophic factors (7), receptors for neurotrophic factors, and
modifiers of receptors for the growth factors (8-10). Furthermore, gangliosides systemically administrated into the body (11, 12) could
show neurotrophic effects in various pathological situations (13).
However, molecular mechanisms by which gangliosides affect the
functions of nervous system and regulate the cell growth and differentiation have not been well clarified. This is mainly because there have been no effective approaches to address those issues other
than to observe the effects of exogenously added gangliosides to
cultured cells or into experimental animals.
These difficulties can now be dramatically overcome since a number of
glycosyltransferase genes have been isolated (14-17), and manipulation
of those genes to modulate the glycosylation pattern of cells has
become possible (18-20). Remodeling of carbohydrates in cultured cells
and genetic modification of carbohydrates in experimental animals have
been tried (21-23), and results of those studies have demonstrated
novel functions of carbohydrates which have never been expected or
definitely demonstrated.
Among complex gangliosides,
GM11 has been
most rigorously studied since it is one of major gangliosides in
vertebrate brain, and shows specific binding with cholera toxin B
subunit resulting in important biological events such as cAMP response
(24). It has also been reported that GM1 was involved in
the receptor functions for peptide hormones such as
follicle-stimulating hormone and luteinizing hormone (25), and was
effective in the therapeutic applications of many pathological status
of nervous system. For detailed analysis of the biological functions of
complex gangliosides, the availability of genes of glycosyltransferases
responsible for the synthesis of individual structures are
essential.
In the present study, we isolated a cDNA clone of
MATERIALS AND METHODS Mouse cell line KF4C is a stable
transfectant of KF3027 (26) with plasmid pMIK-Hyg/M2T1-1
(GM2/GD2 synthase cDNA inserted in pMIK/Hyg
vector). B78-2 is a stable transfectant of B78 (a subline of B16) with
pM2T1-1 (26). These cells were maintained in Dulbecco's modified
Eagle's minimal essential medium containing 7.5% fetal calf serum and
300 µg/ml of G418 (Sigma) with or without hygromycin (250 µg/ml)
(Calbiochem-Novabiochem, La Jolla, CA). L1-17 was a L cell transfectant
line with GM2/GD2 synthase cDNA clone,
pM2T1-1 in pCDM8 (26) with pSV2neo expressing asialo-GM2 (GA2) (27). Monoclonal antibodies (mAbs) specifically reactive with GD1b (mAb 370) and GA1 (mAb 229) were
generated in our laboratory and will be described
elsewhere.2
A cDNA library of
rat brain was prepared using poly(A)+ RNA and the
pcDNAI expression vector (Invitrogen, San Diego, CA). This library
contained 3.5 × 106 independent colonies. The strain
of bacterial host used was Escherichia coli MC1061/P3.
Plasmid pMIK/D3T-31 was constructed by inserting XhoI
fragment of GD3 synthase cDNA clone, pD3T-31 (28), into pMIK/Neo expression vector (kindly provided by Maruyama at Tokyo Medical Dental School).
Plasmids of the cDNA library
were once amplified and transfected into KF4C cells together with
plasmid pMIK/D3T-31 using DEAE-dextran (Pharmacia Biotech, Uppsala,
Sweden) as described previously (29). Subconfluent KF4C cells, 1.5 × 106 in 10-cm dishes (Corning, Corning, NY), were
co-transfected with 8 µg each of cDNA library plasmid and
pMIK/D3T-31. After 60 h, the transfected cells were detached from
plates and incubated with mAb 370 at a 1:200 dilution on ice for 45 min. Cells were plated on dishes coated with goat anti-mouse IgM
(Cappel, Durham, NC) as described previously (30). Plasmid DNA was
rescued from the panned cells by preparing Hirt extracts and
transformed into MC1061/P3. Expanded plasmid DNA was transfected again,
and the same procedure was repeated four times more. Thereafter 96 pools containing 30 colonies each were prepared and screened by the expression of mAb 370 binding activity. Finally, 17 clones from two
positive pools were screened, and three single colonies that directed
the expression of GD1b on KF4C were isolated using
microscale DEAE-dextran transfection and immunofluorescence assay.
Isolated cDNA plasmid was digested by
XhoI and HindIII and cloned into phagemid
BlueScript (pBSK) KS(+) vector. Deletion mutants of this clone were
prepared with a Kilo-Sequence deletion kit (Takara, Kyoto).
Dideoxynucleotide termination sequencing was performed by either T3/T7
dye primers or four additional custom dideoxy terminators with the
PRISM dye terminator cycle sequencing kit and model 377 DNA sequencer
(Applied Biosystems, Foster City, CA).
The mouse melanoma cell
lines, KF4C and B78-2 expressing GM2, were used as
recipient cells in the transient and stable expression systems. For
transient expression of GM1, pM1T-9/cDNAI vector was
transfected into KF4C by DEAE-dextran. For transient expression of
GD1b, co-transfection of KF4C with pMIK-neo/D3T-31 and
pM1T-9/cDNAI was performed. For transient expression of
GA1, L1-17 cells expressing GA2 were
transfected with pM1T-9/cDNAI. Expression of these gangliosides was
detected by an immunofluorescence assay and flow cytometry. To prepare
stable transfectants expressing GM1 and GD1b,
pM1T-9/cDNAI and pMIK-Hyg/D3T-31 were co-transfected into B78-2 by
calcium phosphate precipitation as described previously (31). To select transfectants, the cells were cultured in Dulbecco's modified Eagle's
minimal essential medium, 7.5% fetal calf serum containing G418 (300 µg/ml) and hygromycin (250 µg/ml). Expression of these gangliosides
on stable transfectants was checked by immunofluorescence assay and
flow cytometry.
Ganglioside expression was analyzed
using mouse mAb 370 (anti-GD1b), mAb 229 (anti-GA1), and FITC-conjugated cholera toxin B subunit
(List Biological Laboratories, Campbell, CA). Cells were incubated with
mAbs for 45 min on ice and stained with FITC-conjugated rabbit
anti-mouse IgM for mAbs 370 and 229 for 45 min on ice. To analyze
GM1 on cell surface, cells were incubated with
FITC-conjugated cholera toxin B subunit for 45 min. Cells were analyzed
by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA). 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 only second antibody for
GD1b and GA1. For GM1 expression,
transfectants with vector only were used after staining with
FITC-conjugated cholera toxin B subunit.
RNA was extracted from
adult rat tissues and embryonic rat brains using acid phenol (32).
Total RNA (20 µg) was separated on 1.2% agarose-formaldehyde gel,
then transferred onto a GeneScreen Plus membrane (DuPont, Boston, MA).
Hybridization with [ Stable transfectants of the The enzyme activity of 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 analysis were performed with a software GENETYX-MAC version
6.1.0 (Software Development, Tokyo).
RESULTS To isolate cDNA clone of GD1b synthase
gene, we prepared transfectant lines of KF3027 with
After 5 cycles of transfection of cDNA library, panning
by anti-GD1b mAb 370 and Hirt extraction, a cDNA clone
designated as pM1T-9 was isolated. After co-transfection of pM1T-9 with
pD3T-31 into KF4C, new expression of GD1b was observed as
shown in Fig. 2B. In these
transient transfectant cells, GM1 was also newly expressed
when stained by FITC-conjugated cholera toxin B as shown in Fig.
2A. Therefore, it was strongly suggested that
GD1b synthase and GM1 synthase were identical
and coded by a single gene as previously reported by Sandhoff's group
(36). To confirm this point, KF4C was transfected with pM1T-9 alone,
then examined the expression of GD1b and GM1.
These transfectants expressed GM1, but not
GD1b, corresponding with the proposed pathway of
ganglioside synthesis (36). Furthermore, this enzyme has been thought
to be identical with GA1 synthase which catalyze the
synthesis of GA1 from GA2. L cell transfected
with pM2T1-1 was, therefore, used as a recipient cell of pM1T-9 to
examine the synthesis of GA1 by the transfection of this
cDNA, since L cell lacked all complex gangliosides due to the lack
of GM3 synthase activity (27). As shown in Fig. 2, L cells
transfected by pM2T1-1 (stable) and pM1T-9 (transient) definitely
expressed GA1 as demonstrated by a GA1 specific
mAb 229.
We
established stable transfectant cells of pM1T-9 or pM1T-9 and
pMIK/D3T-31 using B78-2 (a B78 transfectant line with pM2T1-1). Flow
cytometry analysis clearly demonstrated new and significant levels of
synthesis of GM1 (Fig.
3B) or
GM1/GD1b (Fig. 3D).
Glycosphingolipids extracted from the parent cell (B78/M2T1-1), and a
double transfectant (B78/M2T1-1/M1T-9/D3T-31) were analyzed by TLC, and
conversion of glycolipid components as expected were observed (Fig.
4). Specific GD1b bands were
demonstrated in the double transfectant cells by TLC immunostaining.
These results certainly indicated that the cloned cDNA pM1T-9
actually derived from the
GD1b/GM1/GA1 synthase gene.
Fig.
5 shows whole sequence of the insert of
pM1T-9 determined by sequence analysis of the constructs of pBSK
containing the HindIII-XhoI fragment of clone
between HindIII and XhoI sites. Compared with
other glycosyltransferase cDNAs isolated so far, this cDNA was
relatively small in size, i.e. total size was 1613 base
pairs comprising a 194-base pair 5
Based on
the results of transient and stable transfection of pM1T-9, it was
strongly expected that this gene codes for a single
Expression levels of the
DISCUSSION Since Narimatsu et al. (15) isolated cDNA of the
Based on competitive enzymologic studies, Sandhoff and collegues (36)
suggested that GM1/GD1b/GA1
synthases were identical, as were
GM2/GD2/GA2 synthases (46). They,
therefore, proposed that the synthesis of complex gangliosides was
regulated mainly at the levels of GM3 As mentioned above, gangliosides have been expected to play important
roles in the development of vertebrate nervous system (4).
GM1 in particular has been rigorously studied for its biological effects in vivo and in vitro. Its
effects on the repairment of lesioned nerves with toxic agents,
ischemic damages, degenerative diseases, and mechanical operations have
been reported (5, 12, 13, 48). In the in vitro culture
systems, addition of GM1 ganglioside could induce and/or
augment neurite extension (49-52) in neuronal cells. However, the
biological significance of gangliosides can be investigated more
physiologically by the genetic modification of their
glycosyltransferase genes. Mice lacking complex gangliosides following
the disruption of the FOOTNOTES The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB003478. ACKNOWLEDGEMENTS We thank Dr. Kenneth O. Lloyd at Memorial
Sloan-Kettering Cancer Center for carefully reading the manuscript and
Y. Honda and T. Shimomura for excellent technical assistance.
REFERENCES 
Department of Biochemistry II,
Department of
Pediatric Dentistry,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C,
mouse melanoma B16 transfected with polyoma T antigen gene, and
GM2/GD2 synthase cDNA was used as a
recipient cell line for the cDNA library transfection. A cDNA
clone of GD3 synthase, pD3T-31 was co-transfected with a
cDNA library prepared from rat brain RNA using the pcDNAI
expression vector. The isolated cDNA clone pM1T-9 predicted a type
II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino
acids of transmembrane region, and a large catalytic domain with 346 amino acids. Introduction of the cDNA clone into a mouse melanoma
line B16 previously transfected with a GM2/GD2
synthase gene resulted in the neo-synthesis of GM1.
Co-transfection of the cell line with pM1T-9 and a GD3
synthase cDNA resulted in the expression of GD1b as
well as GM1. Moreover, introduction of pM1T-9 into L cell
(lacking GM3 synthase), previously transfected with
GM2/GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that
GD1b/GM1/GA1 synthases were
identical, as previously suggested based on enzymological analysis. In
Northern blots of the 1,3-galactosyltransferase gene with total RNA
from various rat tissues, a 1.6-kilobase mRNA was strongly
expressed in spleen, thymus, kidney, and testis. However, the
expression level of the gene in the adult brain tissue was not
especially high. On the other hand, this gene was expressed at high
levels in the rat brain of embryonal day 12, and reached a peak at
around birth, then fell to low level in the adult brain.
1,3-galactosyltransferase (EC 2.4.1.-) (1,3Gal-T) gene, products
of which determine the expression of GD1b. We demonstrated
that this enzyme also catalyzes the synthesis of GM1 and
asialo-GM1 (GA1) by introducing the cDNA
into various cell lines with appropriate precursor structures, and by
in vitro assay using extracts from the cDNA transfectant cells. The expression pattern of the gene among rat tissues and in rat
brain during development was also investigated.
1,3-Galactosyltransferase
1,3Gal-T Gene
-32P]dCTP-labeled rat 1,3Gal-T
cDNA(pM1T-9), or -actin cDNA (nucleotides 69-814) probes
was performed as described previously (26), then analyzed by BAS 2000 Bio-Imaging Analyzer (Fuji Film, Tokyo).
1,3Gal-T gene were
obtained by co-transfecting KF4C melanoma with the cloned plasmid and
pMIK-Hyg/D3T-31 (2,8-sialyltransferase (2,8S-T) cDNA) by
calcium phosphate precipitation. Among G418- and hygromycin-resistant
clones, GD1b-expressing clones were selected based upon the
results in immunofluorescence assay and flow cytometry. Glycolipids
were isolated as described previously (33). Briefly, cells were
extracted from about 300 µl of packed cells of transfectants and
control clones containing the Hyg gene alone using chloroform/methanol
(2:1, 1:1, 1:2) sequentially. After desalting, gangliosides were
isolated by DEAE-Sephadex A-50 (Pharmacia Biotech Inc.) ion exchange
chromatography. TLC was performed on a high performance TLC plate
(Merck, Darmstadt) using chloroform, methanol, 2.5 N
NH4OH (60:35:8). The components were visualized by spraying
with resorcinol. The identity of new gangliosides was confirmed by TLC
immunostaining using aluminum-backed silica plates (Merck) as described
previously (33). After TLC, the plate was air-dried and transferred
onto polyvinylidene difluoride membrane. The plates were incubated with
1% bovine serum albumin in phosphate-buffered saline for 1 h to
block nonspecific binding. Then, the membrane was incubated with mAbs
for 1 h at room temperature, then with rabbit biotinized
anti-mouse IgG and avidin-biotin complex, Vectastain ABC-PO Kit (Vector
Laboratories) was used. To visualize specific binding of mAbs, Konica
stain kit (Konica, Tokyo) was used.
1,3Gal-T was
measured as described previously (34). 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 the enzyme activity of 1,3Gal-T, the reaction mixture
contained the following in a volume of 50 µl: 150 mM
sodium cacodylate-HCl (pH 7.0), 15 mM MnCl2,
0.375% Triton CF-54 (Sigma), 325 mM GM2 (for
GM1 synthesis), 400 mM UDP-Gal (Sigma),
UDP-[14C]Gal (2.0 × 105 dpm) (NEN Life
Science Products, Boston, MA), and membranes containing 100 µg of
protein. After incubation for 2 h at 37 °C, the products were
isolated by a C18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by TLC and fluorography as described (35).
1,4-N-acetylgalactosaminyltransferase (1,4GalNAc-T)
cDNA, pM2T1-1, resulting in a new acceptor cell line expressing
GM2. This line was named KF4C. Thus, we can expect expression of GD1b after co-transfection of 2,8S-T
cDNA, pD3T-31, and 1,3Gal-T cDNA in the library as shown in
Fig. 1.
Fig. 1.
Strategy of cDNA cloning of 1,3Gal-T
using anti-GD1b mAb. Recipient cell line KF4C was
prepared as described under "Materials and Methods" and expressed
polyoma T antigen and GM2 as well as GM3. When
2,8S-T and 1,3Gal-T cDNAs were introduced together into a
single cell, GD1b expression could be expected.
[View Larger Version of this Image (23K GIF file)]
Fig. 2.
Transient expression of new gangliosides in
B78-2 and L1-17. Cloned cDNA pM1T-9 was transfected by
DEAE-dextran with (right panel) or without (left
panel) GD3 synthase cDNA, pD3T-31. Expression of
GM1, GD1b, and GA1 were analyzed
after 60 h using specific probes in flow cytometry as described
under "Materials and Methods." Thin lines are with
specific probes and solid lines are controls.
[View Larger Version of this Image (27K GIF file)]
1,3Gal-T Activity and Its Products
Fig. 3.
Expression of
GM1/GD1b on the stable transfectant cells.
B78-2 cell was transfected with pM1T-9 alone (left panel) or
with pM1T-9 and pD3T-31 (right panel) as described under
"Materials and Methods." GM1 was expressed in both
A and B, whereas GD1b was expressed
only in D. Thin lines are samples added by mAbs and
solid lines are those treated by the second antibody
alone.
[View Larger Version of this Image (29K GIF file)]
Fig. 4.
TLC of gangliosides extracted from stable
transfectant cells. A, gangliosides were extracted as
described under "Materials and Methods" from B78 (lanes
1 and 2), B78-2 (lane 3), and
B78-2/M1T-9/D3T-31 (lane 4), then separated in TLC.
Lane 1 was gangliosides from 10 mg of wet tissue, and
lanes 2-4 were from 5 mg. St, ganglioside mixture from bovine brain as a standard. Solvent used was chloroform, methanol, 2.5 N NH4Cl (60:35:8). Resorcinol
spray was performed for the detection of bands. B, TLC
immunostaining of ganglioside fractions. Lanes 3 and
4 were prepared as in A, then blotted and stained
by anti-GD1b mAb 370 as described under "Materials and Methods".
[View Larger Version of this Image (37K GIF file)]
-untranslated region, a continuous
open reading frame of 1113 base pairs, and a 306-base pair
3-untranslated region. A single polyadenylation signal and a
polyadenylation region of more than 70 As were present. The initiation
codon at the beginning of the open reading frame is embedded within a
sequence similar to the Kozak consensus initiation sequence (37, 38).
This open reading frame predicts a 371-amino acid protein with a
molecular mass of 40,976.1 daltons. Search of currently available
protein and nucleic acid data bases identified no other gene with
significant sequence homology to this cDNA. Even the sequences of
four other galactosyltransferase genes, i.e. 1,3Gal-T
(14, 39-41), 1,4Gal-T (15, 42, 43), GalCer-Gal-T (16, 44), and
blood type B synthase 1,3Gal-T (17) showed only 10~16% homology
in amino acid sequence. Inspection and hydropathy of the predicted
protein sequence, however, suggested that this protein has a similar
structural organization to those of known glycosyltransferases (Fig.
5). There is a single hydrophobic segment near the amino terminus which
comprised of 21 amino acids. This putative signal anchor sequence would
place 4 residues within the cytosolic compartment and 346 amino acids
within the Golgi lumen as a catalytic domain. The predicted amino acid
sequence indicated the presence of single N-glycosylation
site at 143-145, and a proline-rich region at 42-71 (11/30) amino
acid residues.
Fig. 5.
Nucleotide sequences of cloned 1,3Gal-T
pM1T-9. Top, deduced amino acid sequences were shown for the
single open reading frame. The putative transmembrane region with 21 amino acids was double underlined. A candidate of
N-glycosylation site was marked by a single
underline. A polyadenylation signal was enclosed by a
square. Bottom, hydropathy analysis of the coding region
based on the deduced amino acids according to Kyte and Doolittle
(53).
[View Larger Version of this Image (53K GIF file)]
1,3Gal-T cDNA Can Transfer Galactose onto
GD2, GM2, and GA2
1,3Gal-T
catalyzing the synthesis of three structures, i.e.
GD1b, GM1, and GA1. The membrane
fraction of the stable transfectant cells were examined for enzyme
activity. As shown in Fig. 6A, the membrane fraction from a stable transfectant of B78 with pMIK/M1T-9 and pD3T-31 showed ~20,000 units (pmol/h/mg of protein) of
GM1 synthase activity, while it showed no significant
products when no acceptor was added. As expected, that from a
transfectant with pMIK vector alone showed almost null activity. When
GD2, GA2, GlcCer, GD1b,
GM3, or GD1a were examined as substrates,
GD2 and GA2 showed significant levels of
incorporation of UDP-Gal (Fig. 6B).
Fig. 6.
1,3Gal-T activity in the extracts from a
stable transfectant of cDNA. A, enzyme activity with
GM2 acceptor. Membrane fractions were prepared from the
parent B78-2 and B78-2/M1T-9/D3T-31 and the enzyme activity was
determined using GM2 as an acceptor as described under
"Materials and Methods." As shown in the inset, a
GM1 band was observed in the TLC of the products.
B, relative 1,3Gal-T activity for various acceptor
structures, with that for GM2 acceptor as 100(%).
[View Larger Version of this Image (19K GIF file)]
1,3Gal-T
gene in various rat tissues were examined by Northern blots using total
RNA. Among various tissues, kidney, testis, spleen, and thymus showed
relatively high expression levels, whereas almost all tissues examined
contained some levels of the gene transcripts (Fig.
7A). Gene expression in the
brain tissue of adult rat was not especially high. When the gene
expression was analyzed during the development of rat brain, this gene
was already detected at day 12 of embryonic brain, and the expression
level was maintained at high levels until birth (Fig. 7, B
and C). In the adult brain, the expression level was maintained at low levels.
Fig. 7.
Northern blots of 1,3Gal-T gene.
A, 20 µg each of total RNA from rat tissues was separated in
agarose gel, then blotted onto nylon membrane. Hybridization with
32P-labeled probes derived from pM1T-9 was performed as
described under "Materials and Methods." B, 20 µg each
of total RNA from whole fetus (lane 1), fetal brain
(lanes 2-3), or mouse brain (lane 4) was
separated and blotted as in A. Lanes 5 and 6 were with 30 µg of RNA from brain. The intensities of bands were corrected by the intensities of 18 S rRNA bands stained by ethidium bromide using
densitography (Signal Analysis, Vienna, VA) and plotted in
C. Results of repeated experiments were presented with
±S.D.
[View Larger Version of this Image (37K GIF file)]
1,4-galactosyltransferase gene in 1986, a number of
glycosyltransferase genes of mammals and birds have been cloned. Except
for ceramide:1,4-galactosyltransferase (16, 44) and
ceramide:1,4-glucosyltransferase (45), the majority of them were
type II membrane proteins. The 1,3Gal-T reported in this paper which
uses glycolipid acceptors also showed a typical type II orientation
with a very short cytoplasmic tail of 4 amino acids.
GD3
GT3 synthesis. The identity of
GM2/GD2/GA2 synthase genes was
previously demonstrated by us (26, 27, 47) using substrate specificity
analysis of the 1,4GalNAc-T gene product and by transfections of the
cDNA. The identity of GD1b/GM1/GA1 synthase gene was
demonstrated in this study by isolation of GD1b synthase
cDNA and the demonstration that its product can use glycolipids
containing GalNAc14Gal-, GalNAc14(NeuAc23)Gal-, or
GalNAc14(NeuAc28NeuAc23)Gal-terminal sequences
as acceptors. In the case of GM2/GD2
synthase (27), GA2 synthesis was relatively low when
compared on the basis of substrate specificity analysis. On the other
hand, acceptor activity of 1,3Gal-T with GM2,
GD2, and GA2 was not so different (Fig.
6B), although further analysis remains to be performed.
1,4GalNAc-T gene showed definite dysfunctions
in the nervous systems such as reduced nerve conduction velocity (23),
and in some subtle aspects of their
behavior.3 The high
expression of 1,3Gal-T gene observed only during the developmental
stage of rat brain suggests an important role for its ganglioside
products in the formation and differentiation of brain tissues. Genetic
manipulation of 1,3Gal-T gene would enable us to finely analyze the
roles of individual complex gangliosides higher than
GD1b/GM1/GA1.
¶
Contributed equally to the results in this report.
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.
1
The abbreviations used are: GM1,
Gal13GalNAc14(NeuAc23)Gal14Glc-Cer;
GD1b,
Gal13GalNAc14(NeuAc28NeuAc23)Gal14Glc-Cer; GM2,
GalNAc14(NeuAc23)Gal14Glc-Cer;
GA1 (asialo-GM1), Gal13GalNAc14 Gal14Glc-Cer; GA2 (asialo-GM2),
GalNAc14Gal14Glc-Cer; GD2, GalNAc1 4(NeuAc28NeuAc23)Gal14Glc-Cer;
GD3, NeuAc28NeuAc23Gal14Glc-Cer (ganglioside nomenclature is based on that of Svennerholm (54)); Gal-T,
galactosyltransferase; mAb, monoclonal antibody; pBSK, phagemid
BlueScript; 2,8S-T, 2,8-sialyltransferase (GD3
synthase), 1,4GalNAc-T,
1,4-N-acetylgalactosaminyltransferase
(GM2/GD2 synthase); FITC, fluorescein
isothiocyanate.
2
K. Furukawa, S. Fukumoto, and T. Shimomura,
manuscript in preparation.
3
K. Takamiya, M. Kishikawa, S. Fukumoto, and K. Furukawa, manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 Q. Zhang, K. Furukawa, H.-H. Chen, T. Sakakibara, T. Urano, and K. Furukawa Metastatic Potential of Mouse Lewis Lung Cancer Cells Is Regulated via Ganglioside GM1 by Modulating the Matrix Metalloprotease-9 Localization in Lipid Rafts J. Biol. Chem., June 30, 2006; 281(26): 18145 - 18155. [Abstract] [Full Text] [PDF]
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M. Nishio, S. Fukumoto, K. Furukawa, A. Ichimura, H. Miyazaki, S. Kusunoki, T. Urano, and K. Furukawa Overexpressed GM1 Suppresses Nerve Growth Factor (NGF) Signals by Modulating the Intracellular Localization of NGF Receptors and Membrane Fluidity in PC12 Cells J. Biol. Chem., August 6, 2004; 279(32): 33368 - 33378. [Abstract] [Full Text] [PDF]
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A. Tsuchida, T. Okajima, K. Furukawa, T. Ando, H. Ishida, A. Yoshida, Y. Nakamura, R. Kannagi, M. Kiso, and K. Furukawa Synthesis of Disialyl Lewis a (Lea) Structure in Colon Cancer Cell Lines by a Sialyltransferase, ST6GalNAc VI, Responsible for the Synthesis of {alpha}-Series Gangliosides J. Biol. Chem., June 13, 2003; 278(25): 22787 - 22794. [Abstract] [Full Text] [PDF]
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R. Muller, F. Altmann, D. Zhou, and T. Hennet The Drosophila melanogaster brainiac Protein Is a Glycolipid-specific beta 1,3N-Acetylglucosaminyltransferase J. Biol. Chem., August 30, 2002; 277(36): 32417 - 32420. [Abstract] [Full Text] [PDF]
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T. Schwientek, B. Keck, S. B. Levery, M. A. Jensen, J. W. Pedersen, H. H. Wandall, M. Stroud, S. M. Cohen, M. Amado, and H. Clausen The Drosophila Gene brainiac Encodes a Glycosyltransferase Putatively Involved in Glycosphingolipid Synthesis J. Biol. Chem., August 30, 2002; 277(36): 32421 - 32429. [Abstract] [Full Text] [PDF]
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T. Mitsuda, K. Furukawa, S. Fukumoto, H. Miyazaki, T. Urano, and K. Furukawa Overexpression of Ganglioside GM1 Results in the Dispersion of Platelet-derived Growth Factor Receptor from Glycolipid-enriched Microdomains and in the Suppression of Cell Growth Signals J. Biol. Chem., March 22, 2002; 277(13): 11239 - 11246. [Abstract] [Full Text] [PDF]
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X. Bai, D. Zhou, J. R. Brown, B. E. Crawford, T. Hennet, and J. D. Esko Biosynthesis of the Linkage Region of Glycosaminoglycans. CLONING AND ACTIVITY OF GALACTOSYLTRANSFERASE II, THE SIXTH MEMBER OF THE beta 1,3-GALACTOSYLTRANSFERASE FAMILY (beta 3GalT6) J. Biol. Chem., December 14, 2001; 276(51): 48189 - 48195. [Abstract] [Full Text] [PDF]
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 S. Yoshida, S. Fukumoto, H. Kawaguchi, S. Sato, R. Ueda, and K. Furukawa Ganglioside GD2 in Small Cell Lung Cancer Cell Lines: Enhancement of Cell Proliferation and Mediation of Apoptosis Cancer Res., May 1, 2001; 61(10): 4244 - 4252. [Abstract] [Full Text]
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T. Okajima, H.-H. Chen, H. Ito, M. Kiso, T. Tai, K. Furukawa, T. Urano, and K. Furukawa Molecular Cloning and Expression of Mouse GD1alpha /GT1aalpha /GQ1balpha Synthase (ST6GalNAc VI) Gene J. Biol. Chem., March 15, 2000; 275(10): 6717 - 6723. [Abstract] [Full Text] [PDF]
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S. Fukumoto, T. Mutoh, T. Hasegawa, H. Miyazaki, M. Okada, G. Goto, K. Furukawa, T. Urano, and K. Furukawa GD3 Synthase Gene Expression in PC12 Cells Results in the Continuous Activation of TrkA and ERK1/2 and Enhanced Proliferation J. Biol. Chem., February 25, 2000; 275(8): 5832 - 5838. [Abstract] [Full Text] [PDF]
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T. Okajima, S. Fukumoto, H. Ito, M. Kiso, Y. Hirabayashi, T. Urano, K. Furukawa, and K. Furukawa Molecular Cloning of Brain-specific GD1alpha Synthase (ST6GalNAc V) Containing CAG/Glutamine Repeats J. Biol. Chem., October 22, 1999; 274(43): 30557 - 30562. [Abstract] [Full Text] [PDF]
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S. Isshiki, A. Togayachi, T. Kudo, S. Nishihara, M. Watanabe, T. Kubota, M. Kitajima, N. Shiraishi, K. Sasaki, T. Andoh, et al. Cloning, Expression, and Characterization of a Novel UDP-galactose:beta -N-Acetylglucosamine beta 1,3-Galactosyltransferase (beta 3Gal-T5) Responsible for Synthesis of Type 1 Chain |
