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(Received for publication, November 14, 1994; and in revised form, December 21, 1994) From the
The substrate specificity of
Carbohydrate structures on glycoproteins and glycolipids are
synthesized by the sequential addition of monosaccharides by
glycosyltransferases. Since specific glycosyltransferases are
considered to be needed for the individual combination of sugar donors,
acceptors, and modes of linkage, 100 Recently we have cloned a cDNA of G Glycolipids-The derivation of
glycosphingolipids used as acceptors of enzyme reaction was as follows:
G
Figure 1:
Expression of soluble
When B78
melanoma cells were transfected with pM2T1-1/PROTA, the
conditioned media showed increasing activity of G Using protA, the substrate specificity of soluble
Figure 2:
Transfer of GalNAc onto SPG by the
extracts from pM2T1-1 transfectant cells and the fusion protein
protA. A, TLC of the enzyme products with a MeWo transfectant
line, C7. Both (NeuAc)SPG and (NeuGc)SPG showed generation of
GalNAc-SPG. These components were resistant to neuraminidase treatment,
whereas G
The glycolipids in the
cell lines were also extracted and analyzed by TLC and TLC
immunostaining, with a special focus on the levels of precursors
present. All cell lines, except for L cells, contained high levels of
G
Figure 3:
Changes of glycolipid components in cells
before and after expression of stably transfected
Figure 4:
Double staining of G
Figure 5:
Asialo-G
Figure 6:
Effects of gangliosides on asialo-G
The data presented in this study support the identification
of the cDNA previously reported (3) as coding for the
UDP-GalNAc:G The synthesis of GalNAc-SPG by
melanoma GalNAc-T can be compared with the activity of the enzyme from
normal stomach extracts previously studied by Dohi et
al.(15) . These investigators showed that the stomach
enzyme synthesizes the NGM-1 antigen, which may be related to the
Sd Whether a separate
enzyme is also responsible for asialo-G Another factor that can
apparently influence the ability of GalNAc-T to utilize LacCer as
substrate is the level of other gangliosides, particularly
G The glycolipid composition of cells may also be determined
by the differential compartmentation of the various
glycosyltransferases in the biosynthetic machinery of the cell. The
possibility for differential compartmentation of these enzymes is
suggested by organelle fractionation studies in which
In brief,
factors that determine ganglioside composition in cells are quite
complex, but glycosyltransferase levels, substrate specificities of the
enzymes, and the levels of precursors and other glycolipids in the cell
clearly play important roles. Fig. 7summarizes the ganglioside
expression in the cell lines studied and the effect of transfection
with the
Figure 7:
Ganglioside patterns in cell lines before
and after transfection with
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6149-6155
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
1,4-N-Acetylgalactosaminyltransferase in Vitro and in cDNA-transfected Cells
G
/G
SYNTHASE EFFICIENTLY GENERATES
ASIALO-G IN CERTAIN CELLS (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1,4-N-acetylgalactosaminyltransferase has been analyzed
using a fusion enzyme which consisted of the catalytic domain of the
enzyme and the IgG binding domain of protein A, and also by extracts
from cDNA transfectants. Both enzyme sources were capable of producing
not only G and G, but also
asialo-G, GalNAc-sialylparagloboside, and GalNAc-G
from appropriate acceptors, although the efficiencies were at
most 1-3% of those of G/G. The
biological significance of these low specificities was studied with
transient and stable transfectant cells. From the results of transient
expression of the cDNA, asialo-G expression appeared to
inversely correlate with G
synthase levels in those lines.
Consequently, G seemed to be preferentially synthesized
when both G and lactosylceramide are available, and
asialo-G is synthesized in the absence of G
synthesis. However, the results of double immunostaining of CHO
transfectants with anti-G and anti-asialo-G
antibodies indicated that another factor may be involved in
asialo-G synthesis. From the in vitro assay using
mixed acceptors, it was concluded that the presence of certain levels
of G might enhance the asialo-G synthesis.
These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the
availability of precursors, expression levels of other gangliosides, as
well as the kinetic properties of the enzyme, and the compartmentation
of the glycosylation machineries in the cells.
150 or more different
glycosyltransferases are required for synthesis of carbohydrate
structures present in mammalian cells(1, 2) . The
expression of these carbohydrate structures are regulated by the
expression of each glycosyltransferase during differentiation,
development, or malignant transformation. In order to further
understand the regulatory mechanisms of carbohydrate expression, it is
critical to establish the identity of each glycosyltransferase. /G synthase gene (1,4GalNAc-T)(
)(3) . We
successfully detected the GalNAc-T activity in the culture medium of
cells transfected with a plasmid containing the catalytic domain of
1,4GalNAc-T cDNA fused to the IgG binding domain of the protein A
gene. In this study, substrate specificity of the 1,4GalNAc-T was
analyzed using this fusion enzyme (protA) as well as extracts from cDNA
transfectants. As reported previously, major specificity of the enzyme
was found in G and G
(3) . However,
very low but definite incorporation of GalNAc was also detected on
G, lactosylceramide (LacCer), and sialylparagloboside
(SPG). Despite low specificity in vitro, LacCer could be
converted to asialo-G efficiently depending on the cell
lines into which cDNAs were introduced. Regulatory factors governing
the carbohydrate structures synthesized by this enzyme in cells were
investigated.
and G are purchased from Supelco Co. Inc.
(Bellefonte, PA). G
, G, and GalCer were from
Sigma; LacCer, GlcCer, and G were from Snow Brand Milk
Products Co. (Tokyo); G was obtained from BioCarb
Chemicals (Lund, Sweden); N-glycolylneuraminic acid
(NeuGc)-containing G was extracted and purified from horse
red blood cells as described(4) ; N-acetylneuraminic
acid (NeuAc)-containing SPG and NeuGc-type SPG were also purified from
human and bovine red blood cells, respectively, as described previously (4) ; concentration of purified gangliosides was determined
according to Warren(5) .Cell Culture and Establishment of Transfectants
Mouse
melanoma line B16 (B78) and fibroblast cell line L cell were donated by
Dr. A. Albino at the Sloan-Kettering Cancer Center (New York) and were
maintained in Dulbecco's minimum essential medium supplemented
with 7.5% fetal bovine serum. MeWo was from Dr. L. J. Old at the
Sloan-Kettering Cancer Center. Rat-1 was a rat fibroblast line provided
by Dr. Kondo in Ohsaka University. CHO K1 was obtained from RIKEN cell
bank (Tsukuba, Japan). Stable transfectants of 1,4GalNAc-T gene
were prepared by co-transfection with 1,4GalNAc-T cDNA
(pM2T1-1) and pSV2neo as described previously(3) . After
selection of neoresistant colonies, G-positive clones were
picked up by immunofluorescence assay and used for limiting dilution
cloning.Flow Cytometry
Cell surface expression of
glycolipid antigens was analyzed by flow cytometry with FACScan
(Becton-Dickinson) as described(3) . G was
analyzed using mAb 10-11 ((6) ) which was kindly donated
by Dr. P. O. Livingston at the Sloan-Kettering Cancer Center. Human mAb
KM966 was also used as an anti-G mAb, which was a generous
gift from Dr. N. Hanai at the Kyowa Hakko Research Institute.
Asialo-G expression was detected by mAb 2D4 ((7) )
which was obtained from American Type Culture Collection.Construction of pM2T1-1/PROTA
The SmaI-ScaI fragment of pM2T1-1 extending from
the 3` side of the transmembrane region to the 3`-uncoding region
(coding 505 amino acids) was purified from agarose gel using GeneClean
II (BIO 101 Inc., La Jolla, CA) and inserted into the unique EcoRI site of pPROTA vector (8) (a generous gift from
Dr. R. Breathnach, at INSERM/CNRS, Cédex,
France), after blunt-ended with the Klenow fragment DNA polymerase I.
One clone, named pM2T1-1/PROTA with correct orientation was
obtained by confirming the DNA sequence across the vector and insert
junction.Expression and Purification of the Soluble
Enzyme
B78 or COS7 cells were transfected with
pM2T1-1/PROTA or pPROTA alone by the DEAE-dextran
method(3) . Regular culture medium was replaced by serum-free
ITS (insulin, transferrin, and selenious acid) medium (Collaborative
Research) on the next day, and the culture medium was daily collected
at day 25 after transfection. Collected medium was concentrated
100-fold using Molcut-L (Nihon Millipore Ltd., Yonezawa, Japan) and
dialyzed against 0.1 M sodium cacodylate-HCl (pH 7.2). It was
stored in a frozen tube at -80 °C without significant loss of
the activity. A nonrelevant mouse IgG (mAb 229 reactive with nm23,
IgG2a subclass) was purified and conjugated to Sepharose 4B (Pharmacia,
Uppsala, Sweden) according to the manufacturer's instruction, and
packed in a small column. The concentrated culture supernatant was
applied twice to the column and unbound proteins were removed by
washing rigorously with phosphate-buffered saline. Bound fusion protein
was stored in 25% glycerol of cacodylate buffer at -80 °C
until used.
The enzyme activity
of 1,4GalNAc-T Enzyme Assay1,4GalNAc-T was measured basically according to the method
previously described(9) . The membrane fractions of cells were
prepared as described by Thampoe et al.(10) . Briefly,
cells were lysed using a nitrogen cavitation apparatus. Nuclei were
removed by low centrifugation and supernatant was centrifuged at
105,000 
g for 1 h at 4 °C. The pellet was
resuspended in ice-cold cacodylate-HCl buffer. The reaction mixture
contained in a volume of 50 µl: 100 mM sodium
cacodylate-HCl (pH 7.2), 10 mM MnCl
, 0.3% Triton
CF-54 (Sigma), 325 µM G (for G synthesis), 400 µM UDP-GalNAc (Sigma),
UDP-[
C]GalNAc (3.5 10
dpm)
(DuPont NEN), 10 mM CDP-choline (Kojin Co., Tokyo), and
membranes containing 200 µg of protein. This mixture was incubated
at 37 °C for 2 h. The products were isolated by C18 Sep-Pak
cartridge (Waters, Millford, MA) and analyzed by thin layer
chromatography (TLC) and fluorography as described(11) .
2,3-Sialyltransferase
Assay2,3-Sialyltransferase activity was determined as
described previously(9, 10) . Briefly, the reaction
mixture contained in a volume of 50 µl: 50 mM sodium
cacodylate-HCl (pH 6.0), 0.3% Triton CF-54, 5 mM MgCl, 500 µM LacCer, 500 µM CMP-NeuAc (Sigma), CMP-[C]NeuAc (3
10 dpm, 1200 dpm/nmol) (DuPont NEN), and membranes
containing 200 µg of protein. The reaction products were applied to
a Sep-Pak C18 column and analyzed as described for the 1,4GalNAc-T
assay.Neuraminidase Treatment
Enzyme reaction products
were dried in small tubes and dissolved in 50 µl of 0.1 M acetate buffer (pH 6.0). Neuraminidase from Clostridium
perfringens (Worthington) or neuraminidase from Vibrio
cholerae (Calbiochem Corp.) was added (final concentration, 0.5
unit/ml) and incubated for 1 h at 37 °C. The reaction was stopped
by adding 4 volumes of chloroform/methanol (1:1) and the organic phase
was removed by partition, and used for TLC.Transient Expression and Flow Cytometric
Analysis
For the transient expression, cells were transfected
with pM2T1-1 using the DEAE-dextran method(3) .
Expression of G and asialo-G was examined by
flow cytometry as described above.Determination of Optimal Condition for Asialo-G
For investigation of optimal pH, sodium
cacodylate buffers containing MnCl Synthase Assay and Triton CF-54 were
prepared by adjusting the pH to the indicated values. In order to
determine the best cation for the enzyme assay, MgCl,
MnCl, CaCl, and CuSO
were dissolved
in the cacodylate buffer with Triton CF-54, and adjusted to pH 7.2 with
NaOH prior to using for the assay. Different detergents (Triton X-100,
Tween 20, deoxycholate, and Triton CF-54) were tested at appropriate
concentrations. The membrane fraction of a MeWo transfectant, C7,
expressing a high level of 1,4GalNAc-T was used as an enzyme
source.Extraction of Glycolipids, TLC, and TLC
Immunostaining
Glycolipids were extracted as described
previously (12) . Briefly, lipids were extracted by
chloroform/methanol 2:1, 1:1, then 1:2 sequentially. Glycolipids were
isolated by Florisil column after acetylation, then neutral and acidic
fractions were separated by DEAE-Sephadex (A-50) column chromatography.
TLC and TLC immunostaining were performed as described
previously(12) . Bands in TLC were analyzed by Nu200 CCD Camera
System (Photometrics, Tucson, AZ) and IPLab Gel (Signal Analytics
Corp., Vienna, VA). MAb M2590 was used for detection of G(13) and mAb 81-87 (donated by Dr. D. Scheinberg in
Sloan-Kettering Cancer Center) was used for LacCer(14) .Enzyme Kinetics Assay
K
values of the 1,4GalNAc-T for G and LacCer in
the cDNA transfected cells or the protA were investigated under the
standard assay conditions. In order to analyze the preferred use of
G or LacCer as the enzyme acceptors, and to investigate
the effect of gangliosides on the synthesis of asialo-G,
G, G, or G were mixed with
LacCer in various ratios and used as acceptors in the enzyme assay.
Initial Analysis of GalNAc-T in Soluble Enzyme and Cell
Extracts
In order to demonstrate that the product of the
putative 1,4GalNAc-T cDNA we recently isolated (3) really
catalyzes the synthesis of G and/or G, the
predicted catalytic domain, containing the carboxyl-terminal 505 amino
acids, was fused to a secreted form of the IgG-binding domain of Staphylococcus aureus protein A in the mammalian expression
vector pPROTA (8) (Fig. 1A).
1,4GalNAc-T
fused with protein A. A, construction of the fusion enzyme. SmaI-ScaI fragment of pM2T1-1 was inserted in
the EcoRI site of pPROTA as described under ``Materials
and Methods.'' B, detection of G synthase
activity in the culture medium of B78 transfected with
pM2T1-1/PROTA. Inset shows the products (G)
of the enzyme assay at the time points
indicated.
synthase
as expected and reached plateau levels at 3 days after transfection (Fig. 1B), whereas transfectants with pPROTA or
pM2T1-1/CDM8 demonstrated no or very low levels of the enzyme
activity, respectively. In contrast to transfectants with
pM2T1-1/CDM8, B78 transfected with pM2T1-1/PROTA showed no
surface expression of G (data not shown). COS7 cells
transfected with pM2T1-1/PROTA also produced secreted enzyme
(data not shown). In order to exclude the possible effects of
contaminants in the culture medium, the concentrated medium was
affinity-purified using an IgG-Sepharose column. For this purpose,
culture medium from COS7 transfectants were used because they contained
higher levels of enzyme activity than those from B78 transfectants. 1,4GalNAc-T
was examined and compared with the enzyme activity of SK-MEL-31 (a
highly expressing melanoma line, (11) ) extracts and extracts
from a stable MeWo transfectant. As shown in Table 1A, these
sources of enzyme demonstrated very similar substrate specificities, i.e. very high activity with NeuAc- and NeuGc-type G and NeuAc-G as substrates. In addition, they showed
very low but definite activity using LacCer, G, and SPG
as substrates. The possibility that the asialo-G was a
product formed by an endogenous neuraminidase in the melanoma cells was
examined by adding a melanoma extract to the enzyme assay; no effect
was observed (data not shown).
GalNAc-SPG Synthesis by the
The
ability of 1,4GalNAc-T1,4GalNAc-T to use SPGs was examined in more detail and
the activity was compared to the 1,4GalNAc-T from stomach, since
Dohi et al.(15) reported the presence of a similar
enzyme for GalNAc-SPG synthesis in normal stomach. Both (NeuAc) SPG and
(NeuGc)SPG were used as acceptors in assays with either C7 or protA (Fig. 2). When treated by neuraminidase without detergent, the
products corresponding to GalNAc-SPG were stable, whereas G bands, which were apparently generated from G contaminating the substrates, were completely converted to
G. When membrane extracts from normal stomach tissue were
examined, GalNAc-SPGs with both type of sialic acids were produced (Fig. 2C). In the products from normal stomach
extracts, no G bands were seen, indicating that this
1,4GalNAc-T is distinct from G/G synthase.
, apparently formed from G contaminating the SPG samples, was converted to G. B, results with the purified protA. G synthesis
was demonstrated in this assay also. C, synthesis of
GalNAc-SPG with extracts from normal stomach. GalNAc-SPG showed similar
migration and sensitivity to neuraminidase as in A and B, although no other bands were detected in this
assay.
Similar Assay Conditions Are Required for the Maximal
Enzyme Activity to Synthesize G
As Pohlentz et al.(16) had reported that G and
Asialo-G/G synthase and asialo-G synthase were identical
whereas we found that LacCer was used at only 1-2% the rate of
G, we suspected that the conditions of enzyme assay used
in our study might not be optimal for asialo-G synthesis
although they may be suitable for G/G synthesis. Therefore, various kinds of pH, metal ions, and
detergents were used for synthesis of asialo-G using C7
cell extracts as an enzyme source. The optimal pH was 7.2, and
Mn
was the most effective metal ion for
asialo-G synthesis (data not shown). Triton CF-54 was the
best among detergents tried. These results were basically the same as
for G synthesis, indicating that our assay conditions were
suitable for both enzyme activities(9) .Comparison of Affinities of G
In order to examine the basis for
difference in the activity of and LacCer as
Acceptors for 1,4GalNAc-T1,4GalNAc-T with G and
LacCer, we analyzed the enzyme kinetics for the reaction using G and LacCer as acceptors and cell extracts as the enzyme source.
As shown in Table 1B, G showed very high acceptor
activity for 1,4GalNAc-T in comparison to LacCer. Although K with LacCer was smaller than that with
G, the apparent V
with G was much higher than that with LacCer by more than 2 orders of
magnitude. We were unable to determine kinetic data for G using the protA because of nonlinear data. The V/K ratio with G was about 10 times higher than with LacCer, providing a
reasonable explanation for the results of the substrate specificity
analysis.Glycolipid Products in Cells Transiently Transfected with
The expression of glycolipids in cell
lines transiently transfected with 1,4GalNAc-T cDNA1,4GalNAc-T cDNA was examined
by flow cytometry. Five cell lines derived from mouse melanoma (B78),
fibroblast (L cell), rat fibroblast (Rat-1), human melanoma (MeWo), and
Chinese hamster ovary (CHO) were used because of the lack of
1,4GalNAc-T gene expression and high efficiency of transfection.
Transfected B78, MeWo, and Rat-1 showed G expression but
no asialo-G (Table 2). On the other hand,
transfected L cells expressed only asialo-G and
transfected CHO cells expressed both G and
asialo-G, in a ratio of 16:1.
(Table 2). On the other hand, L cells showed no
ganglioside bands as detected by resorcinol spraying. In the neutral
fractions, all the cell lines showed doublet bands corresponding to
LacCer; its identity was confirmed by TLC immunostaining with mAb
81-87 (data not shown) with various intensities. When the levels
of G synthase activity in these 5 cell lines were
measured, B78, MeWo, and Rat-1 showed very high activity as expected,
whereas CHO and L cell showed very low or no G synthase
activity (Table 2).Glycolipid Composition of Stable Transfectant Cell
Lines
Glycolipids were extracted from B78 and L cells and their
stable transfectants and analyzed by TLC. As shown in Fig. 3,
the majority of LacCer was converted to asialo-G in L
cells. The B78 transfectant showed a very faint line corresponding to
asialo-G and the LacCer band was reduced. As for the
ganglioside composition, the majority of G in B78 was
converted to G in the transfectants. No ganglioside bands
were seen either in the parent L cell or in its transfectant.
1,4GalNAc-T
cDNA. Acidic glycolipids (G and G) and
neutral glycolipids (CMH, CDH, CTH, and asialo-G) of B78
and L cell were detected using a resorcinol spray and orcinol spray,
respectively. Solvents for TLC were chloroform/methanol, 2.5 N NHOH (60:35:8) for acidic fraction, and
chloroform/methanol/HO (60:35:8) for neutral. Relative
intensities of bands in each fraction were analyzed as described under
``Materials and Methods.'' CTH includes glycolipids migrating
at the CTH region.
Two-dimensional Flow Cytometry of CHO Transfectants with
Anti-G
In order
to examine whether the G and Anti-asialo-G MAbs positive CHO transfected cells
were a population distinct from asialo-G positive cells,
double staining of bulk CHO transfectants with anti-G and
anti-asialo-G mAbs was performed. Contrary to expectation,
almost all asialo-G positive cells belonged to the
G positive population as shown in Fig. 4.
and
asialo-G in CHO transfected with pM2T1-1. CHO cells
were co-transfected with pM2T1-1 and pSV2neo, then selected with
G418. Neo-resistant cells were applied for flow cytometry by staining
with mAb 2D4 plus fluorescein isothiocyanate-conjugated anti-mouse IgM,
and human mAb KM966 plus phycoerythrin-conjugated protein A. A, results of transfectant cells. B, parent
cells.
Efficiency of Asialo-G
To investigate the
preferential use of G Synthesis Was
Affected by Coexisting Gangliosides and LacCer by 1,4GalNAc-T, we
performed the enzyme assay using variable mixtures of the two
substrates. G synthesis increased as the
G/LacCer ratio increased (Fig. 5). Unexpectedly,
asialo-G synthesis also increased as the
G/LacCer ratio increased, although there was a big
difference in the amounts of synthesized G and
asialo-G. These results suggested that the presence of
G might enhance the asialo-G synthesis, or
that the LacCer preparation might contain neuraminidase activity which
may convert G to asialo-G. The latter
possibility was excluded by an experiment in which LacCer was added to
the labeled G in the assay mixture (data not shown). The
effect of G or G on asialo-G synthesis was then examined. As shown in Fig. 6, the
presence of G markedly enhanced the synthesis of
asialo-G, whereas G had less effect. With
higher amounts of G or G, the efficiency of
asialo-G synthesis from LacCer was diminished. These
results suggest that the presence of G or G at appropriate levels may enhance the asialo-G synthesis by 1,4GalNAc-T.
and G synthesis are not competitive in in vitro assays. A, TLC of the reaction products of protA enzyme (22 µg of
fusion protein-IgG complex). Mixtures of G and LacCer at
indicated molar ratios were used as acceptors. Separated products were
analyzed by TLC with solvent of chloroform, methanol, 0.22% CaCl (55:45:10). B, plots of the bands shown in A.
synthesis. LacCer was mixed with G, G,
or G at the indicated molar ratios and used as acceptors. Inset demonstrates the asialo-G bands (arrows) synthesized in the presence of G (a) or G (b). Molar ratios of
ganglioside to LacCer of the individual points were 5:95 (lane
1), 20:80 (lane 2), 50:50 (lane 3), and 83:17 (lane 4) to make a total concentration 325 µM.
These results were plotted for G (
), G (&cjs3409;), and GM1 (
) addition. Only the points at the
right end were 5 times the amount (415/17 molar ratio) of G or G at the point of
83:17.
/G 1,4-N-acetylgalactosaminyltransferase gene and show
that the enzyme efficiently synthesizes G and G in the presence of appropriate acceptors. We also investigated
whether the enzyme could synthesize related compounds, i.e. asialo-G, GalNAc-G, and GalNAc-SPG.
Although the purified enzyme could synthesize all three products, the
efficiency of the reaction was only 1-3% of that for
G/G formation. Changing the assay conditions
did not improve the efficiency. Hashimoto et al.(17) reported that a GalNAc-T purified from mouse liver
also preferentially used G and G as
substrates. This enzyme preparation had only trace (<2%) levels of
activity with LacCer, SPG, and G. As Pohlentz et
al.(16) had reported that G, G,
and asialo-G were produced by the same enzyme in extracts
of rat liver, we examined the specificity of the enzyme in more detail
and determined whether these minor reactions were biologically
significant in cultured cells.
blood group specificity. A cDNA coding for the latter
structure has recently been cloned and is clearly different from
1,4GalNAc-T studied here(18) . Moreover, we show (Fig. 2), that in contrast to our 1,4GalNAc-T, the stomach
enzyme does not synthesize G from G, again
suggesting that it may be a separate enzyme. synthesis is not
clear. It appears that even though the G/G synthase use LacCer very inefficiently as a substrate in
vitro, it is able to produce substantial amounts of
asialo-G in certain cells. Analysis of the glycolipid
content of cells transiently and stably transfected with
1,4GalNAc-T cDNA showed that synthesis of G or
asialo-G was highly influenced by the precursors available
in the cells. Thus, asialo-G, and no G, was
produced in transfected L cells. Since high expression of
asialo-G was observed even in transiently transfected
cells, the high level should reflect the actual rate of synthesis and
not be due to the slow accumulation of asialo-G in the
cells. L cells have no G; moreover, in all the transfected
cell lines studied, levels of 2,3-sialyltransferase were inversely
correlated with expression of asialo-G. These results show
that the levels of appropriate precursors (G or LacCer)
influence the propensity of the cell to produce G or
asialo-G. Transfected CHO cells, in contrast to
transfected L cells, produced both asialo-G and
G. This result may be explained by the synthesis of
smaller amounts of G in CHO cells than in the other cell
lines studied (except L cells). The preference of 1,4GalNAc-T for
G over LacCer as substrate also influences the ratio of
G/asialo-G produced by a cell. This enzyme
had a lower K and lower V for LacCer than it did for G (Table 1B). The
low V/K value for LacCer in
comparison to G as substrate observed in vitro is
consistent with the cell line ganglioside composition data. These data
show that the ability of a cell to synthesize asialo-G is
determined by G levels as well as the preferential use of
G over LacCer by 1,4GalNAc-T. G levels,
in turn, are controlled by 2,3-sialyltransferase levels and
possibly by other factors(19) ., in the cell. This was demonstrated in CHO transfected
cells in which asialo-G was detected only in
G-expressing cells. The presence of G in
certain concentrations, but not G, also enhanced
asialo-G synthesis in vitro. From these data, it
is also suggested that G stimulates its own synthesis as
well as asialo-G. If this is true, it may explain the
sigmoid reaction curve and nonlinear double reciprocal plots obtained
in the kinetics of protA for G (data not shown). This fact
would also be consistent with the sigmoidal character of Fig. 5B, upper panel. The mechanism for the
effect is at present not known. We suspect that the presence of
G has an effect in modifying the affinity of
1,4GalNAc-T, but this effect would have to be
concentration-dependent. It is also possible, but unlikely, that
G is simply stabilizing the protA enzyme during the
reaction.2,3-sialyltransferase was recovered in different gradient regions
containing cis-Golgi cisternae, from other sialyltransferase
existing in trans-Golgi-containing fractions(20) .
Thus, the preferential use of G may also be because
2,3-sialyltransferase is located in an earlier compartment than
GalNAc-T and may utilize most of the LacCer before it can reach the
GalNAc-T compartment. The use of brefeldin A also suggested that
2,3-sialyltransferase was located within the Golgi stacks
(brefeldin A resistant) while GalNAc-T was located beyond the brefeldin
A block (brefeldin A sensitive)(21, 22) .1,4GalNAc-T gene on the ganglioside patterns. These
studies suggest that even acceptors showing low efficiencies with the
enzyme might be used in certain cells. The existence of other GalNAc-T
species which would preferentially glycosylate LacCer or other
gangliosides containing a similar terminal Gal residue cannot, however,
be excluded. On-going studies to knock-out the 1,4GalNAc-T gene in
either mice or cultured cells should clarify these issues.
1,4GalNAc-T
cDNA.
), NeuAc (or
NeuGc)2,3Gal1,4Glc-Cer; G,
GalNAc1,4(NeuAc2,3)Gal1,4Glc-Cer; G,
NeuAc2,8NeuAc2,3Gal1,4Glc-Cer; G,
GalNAc1,4(NeuAc2,8NeuAc2,3)Gal1,4GlcCer;
G,
NeuAc2,3Gal1,4GalNAc1,4(NeuAc2,8NeuAc2,3)Gal1,4Glc-Cer;
sialyl-paragloboside, NeuAc or
NeuGc2,3Gal1,4GlcNAc1,3Gal1,4Glc-Cer; other
glycolipids were: asialo-G (gangliotriaosylceramide),
GalNAc1,4Gal1,4Glc-Cer; lactosylceramide or CDH (ceramide
dihexoside), Gal1,4Glc-Cer; CMH (ceramide monohexoside), Glc-Cer;
CTH (ceramide trihexoside), Gal1,4Gal1,4Glc-Cer.
We are grateful to Drs. T. Maita and T. Hayashibara
for valuable discussion. We thank Dr. R. Breathnach for pPROTA, and Dr.
N. Hanai for mAb KM966. We also thank T. Shimomura for excellent
technical assistance and Y. Nakaji for secretarial help.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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