(Received for publication, November 14, 1994; and in revised form, December 21, 1994)
From the
The substrate specificity of
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.
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, 100150 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.
Recently we have cloned a cDNA of G/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.
Glycolipids-The derivation of
glycosphingolipids used as acceptors of enzyme reaction was as follows:
G 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) .
Figure 1:
Expression of soluble 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.
When B78
melanoma cells were transfected with pM2T1-1/PROTA, the
conditioned media showed increasing activity of G 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.
Using protA, the substrate specificity of soluble 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).
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, 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.
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 (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).
Figure 3:
Changes of glycolipid components in cells
before and after expression of stably transfected 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 NH
OH (60:35:8) for acidic fraction, and
chloroform/methanol/H
O (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.
Figure 4:
Double staining of G 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.
Figure 5:
Asialo-G 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.
Figure 6:
Effects of gangliosides on asialo-G 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.
The data presented in this study support the identification
of the cDNA previously reported (3) as coding for the
UDP-GalNAc:G/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.
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 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.
Whether a separate
enzyme is also responsible for asialo-G 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) .
Another factor that can
apparently influence the ability of GalNAc-T to utilize LacCer as
substrate is the level of other gangliosides, particularly
G, 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.
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
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) .
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 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.
Figure 7:
Ganglioside patterns in cell lines before
and after transfection with 1,4GalNAc-T
cDNA.