(Received for publication, August 14, 1995)
From the
LEC29.Lec32 is a glycosylation mutant that was isolated from a selection of mutagenized Chinese hamster ovary (CHO) cells for lectin resistance. Compared with LEC29 CHO cells, the double mutant exhibited an unusually high sensitivity to the toxic lectin, ricin, indicating increased exposure of galactose residues on cell surface carbohydrates. Structural analysis of LEC29.Lec32 cellular glycoproteins showed a nearly complete lack of sialic acid residues. Genetic analysis demonstrated that the lec32 mutation is recessive and novel. Biochemical analysis showed that the mutant cells contained less than 5% of the cytidine 5`-monophosphate N-acetyl-neuraminic acid (CMP-NeuAc) present in parental CHO cells (1.6 nmol/mg of cell protein). A sensitive radiochemical assay used to measure CMP-NeuAc synthetase activity showed that the properties of this enzyme in parental CHO cells were essentially identical to those of CMP-NeuAc synthetase in various mammalian tissues. However, no CMP-NeuAc synthetase activity was detected in LEC29.Lec32 extracts. Mixing experiments provided no evidence for an inhibitor in the mutant CHO cells, and two revertants, which expressed only the LEC29 phenotype, had normal CMP-NeuAc synthetase levels. The combined evidence indicates that the lec32 mutation resides in either the structural gene encoding CMP-NeuAc synthetase or in a gene that regulates the production of active enzyme.
The addition of sialic acid to glycoproteins and glycolipids has
important physiological consequences and appears to be vital for
intercellular adhesion events involved in embryonic development and
differentiation(1, 2, 3) ;
myelination(4) ; the cell-mediated immune response via binding
to selectins, sialoadhesin, or
CD22(4, 5, 6, 7, 8, 9, 10) ;
and oncogenic transformation and metastasis(11, 12) .
The regulation of this terminal event in oligosaccharide synthesis is
mediated through 1) the synthesis of cytidine 5`-monophosphate N-acetylneuraminic acid (CMP-NeuAc) ()by N-acetylneuraminic acid cytidyltransferase (CMP-NeuAc
synthetase; EC 2.7.7.43), 2) the transport of CMP-NeuAc into the
appropriate cellular compartments, 3) the action of a
sialyltransferase, and 4) the action of neuraminidases that may remove
NeuAc residues.
A KB cell mutant lacking a sialyltransferase
activity has been isolated by selecting for resistance to the cytotoxic
effects of ultraviolet-inactivated Sendai virus(13) , and
numerous attempts have been made to obtain additional mammalian cell
mutants, defective in any of the above steps, by selection for
resistance to the sialic acid-binding and cytotoxic plant lectin, wheat
germ agglutinin (WGA) (12, 14, 15, 16, 17) . This
latter strategy has primarily yielded mutants with defects in CMP-NeuAc
transport, including three different Chinese hamster ovary (CHO) cell
lines(12, 18) . Two other types of WGA-resistant
mutant have either undergone derepression of a gene encoding an
(1,3)fucosyltransferase (19, 20, 21) or
alteration of a gene that leads to increased levels of CMP-NeuAc
hydroxylase(22, 23, 24) . These latter two
phenotypes behave dominantly in somatic cell hybrids, whereas the three
other WGA-resistant mutants exhibit a recessive phenotype.
In this
report we describe the isolation and properties of LEC29.Lec32, a cell
line obtained from a mutagenized population of CHO cells following
selection with WGA. The dominant LEC29 phenotype is due to expression
of an (1,3)fucosyltransferase activity, apparently identical to
that previously reported in LEC29 CHO cell extracts(21) . The
Lec32 phenotype is recessive and novel. We show in this paper that the lec32 mutation reduces CMP-NeuAc synthetase activity to
undetectable levels and reduces NeuAc on glycoproteins and glycolipids
by 95%.
To effect mutagenesis, a suspension culture of
Pro5 CHO cells at 2
10
cells/ml
was treated with MNNG as described (28) . After an expression
time of 8 days, mutagenized cells were plated at 1
10
cells/100-mm tissue culture dish in
10%
FCS containing 13.3 µg/ml WGA. Colonies present after 12 days were
screened for the expression of the Lewis-X (Le
) determinant
on cell surface carbohydrates using the stage-specific embryonic
antigen-1 (SSEA-1)/sheep red blood cell conjugate as
described(29) . Colonies that bound the SSEA-1/sheep red blood
cell conjugate were picked and cloned by limiting dilution, and lectin
resistance phenotypes were determined as
described(21, 26) . Somatic cell hybridization
analysis was performed by fusion of CHO cells using the polyethylene
glycol/dimethyl sulfoxide method(26) , and characterization of
the resulting hybrids was as described(21) .
CMP-NeuAc synthetase
activity was determined initially by a thiobarbituric acid
procedure(31) , and subsequently, a radiochemical assay was
used that was a modification of the method of Edwards and
Frosch(32) . Unless otherwise indicated, reaction mixtures
contained 90 mM Tris-HCl (pH 9.0), 20 mM
MgCl, 5 mM CTP (adjusted to pH 7.0 with 1 M KHCO
), 125 nmol of [
C]NeuAc
(560 cpm/nmol), and 5 µl of extract containing 40-140 µg
of protein. Incubation was at 37 °C for 15 min, and the reaction
was stopped by the addition of 1 ml of ice-cold water. The entire
reaction mixture was loaded onto a 1-ml column of AG 1-X4 anion
exchange resin (Cl
form), which was subsequently
washed with 2 ml of water followed by 3 ml each of 0.05, 0.06, 0.07,
0.08, 0.15, 0.20, 0.25, and 0.30 M NaCl. Under these
conditions, hydrolyzed NeuAc elutes with the water, NeuAc elutes with
0.05-0.08 M NaCl, and CMP-NeuAc elutes with
0.15-0.30 M NaCl (see Fig. 6). All eluates were
mixed with 16 ml of Ecolume and counted on a Beckman LS6800 liquid
scintillation counter.
Figure 6:
A
radiochemical assay for CMP-NeuAc synthetase. Parental and LEC29.Lec32
cell-free extracts were incubated with C-NeuAc (2 nmol;
17,500 cpm/nmol) to generate [
C]CMP-NeuAc as
described under ``Experimental Procedures.'' After 1 h,
reaction mixtures were fractionated on ion exchange columns under the
standard conditions described under ``Experimental
Procedures.'' Authentic [
C]NeuAc and
[
C]CMP-NeuAc eluted at the positions shown. Dotted line, parental; solid line,
LEC29.Lec32.
Figure 1:
Lectin
resistance properties of LEC29, LEC29.Lec32, and two Lec32 revertants.
Resistance to the cytotoxic lectins ricin and WGA was determined as
described under ``Experimental Procedures.'' Results are
shown as -fold difference compared with parental
(Pro5) CHO cells. The actual concentrations of each
lectin necessary to kill 90% of the parental CHO cells are as follows:
ricin, 0.010 µg/ml; WGA, 3 µg/ml. The numbers at the bottom of the figure represent -fold resistance or
-fold sensitivity. The data are the average of two separate
experiments.
The existence of two glycosylation mutations in LEC29.Lec32
cells was further supported by a two-step reversion experiment in which
LEC29.Lec32 cells were selected for resistance to ricin. When 0.4 ng/ml
ricin was used, surviving colonies appeared at a rate of about 4
10
and retained the characteristics of LEC29
cells (i.e. the lec32 mutation had been reverted).
When one of these revertants was exposed to 7 ng/ml ricin, surviving
colonies appeared at a rate of 2
10
and were
indistinguishable from parental CHO cells (i.e. both the LEC29 and lec32 mutations had been reverted).
Attempts to isolate double revertants in a single step using the higher
concentration of ricin were unsuccessful when 3
10
cells were screened, as would be expected for an extremely rare
event occurring at a predicted frequency of
8
10
.
Figure 2:
Binding of monoclonal antibodies to cell
surface antigens. Approximately 10 cells were washed and
assayed for their abilities to bind the monoclonal antibody SSEA-1 (upper panel) or SNH3 (lower panel) in an indirect
binding assay. The results show percentage of
I-Protein A
bound and are from a single experiment that was reproduced once or more
for each cell line. The sugar sequences recognized by each monoclonal
antibody are indicated:
, Gal;
, GlcNAc;
, Fuc;
, NeuAc.
Figure 3:
Binding of cellular glycopeptides to
NeuAc-recognizing affinity columns.
[H]Glc-labeled cellular glycopeptides were
prepared as described under ``Experimental Procedures'' and
chromatographed separately on MAA-agarose or
WGA-agarose.
Figure 4:
NeuAc content of cellular glycopeptides.
The amount of NeuAc in cellular glycopeptides prepared from
10
parental or LEC29.Lec32 cells was determined using
HPAEC-PAD analysis. Authentic NeuAc (2 nmol) was used as a standard.
The data are from a single experiment that was repeated
once.
Figure 5:
Intracellular pools of NeuAc and
CMP-NeuAc. The levels of NeuAc and CMP-NeuAc in cytoplasmic extracts
prepared from 6
10
parental (middle
panel) or LEC29.Lec32 (lower panel) cells were determined
by HPAEC-PAD analysis. Authentic CMP-NeuAc (2 nmol) was used as
standard (upper panel). The peak at 73 min corresponds to
CMP-NeuAc; authentic NeuAc and NeuAc derived from hydrolyzed CMP-NeuAc
eluted at 22 min. The species at 26 and 65 min are unidentified
breakdown products of CMP-NeuAc that do not correspond to either NeuGc
(elutes at 46 min) or CMP-NeuGc (elutes at 90 min). The data are from
one of two experiments that gave similar
results.
CMP-NeuAc synthetase activity has been characterized in
various mammalian tissues(39, 40) , and the activity
in CHO cells was optimized for comparison. The optimum pH for the CHO
enzyme was found to be 8.5-9.0 in the presence of Mg (Fig. 7) and 7.0 with Mn
. However, when
Mn
was used at pH values above 8.0 under conditions
that minimized the formation of Mn(OH)
, a similar optimum
to that observed with Mg
(i.e. pH 9.0) was
found, although the levels of activity were only half those observed
with Mg
(data not shown). Highest levels of activity
were obtained when the Mg
concentration was at least
20 mM (Fig. 7). Divalent cations other than
Mn
were unable to effectively substitute for
Mg
, although some activity (10% of that with
Mg
) was obtained in the presence of Fe
(Table 2). As reported for the rat liver
enzyme(40) , Cu
and Zn
were
inhibitory when added with Mg
.
Figure 7:
Optimum pH and MgCl
concentration for CMP-NeuAc formation. CMP-NeuAc synthetase activity
was determined in cell-free extracts of parental CHO cells (about 88
µg of protein) at various pH values in the presence of 10 mM Mg
(upper panel) under the assay
conditions described under ``Experimental Procedures.''
Similar results were obtained in two experiments. CMP-NeuAc synthetase
activity was also determined in parental CHO extracts (
40 µg
of protein) under conditions described under ``Experimental
Procedures'' except that the MgCl
concentration was
varied (lower panel). Similar results were obtained in three
experiments.
The optimum
temperature over the range tested (22-52 °C) was 37 °C
(data not shown). The reaction was essentially linear for at least 60
min and at protein concentrations from about 40-360
µg/reaction (Fig. 8). Other reports have indicated that (at
least for the partially purified mammalian enzyme) sulfhydryl reagents,
such as dithiothreitol, are stabilizing or
stimulatory(39, 40) . The inclusion of 2.5 mM dithiothreitol in our standard reaction increased CMP-NeuAc
synthetase activity by about 30%. It was also observed that
concentrations of CTP above 5 mM inhibited activity but that
the addition of 0.5 nmol of unlabeled CMP-NeuAc to a reaction mixture
had no effect. Kinetic experiments performed under optimal conditions
(see ``Experimental Procedures'') but in the absence of
dithiothreitol gave apparent K values of about
0.34 mM for NeuAc and 1.3 mM for CTP. In each case,
the value falls within the range of those reported for the CMP-NeuAc
synthetase from other mammalian
sources(39, 40, 41) .
Figure 8: LEC29.Lec32 cells lack CMP-NeuAc synthetase activity. CMP-NeuAc synthetase activity of parental CHO (about 90 µg of protein) and LEC29.Lec32 (about 84 µg of protein) extracts was measured over a range of incubation times (upper panel) and at a variety of protein concentrations, for 15 min (lower panel). All other reaction conditions were as described under ``Experimental Procedures.''
Since both the E. coli(42) and rat liver (40) CMP-NeuAc synthetase enzymes are known to be sensitive to NEM, the CHO enzyme was tested. It was found that, whereas very low levels of NEM were slightly stimulatory, the addition of 1 mM NEM to the reaction mixture reduced the CHO activity to about 12%, while the activity of the purified E. coli enzyme was only reduced to 88% under these conditions (Fig. 9).
Figure 9: Inhibition of CHO CMP-NeuAc synthetase by NEM. The CMP-NeuAc synthetase activity of parental CHO cell extracts (about 100 µg of protein) and purified E. coli CMP-NeuAc synthetase (about 0.03 µg of protein) were assayed in the presence of NEM (0.005-1 mM) under the standard reaction conditions described under ``Experimental Procedures.'' In the absence of NEM, 100% activity for the CHO extract was 3.01 nmol/min/mg protein, and for the E. coli enzyme, it was 46,167 nmol/min/mg protein.
The mammalian CMP-NeuAc synthetase has been reported by many authors to be localized to the nucleus(39, 40, 43, 44, 45, 46) . Nuclei prepared from parental CHO cells were found to contain at least 70% of the total extractable activity. Our usual extraction buffer containing 0.15 M NaCl, 25% glycerol, and 1% Nonidet P-40 solubilized 85-95% of total cellular activity. Reextraction of the pelleted material with the same extraction buffer released the remaining activity. Increasing the detergent concentration to 2% did not significantly increase the amount of enzyme activity initially solubilized.
Although many WGA-resistant, NeuAc-deficient glycosylation mutants have been characterized(47) , the lec32 mutation described in this paper is the first to result in the loss of CMP-NeuAc synthetase activity. Analysis of LEC29.Lec32 glycolipids and cell-surface glycopeptides has demonstrated a reduction of 95% in NeuAc content. Furthermore, the cellular pools of CMP-NeuAc are greatly reduced, while that of free NeuAc is increased about 10-fold in mutant cells compared with parental CHO cells. This latter observation may be the result of two different mechanisms: 1) the lack of CMP-NeuAc synthetase activity should result in a build-up of unused NeuAc substrate; and 2) the deficiency of CMP-NeuAc will lead to release of a previously reported feed-back inhibition of UDP-N-acetylglucosamine 2-epimerase(48) , the enzyme responsible for the synthesis of N-acetylmannosamine, a precursor in the synthesis of NeuAc.
The properties of CMP-NeuAc synthetase in cell-free extracts prepared from parental CHO cells appear to be nearly identical to those previously described for CMP-NeuAc synthetase studied in other mammalian tissues(39, 40, 41) . In addition, the CHO CMP-NeuAc synthetase is predominantly localized to the nucleus, as observed for the mammalian enzyme from other sources(39, 40, 43, 44, 45, 46
Molecular
biological approaches to the study of CMP-NeuAc synthetase have thus
far been confined to the enzyme from bacterial sources such as E.
coli(31, 49, 50) and Neisseria
meningitidis(32) . In both cases, the studies were aimed
at understanding the steps involved in the synthesis of bacterial
virulence factors, such as capsular polysaccharides, which contain
large amounts of (2,8)-linked NeuAc residues, or providing a
source of enzyme for the synthesis of large amounts of CMP-NeuAc. The
nature of the lec32 mutation is currently unknown and is the
subject of further investigation. Critical reagents, such as a
molecular probe for the mammalian CMP-NeuAc synthetase gene and an
antibody that specifically recognizes the enzyme must be developed.
Attempts to detect the CHO enzyme with an antibody directed against E. coli CMP-NeuAc synthetase were unsuccessful. (
)Transfection experiments aimed at correcting the defect in
LEC29.Lec32 cells using two different mammalian expression vectors
containing the cloned E. coli gene (kindly supplied by Dr.
Willie Vann), were also negative (data not shown). The fact that small
amounts of NeuAc are present on both glycoproteins and glycolipids of
LEC29.Lec32 cells suggests that CMP-NeuAc synthetase is not completely
inactivated by the lec32 mutation. This would be consistent
with a mutation in the coding region of the CMP-NeuAc synthetase gene
that affects the activity, stability, or localization of the enzyme. It
is also possible that the lec32 mutation involves a mutation
in an upstream control element, a gene encoding a positive or negative
transcriptional regulatory factor, or a gene rearrangement, all of
which could allow small amounts of enzyme to be synthesized. Although a
partial NH
-terminal amino acid sequence has been reported
for the CMP-NeuAc synthetase from rat liver(40) , there have,
to date, been no reports of the cloning of a mammalian gene that
encodes this enzyme. As an alternative approach, an expression cloning
strategy will be used to correct the defect in LEC29.Lec32 cells by
transfection of a cDNA library. This should result in the cloning of a
CMP-NeuAc synthetase cDNA or a cDNA encoding a factor that regulates
CMP-NeuAc synthetase expression.
As for any enzyme involved in the regulation and synthesis of carbohydrate ligands for cell adhesion molecules, further studies of CMP-NeuAc synthetase may be of considerable importance. The nuclear localization of this enzyme (which has long puzzled investigators) provides yet another incentive for probing both the range of its normal cellular functions and the regulation of the gene that encodes this enzyme. It has been proposed that the compartmentalization of this enzyme may be required to protect its product from the action of a cytoplasmic, membrane-bound hydrolase(39) . Recent work involving the glycosylation of nuclear proteins (51) has suggested that a sialyltransferase, whose activity is critical for the normal functions of the various nuclear proteins that never transit the Golgi apparatus, may exist in the nucleus. This theory is supported by a report in which five glycosyltransferases, including a sialyltransferase, were found to be associated with rat liver nuclei(52) . In this case, the nuclear localization of the enzyme responsible for producing CMP-NeuAc would clearly be of functional importance.