(Received for publication, July 25, 1995)
From the
The dominant Chinese hamster ovary cell glycosylation mutant,
LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells
express altered cell surface carbohydrates with markedly reduced
binding to I-PSA and increased binding to
I-labeled Datura stramonium agglutinin (DSA)
compared with parental cells. Desialylated
[
H]Glc-labeled LEC18 cellular glycopeptides that
did not bind to concanavalin A-Sepharose exhibited an increased
proportion of species that were bound to DSA-agarose. Most of these
glycopeptides bound to ricin-agarose and were unique to LEC18 cells.
This fraction was purified from
10
cells and shown by
H NMR spectroscopy and methylation linkage analysis to
contain novel N-linked structures. Digestion of these
glycopeptides with mixtures of
-D-galactosidases and N-acetyl-
-D-glucosaminidases gave core
glycopeptides that, in contrast to cores from parental cells, were
mainly not bound to concanavalin A-Sepharose or to PSA-agarose.
H NMR spectroscopy, matrix-assisted laser desorption
ionization/time of flight mass spectrometry, electrospray mass
spectrometry, and collision-activated dissociation mass spectrometry
showed that the LEC18 core glycopeptides contained a new GlcNAc residue
that substitutes the core GlcNAc residue. Methylation linkage analysis
of the parent compound provided evidence that the GlcNAc is linked at
O-6 to give the following novel, N-linked core structure.
The N-linked carbohydrates of mammalian cells are
initially synthesized on dolichol-phosphate, transferred en bloc to an Asn-X-(Ser/Thr) sequon, trimmed by glycosidases,
and matured via the sequential action of a series of
glycosyltransferases(1) . The core region of a mature N-linked structure is the
Man(1,6)[Man
(1,3)]Man
(1,4)GlcNAc
(1,4)GlcNAc,
originally synthesized on dolichol. In mammalian cells, only four types
of core have been described: the Man
GlcNAc
Asn
described above; Man
GlcNAc
(Fuc)Asn, where Fuc (
)is linked O-6 to the Asn-linked GlcNAc; and cores, with or
without Fuc, that contain a bisecting GlcNAc(2, 3) .
By contrast, certain plant glycoproteins have N-linked
carbohydrates with Xyl attached at O-2 of the
-1,4-linked Man
and/or Fuc attached at O-3 of the Asn-linked GlcNAc (4, 5) . A glycoprotein from honeybee has been
reported to have two Fuc residues substituting the Asn-linked GlcNAc at
O-3 and O-6(6) . No substitutions of the latter types have been
reported to date in mammalian
glycoproteins(1, 2, 3) . However, an altered N-linked core might be expected to be the basis of cellular
resistance to the toxicity of pea lectin (PSA), because PSA binds
specifically to the fucosylated core region of biantennary and
-1,6-branched, triantennary, N-linked
carbohydrates(7) .
LEC18 Chinese hamster ovary (CHO) cells are rare mutants that were isolated following a selection for resistance to PSA(8) . They are 39-fold more resistant to PSA compared with parental cells. In addition, LEC18 cells are 16-fold more resistant to Lens culinaris agglutinin (LCA), a lectin with a very similar binding specificity to PSA(9) . The properties of LEC18 CHO cells are dominant in somatic cell hybrids formed with parental cells(8) , showing that their lectin resistance arises from a gain-of-function mutation. Other gain-of-function CHO glycosylation mutants include LEC10, LEC11, LEC12, LEC29, and LEC30(10) . Each of these mutants synthesizes cell surface carbohydrates carrying a sugar residue that is not synthesized by parental CHO cells and that confers a distinctive pattern of lectin resistance. In this paper, we show that LEC18 cells also synthesize a species of N-linked carbohydrate that is not present in parental cells, nor in any of the previously described dominant CHO mutants. These carbohydrates have a novel GlcNAc substitution in their core region, which markedly alters the conformation of the trimannosyl core and is likely to be the reason that LEC18 cells are highly resistant to both PSA and LCA.
Samples of glycopeptides were applied
to L-PHA-agarose, RCA-agarose,
RCA
-agarose, tomato-agarose, or DSA-agarose columns in
PBS
(pH 7.4), and the columns were washed with >8
column volumes of the same buffer. The addition of sugar was not
required for elution of glycopeptides from the L-PHA-agarose
column. Buffer containing 100-200 mM lactose was used to
elute bound glycopeptides from RCA
-agarose and
RCA
-agarose columns. The bound glycopeptides from
tomato-agarose and DSA-agarose columns were eluted using a mixture of N,N`-diacetylchitobiose and N,N`,N"-triacetylchitotriose (7-10
mg/ml) in PBS
(pH 7.4). Chromatography was performed
either at room temperature or at 4 °C at flow rates of 6-10
ml/h.
Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) was performed on a
Voyager RP Biospectrometry work station (Perspective Biosystems,
Framingham, MA), using -cyano-4-hydroxycinnamic acid as matrix. A
laser power of 190 attenuator (nitrogen laser at 337 nm) was used, and
an average of 50 scans were taken.
Figure 1:
Binding of I-PSA and
I-DSA. Parent CHO cells
(
), Pro
LEC18.21B (
),
Gat
LEC18.14F (
), Pro
Lec13
(
), and Pro
Lec1 (
) cells were incubated
with
I-PSA or
I-DSA at 4 °C for 1 h as
described under ``Experimental Procedures.'' After
centrifugation through 15% BSA, the amount of bound lectin was
calculated.
Most CHO glycosylation mutants exhibit an increased
sensitivity to and binding of certain lectins(21) . Such
lectins, once identified, can be useful for affinity purification of
novel glycopeptides. In binding experiments with a series of I-lectins, DSA was identified as binding better to LEC18
cells than to parental CHO cells (Fig. 1). DSA binds to
poly-N-acetyllactosamine chains (22, 23) ,
and it can be seen that LEC18 cells bound significantly more
I-DSA than parental cells. Both independent LEC18 mutants
exhibited a similar increase in DSA binding. Lec1 CHO cells were used
as a negative control, because they do not synthesize complex, N-linked carbohydrates(24) .
Figure 2:
Lectin affinity chromatography of H-Glc-labeled glycopeptides.
[
H]Glc-labeled glycopeptides that passed through
ConA-Sepharose were desialylated, and fractionated on tomato-agarose.
The unbound fraction was subsequently fractionated on DSA-agarose (upper panel). In the lower panel, the DSA-retarded
fraction from parental CHO or LEC18 cells was fractionated on
RCA
-agarose.
About 65% LEC18
glycopeptides were retarded on the DSA-agarose column, but 15%
parental glycopeptides were also present in this pool. In order to
separate glycopeptides unique to LEC18 cells, further fractionation of
the DSA-retarded glycopeptides was performed on a series of lectin
columns. A dramatic difference in elution profile was observed on
RCA
-agarose (Fig. 2). LEC18 glycopeptides separated
into late retarded (45%) and bound (55%) species, whereas parent
glycopeptides eluted in the unbound (35%) or early retarded (65%)
fractions, indicating that the glycopeptides derived from LEC18 cells
were indeed unique to the mutant.
Figure 3:
Purification of
[H]Glc-labeled core glycopeptides. The
DSA-retarded glycopeptides of LEC18 and parental CHO cells (Fig. 2) were, exhaustively, digested with bovine testis
-D-galactosidase and Jack bean N-acetyl-
-D-glucosaminidase and chromatographed
on a Bio-Gel P-2 column (1.5 cm
70 cm) as described under
``Experimental Procedures.''
Fractionation on ConA-Sepharose and PSA-agarose revealed a
significant difference between V glycopeptides of
LEC18 and parent cells. As would be expected,
95% of parental CHO V
glycopeptides did bind to ConA-Sepharose,
indicating that they had been converted to trimannosyl cores (Fig. 4). By contrast,
65% of LEC18 V
glycopeptides did not bind to ConA-Sepharose (Fig. 4).
These results suggested that the core glycopeptides from LEC18 cells
have a modified structure. Similarly, for core glycopeptides obtained
following exhaustive digestion with a mixture of
-D-galactosidase and N-acetyl-
-D-glucosaminidase from D.
pneumoniae(27) , a much greater proportion of LEC18 V
glycopeptides did not bind to ConA-Sepharose,
compared with parental glycopeptides.
Figure 4:
ConA-Sepharose and PSA-agarose affinity
chromatography. The V fraction obtained
from Parent and [
H]Glc-labeled LEC18 cells (Fig. 3) was fractionated separately on ConA-Sepharose as
described under ``Experimental Procedures.'' The LEC18 V
glycopeptides were also fractionated on
PSA-agarose (lower panel). Fucosylated trimannosyl core
glycopeptides from parent CHO cells were prepared as described under
``Experimental Procedures'' and chromatographed separately on
PSA-agarose. MM, methyl
-D-mannoside.
A trimannosyl, fucosylated
core structure would be expected to bind to PSA-agarose(7) .
This was found to be the case for a preparation of fucosylated core
glycopeptides from parental CHO cells (Fig. 4). However, the V glycopeptides from LEC18 cells did not bind to
PSA-agarose (Fig. 4), despite the presence of almost one full
Fuc equivalent (Table 1). The monosaccharide compositional
analysis of the V
fraction of LEC18 and parental
CHO cells in Table 1also showed that neither LEC18 nor parent
core glycopeptides had any Gal residues. In addition, parent cores had
only approximately two GlcNAc equivalents showing that the glycosidase
digestion was complete. However, LEC18 core glycopeptides contained an
additional GlcNAc residue that had resisted N-acetyl-
-D-hexosaminidase digestion. This
GlcNAc was postulated to be the reason most LEC18 core glycopeptides
did not bind to ConA-Sepharose or PSA-agarose (Fig. 4).
Figure 5:
H NMR spectroscopy of LEC18
glycopeptides at 23 °C. Approximately 500 µg of the
RCA
-bound fraction (Fig. 2) were prepared from
10
LEC18 cells. They were extensively desalted on Bio-Gel
P-2, passed through Chelex, exchanged with D
O, and
subjected to
H NMR spectroscopy at 500
MHz.
The H NMR spectrum in Fig. 5is
unusually complex in the 4.9-5.2-ppm region, and no resonances
corresponding to known H
or H
chemical shifts
for core Man residues in previously reported structures were present in
the NMR carbohydrate data base (Sugabase, version 1.05; 3). The
resonance at 2.028 ppm is typical of -NAc groups from GlcNAc residues
in poly-N-acetyllactosamine chains(28) . Thus the
glycopeptides are a mixture of complex N-glycans containing
poly-N-acetyllactosamine chains.
After further acid
hydrolysis to remove the small amount of sialic acid, a portion of
these glycopeptides was subjected to methylation linkage analysis. From
the data in Table 2, it is evident that the LEC18 glycopeptides
have terminal Gal(3) , GlcNAc(1) , and Fuc (0.7)
residues and substituted Gal, GlcNAc, and Man residues. The
substitutions of the Man residues are consistent with a mixture of tri-
and/or tetraantennary N-linked carbohydrates. The presence of
Gal substituted at O-3 is consistent with the presence of
poly-N-acetyllactosamine chains, as is the 4-substituted
GlcNAc. The most remarkable finding from this analysis was the presence
of a single, unsubstituted GlcNAc residue and of 1.75 molar residues of
4,6-substituted GlcNAc. The only 4,6-substituted GlcNAc in the usual N-linked structure arises from the presence of Fuc at the O-6
of the Asn-linked GlcNAc. In the LEC18 glycopeptides, Fuc linkage
accounts for 0.7 of the 4,6-linked GlcNAc, leaving one full
residue of 4,6-substituted GlcNAc to be accounted. In fact, it will be
shown subsequently that this residue is located in the altered core
region of LEC18 glycopeptides.
The methylation linkage analysis also
showed the presence of O-4 substituted Gal residues (Table 2), a
substitution not observed previously for N-linked
carbohydrates from mammalian cells (1, 2, 3) . However, the presence of this
residue in glycolipids and in fish egg N-glycans has been
observed previously(29, 30) . Since no direct linkage
analysis has previously been reported for CHO-derived
poly-N-acetyllactosamine containing N-linked
carbohydrates, the O-4-substituted Gal may be a feature of some N-glycans in all Pro CHO cells. Whatever its
origin, this residue was susceptible to the exoglycosidases used,
because Gal residues were absent after digestion (Table 1),
indicating that 4-substituted Gal residues are part of the
poly-N-acetyllactosamine chains and are linked by
-glycosyl residues. Furthermore, the methylation linkage analysis
showed the presence of small amounts of 6-substituted, 4,6-substituted,
and 3,6-substituted Gal residues (Table 2). Finally, the analysis
showed no evidence for 3,4,6-substituted GlcNAc that would be predicted
if the Asn-linked GlcNAc was disubstituted, for 3,4,6-substituted Man
that would be predicted for the presence of a bisecting GlcNAc, nor for
2,3,6-substituted Man or for a significant molar proportion of any
unusual sugar like Xyl.
Figure 6:
Mass
spectrometry of LEC18 V core
glycopeptides. The V
core glycopeptide
fraction (Fig. 3) was isolated from LEC18 glycopeptides shown in Fig. 5as described under ``Experimental Procedures.''
A portion (
5 µg) was subjected to MALDI-TOF-MS (upper
panel). Approximately 5 µg of the same preparation was
subjected to ES-MS (lower panel). The ES-MS spectrum was
recorded with an ionspray voltage of 3300 and the orifice at 35
V.
When ES-MS spectra were recorded at 70 V, a
strong MH ion at 1374.4 atomic mass units along with
some fragment ions were observed (Fig. 7). Attempts were made to
obtain daughter ions by collision-activated dissociation (MS/MS). The
MS/MS spectrum in Fig. 8shows that the MH
ion
at 1374.4 atomic mass units gave daughter ions inter alia at
atomic mass units 1228.4, 1171.4, 889.2, and 686.2. The interpretation
of these ions is shown in Fig. S1. Briefly, the ions at 1228.4
and 1171.4 atomic mass units were derived from the MH
ion with a loss of a Fuc or GlcNAc residue, respectively.
However, the ion at 889.2 atomic mass units corresponds to a loss of
three Man residues from MH
and has a composition of
GlcNAc
(Fuc)Asn, confirming the presence of an extra GlcNAc
residue in the core region. This ion loses a GlcNAc residue to give the
ion at 686.2 atomic mass units.
Figure 7:
ES-MS
spectrum of LEC18 V core glycopeptides
recorded at 70 V. The LEC18 V
core
glycopeptides (
5 µg) shown in Fig. 6were subjected to
ES-MS as described under ``Experimental Procedures'' except
that the orifice used was at 70 V.
Figure 8:
MS/MS spectrum of the MH ion 1374.4 obtained by ES-MS. The ion at 1374.4 atomic mass units
in the ES-MS spectrum of Fig. 6, lower panel, was
subjected to collision-activated dissociation as described under
``Experimental Procedures.''
Figure S1:
Scheme
1MS/MS fragmentation of the MH ion at 1374.4 atomic
mass units. M, Man; Gn,
GlcNAc.
Based on this information, it was
possible to interpret many of the fragment ions observed in the ES-MS
spectrum of Fig. 7. Four fragmentation pathways could be deduced (Fig. S2). It is evident that the glycopeptide undergoes
fragmentation both from the nonreducing end and from the Asn end to
give fragment ions that are interpretable in structural terms. The most
critical ions observed were at atomic mass units 889.2, 686.2, and
483.2 and were composed of GlcNAc(Fuc)Asn,
GlcNAc
(Fuc)Asn, and GlcNAc
(Fuc)Asn,
respectively. The latter shows that the Asn-linked GlcNAc is
substituted with Fuc. This sequence of ions and the MS/MS data provide
strong evidence that the new GlcNAc is attached to the core GlcNAc
residue and not to the Asn-linked GlcNAc.
Figure S2:
Scheme 2ES-MS fragmentation of LEC18 V core glycopeptides. M, Man; Gn, GlcNAc.
H NMR spectra
of the LEC18 V
core glycopeptides shown by mass
spectrometry in Fig. 6to contain one major molecular species
are shown in Fig. 9. It is apparent that the preparation
contained a single glycopeptide species with three GlcNAc residues,
three Man residues, and one Fuc residue. Spectra were recorded at 23
and at 42 °C. At the higher temperature, the HOD peak shifted to
reveal certain resonances obscured at 23 °C. When the chemical
shifts of H
, H
, -NAc, and Fuc regions were
entered into Sugabase version 1.05(3) , no structure was given,
consistent with the evidence that the LEC18 V
core
is a new structure. Since only four other fucosylated core structures
are known to exist in mammalian N-linked carbohydrates (1, 2, 3) and since the ES-MS, MS/MS, and
GLC-MS data provide strong evidence for the proposals in Schemes 1 and
2, the spectra in Fig. 9were tentatively assigned (Table 3). Several reasons argue that these assignments will
prove correct once larger quantities of LEC18 core glycopeptides can be
subjected to chemical analysis. Whereas the presence of the bisecting
GlcNAc causes H
and H
resonances for each core
Man residue to shift, only the H
of the Man
1,4-
resonance was significantly changed in the LEC18 core; whereas the
presence of a bisecting GlcNAc does not alter the H
or -NAc
resonances of the Asn-GlcNAc, both are changed in the LEC18 core. Most
interestingly, the H
and -NAc resonances of the LEC18 core
GlcNAc are different from any other core, including those from
nonmammalian sources. Importantly, none of the latter cores show the
resonance at 5.211 ppm. This is assigned to the new GlcNAc of the LEC18
core because it is very similar to the H
at 5.144 ppm of
the GlcNAc in N,N`-diacetylchitobiose-p-nitrophenyl (33) and to the N-acetyl of this GlcNAc, which occurs
at 2.012 ppm compared with 2.018 ppm for the novel LEC18 GlcNAc
residue. The chitobiose disaccharide was synthesized by a new
GlcNAc-GlcNAc-transferase from snail, and the LEC18 GlcNAc, also in
GlcNAc-GlcNAc linkage, behaves very similarly, although not
identically. The 5.211-ppm resonance at both 23 and 42 °C has a J value of 5.5 Hz, a value intermediate between the J values observed for residues in
-linkage (J =
3-4 Hz; 3) and residues in
-linkage (J =
6.5-8.0 Hz; (3) ). The resonance assigned to the novel
GlcNAc is not present in an equimolar amount, in the same manner that
the Asn-GlcNAc is not. However, there was no evidence for
3,4,6-substituted GlcNAc in the linkage analysis (Table 2), and
the H
and NAc chemical shifts of the Asn-GlcNAc are as
expected. Thus the novel GlcNAc residue in LEC18 cannot be linked to
the Asn-GlcNAc.
Figure 9:
H NMR spectroscopy of LEC18 V
core glycopeptides. The LEC18 V
core glycopeptides (
40 µg in
200 µl D
O) characterized in Fig. 6Fig. 7Fig. 8were subjected to
H
NMR spectroscopy at 500 MHz at 23 and 42 °C. Chemical shifts (ppm)
were assigned based on the acetone signal at 2.225
ppm.
The H NMR spectra and chemical shift
comparisons with all previously assigned core structures from all
sources, along with methylation linkage analysis and the combined
MALDI-TOF-MS, ES-MS, and MS/MS data provide strong evidence that the
new GlcNAc is linked to the nonfucosylated core GlcNAc residue at O-6.
It is not clear whether the configuration of the GlcNAc1,6GlcNAc
linkage is
or
because the
H NMR spectrum of
core glycopeptides revealed an intermediate J value of 5.5 Hz
for the new GlcNAc residue. The structure proposed for the novel core
region of LEC18 N-linked carbohydrates is shown in Fig. S3.
Figure S3:
Scheme 3Proposed structure for the LEC18 V core
glycopeptides.
In this paper, a series of experiments on highly purified
[H]Glc-labeled and unlabeled glycopeptides from
LEC18 CHO cells have provided evidence for a new N-linked core
structure that has not previously been observed in N-glycans
from any source. The novel core structure was found on branched,
polylactosamine-containing, N-glycans from LEC18 cells that
were not found in parent CHO cells and that bound to both DSA and
RCA
(Fig. 2). Parent CHO glycopeptides were present
in the DSA-retarded fraction, but they were shown to have the expected
trimannosyl cores (>95% bound to ConA) after exhaustive digestion
with
-D-galactosidases and N-acetyl-
-D-glucosaminidases ( Fig. 4and Table 1). By contrast, identically treated LEC18 glycopeptides
remained largely unbound to ConA and PSA (Fig. 4). Composition
analysis revealed that the LEC18 core glycopeptides had an extra GlcNAc
compared with core glycopeptides from parent cells (Table 1).
Proof of an extra GlcNAc in LEC18 core glycans was obtained by
preparing 500 µg of the unique LEC18 species that binds to DSA
and RCA
. It was shown by
H NMR spectroscopy to
be a mixture of branched, poly-N-acetyllactosamine-containing
species with a core fucose residue (Fig. 5). This material was
too complex to analyze further by
H NMR, as no
corresponding spectrum is present in Sugabase(3) . Exhaustive
exoglycosidase digestion of this material gave a discreet core
preparation of
45 µg. MALDI-TOF-MS analysis revealed a major
MH
ion at 1374.4 atomic mass units corresponding to
Hex
HexNAc
dHex
Asn (or
Man
GlcNAc
(Fuc
)Asn). Fragment ions
generated during ES-MS revealed the critical ion at 889.2 atomic mass
units that corresponded to a species of
HexNAc
dHex
Asn (or
GlcNAc
(Fuc
)Asn). MS/MS analysis of the 1374.4
atomic mass units ion confirmed the generation of this critical
fragment ion (Fig. S1). In addition, fragment ions generated by
ES-MS confirmed the results of MS/MS. These ions were generated when
the sample was bombarded at 70 V under conditions normally used to
obtain the molecular weight of proteins. Interpretation of the ES-MS
data revealed a fragment ion corresponding to breakage at every
glycosidic linkage and the occurrence of four fragmentation pathways (Fig. S2). Although no examples in the literature describe
fragment ions obtained from glycopeptides using ES-MS(34) , our
experience shows that it is possible to obtain sequence information on
underivatized, native glycopeptides by recording ES-MS using two
different conditions (one at 35 V and the other at 70 V). This is very
useful when dealing with small amounts of samples that are difficult to
derivatize.
Taken together, the H NMR and MS data
provide proof for the existence of a novel core that is characterized
by GlcNAc substitution of the core GlcNAc. The evidence that the new
GlcNAc is linked at O-6 of the core GlcNAc was derived from GLC-MS of
the parent compound (Table 2). However the configuration of the
new linkage is uncertain because the coupling constant of the
H
for the novel GlcNAc is 5.5 Hz (Fig. 9), a value
intermediate between known
- and
-linked
residues(3) . The combined data provide strong evidence for the
core structure showed in Fig. S3.
If the new GlcNAc in the
LEC18 core is in -linkage, as seems likely based on its similarity
to the terminal nonreducing GlcNAc in
GlcNAc
1,4GlcNAc-p-nitrophenol(33) , it was
nevertheless not hydrolyzed by two N-acetyl-
-D-hexosaminidases. This may be because
the new GlcNAc residue is internal and hence crowded and not accessible
to the enzymes. An analogous situation exists in the case of
ganglioside GD
and also in lipopolysaccharides from Campylobacter jejuni, which mimic the GD
structure. These molecules contain two, nonreducing, terminal
sialic acid residues, one linked to the penultimate Gal, and the other
linked to the internal Gal. Known sialidases are able to remove only
the sialic acid residue that is linked to the penultimate Gal, but the
sialic acid linked to the internal Gal remains resistant(35) .
The combined data presented in this paper strongly suggest that the
addition of the new GlcNAc residue is the result of the dominant
mutation in LEC18 cells. The novel core is in species of LEC18
glycopeptides that are not synthesized by parent CHO cells;
the novel core is fucosylated, gives a new NMR spectrum, and does not
bind to ConA or PSA (Fig. 3); and the novel core correlates with
the high degree of resistance of LEC18 cells to PSA and LCA. Other
dominant CHO mutants have analogous properties: LEC11, LEC12, LEC29,
and LEC30 CHO mutants add (1,3)-Fuc residues to lactosamine chains
on N-glycans (36, 37, 38) while
LEC10 CHO mutants add the bisecting GlcNAc to N-glycans(39) . Parent CHO cells do not synthesize
either
(1,3)-Fuc or bisecting GlcNAc-containing N-glycans. In each dominant mutant, the new carbohydrates are
the result of the action of a glycosyltransferase activity that is
newly expressed and is not present in parent CHO cells. Each newly
expressed transferase is one that is developmentally regulated in
mammals. Previous studies have provided biochemical evidence for the
absence of these specialized transferase activities in CHO cells (36, 37, 38, 39) , but more recently
we have shown by Northern analysis that CHO cells also lack RNAs for
the relevant glycosyltransferase, whereas the dominant mutants possess
the expected RNA species. (
)Based on our experience with
previous dominant CHO mutants, it was expected that LEC18 cell extracts
would have a transferase activity that adds the new GlcNAc residue to a
suitable acceptor and that such an activity would be absent from parent
CHO cells. In fact, transfer of a GlcNAc residue from UDP-GlcNAc to a
biantennary, N-linked glycan that terminates in GlcNAc has
been achieved with LEC18 cell extracts, (
)whereas no such
activity is detected in parent cell extracts(36) . It will now
be necessary to determine optimal conditions for assaying this
transferase activity and to characterize its product. It is predicted
to be a medial Golgi GlcNAc-transferase that is developmentally
regulated in vivo.