From the Department of Cell Biology, Albert Einstein
College of Medicine, New York, New York 10461, the
§ Oncology Center, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21287, and ¶ Analytical Chemistry,
Genentech Inc., South San Francisco, California 94080
Received for publication, November 3, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Six A gene that encodes For glycolipid acceptors, all To identify in vivo functions of each To identify glycosyltransferases and other factors that regulate
glycosylation in mammals, we have isolated a number of lectin-resistant CHO glycosylation mutants (20). Two independent CHO mutants that were
selected for resistance to PHA-E belong to the Lec20 complementation
group and behave as loss-of-function mutants in somatic cell hybrids
(21). In this study, we show that they are both defective in the
Materials--
D-[6-3H]Gal (31.5 Ci/mmol), D-[6-3H]GlcN hydrochloride (31.5 Ci/mmol), UDP-[6-3H]GlcNAc (41.60 Ci/nmol),
UDP-[6-3H]Gal (10 Ci/mmol), and ConA-Sepharose were from
Amersham Pharmacia Biotech. PHA-E lectin, PHA-L-agarose, and
RCAII-agarose were from Vector Laboratories, Inc. Bio-Gel
P-2 (45-95 mesh), the detergent compatible protein assay
reagent, and AG 1-X4 resin (200-400 mesh, Cl Cell Lines and Cell Cultures--
Pro Preparation of Radiolabeled VSV Glycopeptides--
Cells growing
in suspension were infected with VSV and subsequently cultured in
Lectin Affinity Chromatography of Radiolabeled
Glycopeptides--
VSV glycopeptides desalted on Bio-Gel P-2 were
fractionated on a 5-ml column of ConA-Sepharose as described previously
(22) into branched and biantennary N-glycans. The ConA-bound
fraction was desalted, treated with neuraminidase, and fractionated on a 5-ml column of RCAII-agarose. Lectin chromatography was
performed at room temperature or at 4 °C. Phosphate-buffered saline
containing 100 mM GalNAc or 200 mM lactose was
used to elute bound glycopeptides. Samples of 0.5-1.0 ml were mixed
with Ecolume at a ratio of ~1:10 and counted in a scintillation counter.
Preparation of Cell Extracts--
Post-nuclear supernatant from
Lec20 and parental CHO cells was prepared as described (23). Briefly,
cells (~6 × 107) were washed two times with saline,
followed by one wash with homogenizing buffer (10 mM
Tris-HCl, pH 7.4, and 250 mM sucrose), and incubated in 1 ml of homogenizing buffer containing an EDTA-free protease inhibitor
tablet on ice. After 20 min, the swollen cells were homogenized using a
Balch homogenizer (Industrial Tectonic Inc.) at 4 °C. The lysate was
centrifuged at 3000 rpm for 30 min at 4 °C. Glycerol was added to
the supernatant to a final concentration of 20% before storage at
Preparation of GlcNAc-terminating Glycopeptides--
Biantennary
N-linked glycopeptides with no fucose and terminating with
GlcNAc (GnGn) were isolated from human fibrinogen as described (24).
Triantennary N-linked glycopeptides (GnGn
Desialylated glycopeptides from Enzyme Assays--
Enzyme assays with cell extracts or
microsomal membranes were carried out in 1.5-ml Eppendorf tubes in a
50-µl total volume. For cell extracts, the reaction contained 5 µmol of MES, pH 6.5, 3 µmol of MnCl2, 1.2% Triton
X-100, 25 nmol of UDP-[6-3H]Gal (~10,000 cpm/nmol), and
~100 µg of protein. For microsomal membranes, the reaction
contained 25 nmol of UDP-[6-3H]Gal (~10,000 cpm/nmol),
1.5 µmol of MnCl2, 1.2% Triton X-100, 5 µmol of sodium
cacodylate buffer, pH 6.5, and 20-25 µg of protein. Acceptors were
0.5-1 µmol of GlcNAc, 2 µmol of Glc (and 0.4 mg/ml Release of N-Linked Oligosaccharides by PNGase F--
Cells were
harvested and washed three times with phosphate-buffered saline. The
cell pellet was resuspended in 20 mM Tris-HCl, pH 7.4, to
obtain ~1 × 1010 cells/ml, to which an equal volume
of 3% Triton X-100 was added. The suspension was mixed well and
incubated on ice for 10 min and at room temperature for 10 min. The
suspension was vortexed for ~2 min and then centrifuged at 5000 rpm
for 30 min. The supernatant was removed and stored at Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF-MS)--
MALDI-TOF-MS was performed on a
Voyager DE Biospectrometry workstation (PerSeptive Biosystems) equipped
with delayed extraction. A nitrogen laser was used to irradiate samples
with ultraviolet light (337 nm), and an average of 240 scans were
taken. The instrument was operated in linear configuration (1.2-m
flight path), and an acceleration voltage of 20 kV was used to propel
ions down the flight tube after a 60-ns delay. Samples (0.5 µl) were
applied to a polished stainless steel target to which 0.3 µl of
matrix was added and dried under vacuum (50 × 10 Generation of Northern Blot Analysis--
Total RNA from CHO or mutant cells
was prepared using 1 ml of TRIzol Reagent (Life Technologies, Inc.) for
107 cells to obtain ~100 µg of total RNA, and
poly(A)+ RNA was prepared using an oligo(dT) column. RNA
was electrophoresed on a formaldehyde-agarose gel, transferred to a
Nytran membrane, and cross-linked using a Spectrolinker UV
cross-linker. Blots were hybridized using ULTRAhyb (Ambion Inc.)
according to the manufacturer's instructions. The probe for each
Reverse Transcription (RT)-PCR--
For reverse transcription, 2 µg of poly(A)+ RNA, 0.5 µg of
oligo(dT)12-18 primer, and 0.05 µg of random hexamer
were heated to 70 °C for 10 min and slowly cooled to room
temperature before adding 200-400 units of Superscript II reverse
transcriptase together with first strand buffer (Life Technologies,
Inc.), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 10 mM
dithiothreitol, and 1 unit/µl RNasin (Promega). Reactions were
incubated for 50 min at 42 °C, heated at 70 °C for 15 min, and
stored at
PCR for
For RT-PCR in Fig. 5B, the forward primer for CHO
Glycolipid Extraction and Purification--
About
109 exponentially growing cells were washed twice with cold
phosphate-buffered saline. Glycolipids were extracted with chloroform/methanol (2:1) as described (29). The extract was evaporated
to dryness, redissolved in chloroform/methanol (1:1) at 108
cell eq/ml, and stored at Thin-layer Chromatography--
Purified glycolipids (1 ml) were
dried under nitrogen gas and resuspended in 25 µl of
chloroform/methanol (2:1) and 25 µl of chloroform/methanol (1:2).
Twenty-five µl were spotted on a Silica Gel 60 high-performance TLC
plate (EM Science) with standard GlcCer (20 µg), LacCer (27 µg), and GM3 (4 µg). The plate was developed by ascending
chromatography in chloroform, methanol, and 0.02% CaCl2
(60:40:9). The dried plate was stained by
resorcinol/H2SO4 reagent and scanned.
Reduced Galactosylation of N-Glycans in Pro Lec20 Mutants Have Reduced
The coding region of
Comparison of the Gat Bovine Pro MALDI-TOF-MS Analysis of N-Glycans in CHO Cells Lacking
The mass spectrometry of neutral N-glycans revealed
markedly increased complexity for the
The lack of
In the case of sialylated N-glycans, the picture is somewhat
different (Fig. 7). First, there were
significant differences between the MALDI-TOF-MS spectra from
Gat CHO Mutants Lacking In Vitro
In the mammary gland,
When more complex N-glycans were assayed as acceptors,
Gat
Lactosylceramide synthase activity was equivalent in wild-type
Gat
Finally, both The knowledge that mammals have six A summary of mutants and their properties is given in Table
V. The loss of 4-galactosyltransferase
(
4GalT) genes have been cloned from mammalian sources. We show that
all six genes are expressed in the Gat
2 line of
Chinese hamster ovary cells (Gat
2 CHO). Two independent
mutants termed Pro
5Lec20 and Gat
2Lec20,
previously selected for lectin resistance, were found to have a
galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro
5Lec20 were proportionately less
ricin-bound than similar species from parental CHO cells, and Lec20
cell extracts had a markedly reduced ability to transfer Gal to
GlcNAc-terminating acceptors. Northern blot analysis revealed a severe
reduction in
4GalT-1 transcripts in Pro
5Lec20 cells.
The Gat
2Lec20 mutant expressed
4GalT-1 transcripts of
reduced size due to a 311-base pair deletion in the
4GalT-1 gene
coding region. Northern analysis with probes from the remaining five
4GalT genes revealed that Gat
2 CHO and
Gat
2Lec20 cells express all six
4GalT genes.
Unexpectedly, the
4GalT-6 gene is not expressed in either
Pro
5 or Pro
5Lec20 cells. Thus, in addition
to a deficiency in
4GalT-1, Pro
5Lec20 cells lack
4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry data of N-glycans released
from cellular glycoproteins showed that both the
4GalT-1
(Gat
2Lec20) and
4GalT-1
/
4GalT-6
(Pro
5Lec20) mutants have a similar Gal deficiency,
affecting neutral and sialylated bi-, tri-, and tetraantennary
N-glycans. By contrast, glycolipid synthesis was normal in
both mutants. Therefore,
4GalT-1 is a key enzyme in the
galactosylation of N-glycans, but is not involved in
glycolipid synthesis in CHO cells.
4GalT-6 contributes only slightly
to the galactosylation of N-glycans and is also not
involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo
substrates of different
4GalTs. They may be used in glycosylation
engineering and in investigating roles for
4GalT-1 and
4GalT-6 in
generating specific glycan ligands.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4-Galactosyltransferase
(
4GalT)1 is a
Golgi-localized, type II transmembrane glycoprotein that catalyzes the
transfer of galactose to GlcNAc, forming Gal
1,4GlcNAc (lactosamine)
units in glycoconjugates (1, 2). Lactosamine sequences are important components of carbohydrate ligands that mediate cell recognition events
such as fertilization (3) and lymphocyte trafficking (4). Also,
terminal galactose residues function as ligands for galactose-binding
proteins such as galectins (5), contactinhibin (6), and the hepatic
binding protein (7).
4GalT-1 was initially cloned from bovine kidney
(1) and mammary gland (2) cDNA libraries. Further cloning and data
base analyses identified five additional human genes that have 30-55%
amino acid identity to the original
4GalT-1 (8). Each encodes a
galactosyltransferase that utilizes the donor substrate UDP-Gal and
transfers Gal in a
1,4-linkage to GlcNAc or Glc (8-11).
4GalT-1
and
4GalT-2 show similar activity in the presence of
-lactalbumin
in that their acceptor specificity is changed from GlcNAc to Glc (9).
4GalT-3, -4, and -5 do not synthesize lactose (9-11).
Interestingly, the ability of
4GalT-4 to transfer Gal to GlcNAc is
activated by
-lactalbumin (11), and the activity of both
4GalT-1
and
4GalT-2 with GlcNAc is inhibited by
-lactalbumin (9). It was
reported that
4GalT-4 is most efficient in galactosylating
mucin-type, core 2 branch oligosaccharides (12), whereas
4GalT-1 is
most efficient in galactosylating i/I antigens (13). It was also
suggested that
4GalT-5 may function best in transferring Gal to
O-glycans (14).
4GalTs show a different activity.
Thus,
4GalT-1 has high activity for GlcCer, Lc3, and
nLc5 in in vitro assays (9).
4GalT-3, -4, and
-5 have little activity with GlcCer (9, 11, 14).
4GalT-6 is a
lactosylceramide synthase predicted to be important for glycolipid
biosynthesis (15).
4GalT-3 utilizes Lc3 efficiently, but
not nLc5 (11); and nLc5 is a poor substrate for
4GalT-4 and
4GalT-5 (10, 11).
4GalT, it is
important to consider the tissue expression pattern as well as acceptor specificity. For example,
4GalT-1 is up-regulated in lactating mammary glands (16), whereas
4GalT-2 is
not.2 Furthermore, mice
deficient in
4GalT-1 do not produce lactose in milk (17, 18). The
4GalT-1, -3, -4, and -5 genes are ubiquitously expressed, whereas
the
4GalT-2 and
4GalT-6 genes exhibit a more restricted
expression pattern (8, 10, 11). Although ~80% mice lacking
4GalT-1 die soon after birth, the remainder are viable and fertile
(17, 18). Serum glycoproteins from
4GalT-1
/
mice were found to be
galactosylated to ~10% compared with those from wild-type mice (19),
providing evidence for the existence of other functional
4GalTs.
However, almost nothing is known of the biological roles of these
4GalTs, and their acceptor specificity has not been defined for
in vivo substrates.
4-galactosylation of N-glycans due to independent
mutations in the
4GalT-1 gene. We also report that
Pro
5 CHO cells lack
4GalT-6 transcripts; and
therefore, Pro
5Lec20 mutants derived from
Pro
5 CHO cells lack both
4GalT-1 and
4GalT-6
activities. Analyses of N-glycans and glycolipids
synthesized by these four CHO cell lines identified in vivo
substrates for several
4GalTs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
form) were
from Bio-Rad. Pronase (Streptomyces griseus), EDTA-free protease inhibitor tablets, and
-galactosidase (Diplococcus
pneumoniae) were from Roche Molecular Biochemicals. Triton X-100,
Nonidet P-40, Triton CF-54, CHAPS, sodium deoxycholate,
-galactosidase (bovine testis and jack bean), neuraminidase
(Clostridium perfringens), GlcNAc, UDP-GlcNAc,
UDP-Gal, human fibrinogen, fetuin, human
1-acid glycoprotein, GlcCer, LacCer, and GM3 were from Sigma. Tween 20, Brij
35, and Lubrol-PX were from Pierce. G418, fetal bovine serum, and
-medium were from Life Technologies, Inc. Ecolume was from ICN
Biomedicals. The detergent G3634A was a gift of Dr. Subashu Basu (Notre
Dame University).
5,
Gat
2, Pro
5Lec20 (clone 15C), and
Gat
2Lec20 (clone 6A) CHO cells were isolated as
previously described (21). Cells were grown in suspension at 37 °C
in complete
-medium containing 10% fetal bovine serum.
-medium containing reduced glucose (0.5 mg/ml), 2% Nuserum
(Collaborative Research), and 83 µCi of [3H]GlcN/10 ml
as described previously (22). Virus was purified by gradient
centrifugation and exhaustively digested with Pronase to generate
Pronase glycopeptides.
80 °C. For preparation of microsomal membranes, the post-nuclear
supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. Also, cell-free extract was prepared in 1.5% Triton
X-100 after cell washing, to which glycerol was added to 20% by volume
before storage at
80 °C as described (23).
4Gn) were
prepared from fetuin, and tetraantennary N-linked
glycopeptides (GnGnGnGn) were prepared from
1-acid
glycoprotein. Briefly, desialylated glycopeptides (3.2 mg of
phenol-sulfuric acid-positive material) prepared from fetuin by
exhaustive Pronase digestion and mild acid treatment (0.01 M HCl, 80 °C, 2 h) were digested with
-galactosidases (jack bean, D. pneumoniae, and bovine
testis) separately. Jack bean
-galactosidase digestion was carried
out in 50 mM acetate buffer, pH 3.5, at room temperature
for 48 h with a total of 200 milliunits of enzyme. The desalted
glycopeptides were digested with D. pneumoniae
-galactosidase (a total of 20 milliunits) in 20 mM
cacodylate buffer, pH 6.5, at 37 °C for 48 h. After desalting, glycopeptides were digested with bovine testis
-galactosidase in 50 mM sodium citrate/phosphate buffer, pH 4.3, at 37 °C for 48 h with a total of 40 milliunits enzyme, desalted, and
freeze-dried (~1.8 mg of phenol-sulfuric acid-positive material based
on the standard curve for mannose).
1-acid glycoprotein
(32.8 mg of phenol-sulfuric acid-positive material) were fractionated on ConA-Sepharose (15 × 43 cm), and the desalted ConA-unbound glycopeptides (~14 mg) were fractionated on PHA-L-agarose (1 × 30 cm) in 1-mg aliquots. The PHA-L-retarded glycopeptides (1.5 mg) were
digested with
-galactosidase as described above, desalted, and
freeze-dried (0.55 mg of phenol-sulfuric acid-positive material). The
glycopeptides were checked by monosaccharide analysis and high-performance anion-exchange chromatography with pulsed amperometric detection. Acid hydrolysis (2.5 M trifluoroacetic acid,
100 °C, 4 h) was followed by fractionation on PA-10 eluted with
18 mM NaOH. When the mannose content was normalized to 3 residues, glycopeptides from fetuin had 0.1 Gal and 4.7 GlcNAc
residues, and glycopeptides from
1-acid glycoprotein had
0.4 Gal and 6.3 GlcNAc residues.
-lactalbumin), and 2 µmol of
Gal
3(GlcNAc
6)GalNAc
-O-paranitrophenol (Toronto Research Chemicals) or 0.11 µmol of GnGn, 0.1 µmol of GnGn
4Gn, 0.05 µmol of GnGnGnGn, and 0.05 µmol of GlcCer.
Reactions lacking acceptor were used to determine incorporation into
endogenous acceptors and degradation of donor sugar. After incubation
at 37 °C for 2 h, the reaction was stopped by adding 1 ml of
cold water. Reactions containing simple sugar or glycopeptides were passed through a 1-ml column of AG 1-X4 (Cl
form), which
was subsequently washed with 2 ml of water to obtain unbound product.
Reactions with
Gal
3(GlcNAc
6)GalNAc
-O-paranitrophenol or GlcCer
were passed through a Sep-Pak C18 (Waters), and
radiolabeled product was eluted with 50% aqueous methanol.
Radioactivity was measured in a liquid scintillation counter.
80 °C until
further use. The protein concentration of the supernatant was ~10
mg/ml. N-Linked oligosaccharides were released by PNGase F
treatment of glycoproteins bound to polyvinylidene difluoride
membranes using a high-throughput microscale method as described by
Papac et al. (25). Released oligosaccharides were passed
through a 0.6-ml cation-exchange resin (AG-50W-X8 resin, H+
form, 100-200 mesh, Bio-Rad) to remove salt and protein contaminants prior to analysis by mass spectrometry.
3 torr). Oligosaccharide standards were
used to achieve a two-point external calibration for mass assignment of
ions (26, 27). 2,5-Dihydroxybenzoic acid/5-methoxysalicylic acid and
2,4,6-trihydroxyacetophenone matrices were used in the analysis of
neutral and acidic oligosaccharides, respectively.
4GalT-1 Transfectants--
Different amounts of
plasmid pSVL DNA containing a bovine
4GalT-1 cDNA (a generous
gift of Dr. Joel H. Shaper) were mixed with pSV2neo DNA (5 µg)
separately and transfected into Pro
5Lec20 cells using the
Polybrene method described previously (28). Transfectants were selected
for resistance to G418 (1.5 mg/ml active weight). The transfectants
were expanded and tested for lectin resistance to PHA-E and for
4GalT activity with GlcNAc as acceptor.
4GalT family member was generated by PCR using forward and reverse
primers corresponding to the beginning and end of the corresponding
full-length murine coding sequence (~1 kb). Probes were labeled with
[32P]dCTP to similar specific activities, and the blots
were hybridized overnight at 42 °C. After washing and exposure to
film for ~3 days, a PhosphorImager (Molecular Dynamics, Inc.) was
used to quantitate band intensity. The primer pairs were as
follows: for
4GalT-1, 5'-GATGAGGTTTCGTGAGCAGT-3' (forward) and
5'-TATCTCGGTGTCCCGATGTC-3' (reverse); for
4GalT-2,
5'-ACGTCTATGCCCAGCACCTG-3' (forward) and 5'-TGGGCTGTCCAATGTCCACT-3'
(reverse); for
4GalT-3, 5'-TGGAGAGACCCTGTACATTG-3' (forward) and
5'-TGTGGTTGGCAGTGGGCA-3' (reverse); for
4GalT-4, 5'-CCTTATCACCTCTCCTACAG-3' (forward) and 5'-GCAGTCCAGAAATCCACTGT-3' (reverse); for
4GalT-5, 5'-GGCATAGTGAACACCTACCT-3' (forward) and
5'-GCATCTCAGTACTCAGTCAC-3' (reverse); and for
4GalT-6,
5'-ACGTACCTCTTTATGGTACAAGCT-3' (forward) and 5'-AACCAGTATTTTGGGTGTGT-3'
(reverse). The glyceraldehyde-3-phosphate dehydrogenase probe was
generated by PCR of a mouse cDNA clone. The fragment generated was
250 bp. The forward primer was 5'-CCATGGAGAAGGCTGGGG-3', and the
reverse primer was 5'-CAAAGTTGTCATGGATGACC-3'.
20 °C.
4GalT-1 gene sequencing was performed using the forward
primer 5'-GTAGCCCACMCCCYTCTTAAAGC-3' and the reverse primer 5'-AATGAGAGGGACCAGCCCAG-3'. The primers were designed on the basis of
proximal 5'- and 3'-untranslated region sequences of the human, bovine,
and mouse
4GalT-1 genes. The PCR mixture contained 15 pmol of
primers, 2 µl of reverse transcription product, 1 µl of 10 mM dNTPs, 0.5 µl of Taq DNA polymerase, 5 µl
of 10× PCR buffer, and 3 µl of 25 mM MgCl2
in a total volume of 50 µl. The mixtures were heated at 94 °C for
2 min, followed by 94 °C for 1 min, annealing at 65 °C for 1 min,
and elongation at 72 °C for 2 min through 40 cycles. PCR products
were purified using a QIAquick gel extraction kit (QIAGEN Inc.) and
sequenced, either directly or after subcloning into the pCR2.1 vector
using the Original TA cloning kit from Invitrogen.
4GalT-1 was 5'-TCACAGCCCCGGCACATTTCT-3' from exon III. The
reverse primer in exon VI was 5'-TATCTTGGTGTCCCGATGTC-3'. For
4GalT-6, the forward primer 5'-ATGTCTGCGCTCAAGCGGAT-3' corresponded
to the 5'-end of the coding sequence of CHO
4GalT-6, and the reverse
primer 5'-GTCTTCGATTGGAGCTAACTC-3' corresponded to the 3'-end of the
coding sequence. Each sample was subjected to one cycle at 94 °C for
1 min, followed by 30 cycles at 94 °C for 1 min, 57 °C for 2 min,
and 72 °C for 3 min.
20 °C. For purification, 0.9 ml of this
preparation were evaporated to dryness and saponified with 1 M sodium hydroxide in methanol at 40 °C for 1 h.
After being neutralized with 1 M acetic acid, the solution
was evaporated to dryness, and the residue was dissolved in 2 ml of
methanol and 1.6 M aqueous sodium acetate (1:1). The
solution was applied to a Sep-Pack C18 cartridge, and the
flow-through was collected and reapplied twice. The cartridge was
washed with 40 ml of water, and glycolipids were eluted with 2 ml of
methanol, followed by 10 ml of chloroform/methanol (2:1). The eluant
was evaporated to dryness, dissolved in 1 ml of chloroform/methanol
(2:1), and stored at
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5Lec20 CHO
Cells--
Independently isolated Lec20 CHO mutants are resistant to
the Gal-binding lectins PHA-E, PHA-L, and ricin (21), consistent with a
reduction in cell-surface Gal residues. To rapidly determine if
N-glycans synthesized in Lec20 cells lack Gal residues,
uniformly labeled Pronase glycopeptides of the G-glycoprotein of VSV
grown in parental (Pro
5 CHO) or Pro
5Lec20
cells were subjected to serial lectin affinity chromatography. ConA-Sepharose chromatography showed no difference in the proportion of
branched (~20%) and biantennary (~80%) complex
N-glycans between parental and mutant-derived VSV
glycopeptides. However, when the ConA-bound, biantennary population of
N-glycans was fractionated on RCAII-agarose at
room temperature, a marked difference between parental and mutant
glycopeptides was revealed. Whereas 46% of the Pro
5
CHO/VSV biantennary N-glycans bound to
RCAII-agarose, consistent with the presence of 2 Gal
residues/N-glycan (30), there were no
RCAII-bound glycopeptides among the
Pro
5Lec20/VSV biantennary species (Fig.
1A). This could be due to increased sialylation or decreased galactosylation. After neuraminidase treatment, ~71% of the Pro
5Lec20/VSV biantennary
glycopeptides bound to RCAII-agarose at 4 °C (Fig.
1B) and were eluted with GalNAc, consistent with the presence of only 1 Gal residue/biantennary N-glycan (30).
Proof that this binding was due to terminal Gal was obtained by
-galactosidase treatment, after which no Lec20/VSV glycopeptides
bound to RCAII-agarose (Fig. 1C). Reduced
RCAII-agarose binding of desialylated biantennary N-glycans was also found with [3H]Gal-labeled
glycopeptides from Pro
5Lec20 cellular glycoproteins (data
not shown). The combined data suggest that Pro
5Lec20
cells have a defect in the addition of Gal residues to complex
N-glycans.
View larger version (31K):
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Fig. 1.
RCAII-agarose affinity
chromatography. [3H]Glucosamine-labeled
Pro 5 CHO/VSV (
) and Pro
5Lec20/VSV (
)
Pronase glycopeptides that bound to and were eluted from ConA-Sepharose
were desalted and fractionated on RCAII-agarose at room
temperature (A). These biantennary N-glycans were
also treated with neuraminidase (NAN'ase) (B) or
neuraminidase and
-galactosidase
(
-Gal'ase) (C) and fractionated on
RCAII-agarose at 4 °C. Bound glycopeptides were eluted
with 100 mM GalNAc (open arrow), followed by 200 mM lactose (closed arrow). In A,
lactose was added at slightly different fractions for
Pro
5Lec20 (solid arrow) and Pro
5
CHO (dashed arrow).
4GalT Activity--
4GalT enzyme
assays were performed with detergent cell extracts and GlcNAc or
biantennary GlcNAc-terminating glycopeptide (GnGn) as acceptor.
Pro
5Lec20 and Gat
2Lec20 cell extracts had
10% galactosyltransferase activity compared with parental cells
(Table I). Mixing equal amounts of
parental and mutant cell extracts yielded one-half the level of
4GalT activity (Table I), showing that the reduced activity in Lec20 cells is not due to the presence of an inhibitor.
4GalT activity of Lec20 CHO mutants
4GalT-1 Transcripts Are Altered in Lec20 Mutants--
When a
Northern blot was probed with a mouse
4GalT-1 probe, the CHO
4GalT-1 signal was observed at ~4.1 kb, and it was apparent that
4GalT-1 transcripts were almost absent in Pro
5Lec20
cells (Fig. 2).
4GalT-1 gene
transcripts in Gat
2Lec20 cells were somewhat reduced and
were notably smaller in size (Fig. 2).
View larger version (45K):
[in a new window]
Fig. 2.
4GalT-1 transcripts in CHO and
Lec20 cells. A Northern blot containing ~7 µg of
poly(A)+ RNA was hybridized to an ~1-kb murine
4GalT-1
probe and subsequently to a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe of a 250-bp PCR product.
4GalT-1 cDNAs from Gat
2 CHO
cells was sequenced (Fig. 3A).
The sequence predicts a polypeptide of 393 amino acids, and hydropathy
analysis (31) revealed a single hydrophobic membrane-spanning domain of
20 amino acids near the N terminus, which predicts the type II
transmembrane topology typical of Golgi glycosyltransferases (32). The
sequence also predicts one putative N-glycosylation site.
ClustalW analysis showed that Gat
2 CHO
4GalT-1 is
90.2% identical to mouse, 83.2% to human, 76.7% to bovine, and
61.9% to chicken
4GalT-1 at the amino acid level. The number and
positions of all 7 Cys residues are conserved in Gat
2 CHO
4GalT-1 (Fig. 3A).
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Fig. 3.
A, ClustalW alignment of 4GalT-1 from
CHO cells, mouse, human, bovine, and chicken. A potential
N-glycosylation site (*) and conserved
4GalT-1 cysteine
residues (
) are marked. B, the
4GalT-1 gene deletion
in Gat
2Lec20. Shown is a schematic diagram of a
4GalT-1 cDNA and protein product. Exons I-VI are based on the
human
4GalT-1 gene (9). The Gat
2Lec20
4GalT-1
cDNA lacked nucleotides reflecting deletion of exon III and all of
exon IV except for the last A residue. Shaded bars represent
translated protein with the first and last amino acids.
2 CHO and Gat
2Lec20
4GalT-1 sequences revealed that the Lec20 mutant was identical
except for a 311-bp deletion that results in the production of a
truncated protein of 214 amino acids derived from exons I, II, and V
(Fig. 3B). This deletion includes a significant portion of
the catalytic domain and therefore appears to be responsible for the
lectin resistance phenotype and reduced
4GalT activity of the
Gat
2Lec20 mutant (Table I). The marked reduction of
4GalT-1 gene transcripts in Pro
5Lec20 cells gives rise
to an essentially identical galactosylation-defective phenotype.
4GalT-1 Corrects the Phenotype of Lec20 Cells--
To
confirm that the reduced
4GalT activity and the lectin resistance
phenotype of Lec20 cells result from an absence of
4GalT-1, bovine
4GalT-1 cDNA was transfected into Pro
5Lec20 cells.
Transfectants were obtained by selection with G418 and tested for their
ability to bind the lectin PHA-E, for which the Lec20 mutant shows
7-fold resistance (21), and for their
4GalT activity. All
transfectants were more sensitive to the toxicity of PHA-E and had
increased
4GalT activity (Fig. 4). Two
transfectants reverted almost to the parental phenotype. These results
support the conclusion that the loss of
4GalT-1 is the cause of the
Lec20 mutant phenotype.
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Fig. 4.
A bovine 4GalT-1
cDNA corrects the Lec20 mutant. The
4GalT activity and
PHA-E sensitivity of Pro
5 CHO, Pro
5Lec20,
and bovine
4GalT-1 transfectants (Tf) of
Pro
5Lec20 cells showed an inverse relationship. Tf
20.1 had a phenotype very similar to that of parental cells.
5 CHO Cells Lack
4GalT-6 Transcripts--
The
absence of functional
4GalT-1 in Lec20 CHO mutants clearly does not
lead to a complete loss of
4GalT activity in cell extracts (Table
I). Thus, it was important to determine which of the other five
mammalian
4GalT genes are expressed in CHO and Lec20 cells. Two
Northern blots were prepared with poly(A)+ RNA from
parental and mutant cells and hybridized with probes of ~1 kb derived
by RT-PCR from the coding region of the corresponding murine
4GalT
sequence. The results in Fig.
5A show that
Gat
2 CHO and Gat
2Lec20 cells express the
six
4GalT genes at similar levels.
4GalT-1 transcripts were the
only ones altered in size in Gat
2Lec20 cells. By
contrast, Pro
5 CHO and the Pro
5Lec20 mutant
were missing
4GalT-6 transcripts. Both also had a somewhat reduced
level of
4GalT-3 transcripts (Fig. 5A). A complete lack
of
4GalT-6 transcripts in Pro
5 CHO and
Pro
5Lec20 cells was confirmed by RT-PCR (Fig.
5B). Thus, the Pro
5 CHO cell, considered a
"wild-type" CHO cell, is actually a "mutant" lacking
4GalT-6. Pro
5Lec20 is a double mutant, essentially
missing
4GalT-1 (transcripts were detected by the sensitive RT-PCR
experiment in Fig. 5B, but not by Northern analysis in Fig.
2) and completely lacking
4GalT-6. Changes in galactosylation of
glycoproteins and glycolipids in the three CHO
4GalT mutants must be
interpreted on this basis.
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Fig. 5.
A, shown is the expression of
4GalT-2, -3, -4, -5, and -6 in CHO and Lec20 cells. Two separate
Northern blots (blot-1 and blot-2) containing 7 µg of poly(A)+ RNA from each cell line were hybridized to
an ~1-kb murine probe and subsequently to a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe of a
250-bp PCR product as described under "Experimental Procedures."
B, RT-PCR was performed with specific primers for CHO
4GalT-6 and CHO
4GalT-1 as described under "Experimental
Procedures." The
4GalT-6 cDNA generated from
Gat
2 CHO RNA was confirmed by digestion with
NarI. The predicted 656- and 478-bp products were
generated.
4GalT-1,
4GalT-6, or Both--
MALDI-TOF-MS has been used for
both structural characterization (33) and relative quantitation (34) of
neutral and sialylated N-glycans in a mixture. To examine
in vivo galactosylation of the spectrum of
N-glycans in CHO glycoproteins, N-glycans were released from parental and mutant CHO cellular glycoproteins by PNGase
F and analyzed by MALDI-TOF-MS. Because most N-glycans derived from mammalian glycoproteins are composed of only a few monosaccharides and generate structures with unique masses that are of
the oligomannosyl or bi-, tri-, or tetraantennary complex type, the
nature of the species released by PNGase F may be deduced from their
molecular mass in the context of known N-glycan structures (25-27).
4GalT-1 mutants
Gat
2Lec20 and Pro
5Lec20 compared with the
4GalT-6 mutant Pro
5 CHO and wild-type
Gat
2 CHO cells (Fig. 6),
consistent with the synthesis of a range of immature,
undergalactosylated N-glycans in Lec20 cells.
Analysis of these spectra is presented in Table
II, in which the numbered peaks in Fig. 6
are identified based on the observed mass of [M + Na]+
ions. It can be seen that each CHO cell line synthesizes a similar complement of oligomannosyl structures. Therefore, a lack of one or two
4GalTs did not significantly alter the proportion of these species,
as expected. By contrast, both cell lines lacking a functional
4GalT-1 had an increased proportion of all the possible forms of
undergalactosylated bi- (peaks 4, 6, 7, and 9), tri- (peaks 11, 12, 15, and 18), and tetraantennary (peaks 16, 19, 21, and 22)
N-glycans. They also made significantly less fully
galactosylated bi- (peaks 10 and 14) and triantennary (peak 20)
N-glycans (Fig. 6), consistent with their reduced in
vitro activities with exogenous acceptors (see Table IV).
Nevertheless, fully galactosylated tetraantennary N-glycans
(peak 23) were equivalently represented in wild-type and Lec20 cells
(Fig. 6). Most striking was the fact that fully galactosylated
N-glycans of each branched type, including biantennary, were
synthesized in the absence of functional
4GalT-1. Although radiolabeled, desialylated VSV biantennary G-glycopeptides from Lec20
did not contain 2 Gal residues (see Fig. 1), high-performance anion-exchange chromatography with pulsed amperometric detection analysis of the reduced proportion of [3H]Gal-labeled,
desialylated biantennary glycopeptides from Lec20 cellular
glycoproteins revealed biantennary glycopeptides that eluted in the
position of a digalactosylated species (data not shown), consistent
with the mass spectrometry data obtained from total
N-glycanase-released species.
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Fig. 6.
MALDI-TOF-MS of neutral
N-glycans. N-Glycans were prepared
from cellular glycoproteins of the four cell lines using PNGase F as
described under "Experimental Procedures" and subjected to
MALDI-TOF-MS with the spectrometer in positive mode.
Neutral N-glycans of CHO and CHO 4GalT mutants
4GalT-6 in Pro
5 CHO cells had a very small
effect on the spectrum of galactosylated neutral N-glycans
(Fig. 6). The only truncated N-glycans that appeared to be
slightly increased in cells lacking
4GalT-6 were peaks 3, 9, and 18 (Fig. 6 and Table II). There were almost no partially galactosylated
tri- or tetraantennary N-glycans in the absence of
4GalT-6. Remarkably, although partially galactosylated biantennary
N-glycans were found in wild-type Gat
2 CHO and
in the absence of
4GalT-6 (Pro
5 CHO cells), there was
no evidence of incomplete tri- or tetraantennary structures in
glycoproteins from either of these cells. It appears that once a third
or fourth GlcNAc is added to the trimannose core of an
N-glycan, galactosylation goes to completion. By contrast, there were many partially galactosylated tri- and tetraantennary N-glycans in the absence of
4GalT-1. Most interestingly,
fully galactosylated complex N-glycans (peaks 10, 14, 20, and 23 in Table II) are well represented in the absence of functional
4GalT-1 and
4GalT-6. Thus, it is clear that the combined
activities of
4GalT-2, -3, -4, and -5 cause Gal to be added to all
antennae. It can be concluded that
4GalT-6 plays an insignificant
role in the galactosylation of neutral N-glycans and that
4GalT-1 plays an important role in the efficient completion of their galactosylation.
2 CHO and Pro
5 CHO N-glycans,
indicating some clear consequences of the lack of
4GalT-6 in
Pro
5 CHO cells (Fig. 7). Pro
5 CHO cells had
a reduced proportion of sialylated bi-, tri-, and tetraantennary
species (see particularly peaks 7, 8, 13, 15, 16, and 18 in Fig. 7),
suggesting that
4GalT-6 is involved in the galactosylation of all
sialylated N-glycans, including those with polylactosamine
units such as peaks 16 and 18 (Table
III). Second, it is clear that
4GalT-1
is involved in synthesizing all classes of sialylated
N-glycans and to a much greater extent than
4GalT-6. Most
of the sialylated complex N-glycans present in
Gat
2 CHO cells (peaks 11 and 13-19 in Table III) were
missing in Gat
2Lec20 and Pro
5Lec20 cells.
However, most of the small population of sialylated N-glycans that were present in Lec20 cells contained a full
complement of Gal (peaks 1/2, 6, 9, and 12 in Table III). In
particular, it is notable that equivalent peaks of
SG2Gn2M3Gn2F (where S
is sialic acid, G is galactose, Gn is N-acetylglucosamine, M
is mannose, and F is fucose) (peaks 1/2) were present in the wild type
and cells lacking
4GalT-1,
4GalT-6, or both (Fig. 7 and Table
III). In addition, few of the many partially galactosylated neutral species generated in the absence of
4GalT-1 (Table II) were found as
sialylated N-glycans (Fig. 7 and Table III). The only
exceptions were peaks 3 and 4 in Fig. 7
(SG2Gn3M3Gn2 and
SG1Gn4M3Gn2 in Table III). Interestingly, these species were equally represented in cells
lacking functional
4GalT-1,
4GalT-6, or both. The combined data
suggest that fully galactosylated N-glycans are sialylated more efficiently than N-glycans with a partial Gal
complement.
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Fig. 7.
MALDI-TOF-MS of sialylated
N-glycans. N-Glycans were prepared
from cellular glycoproteins of the four cell lines using PNGase F as
described under "Experimental Procedures" and subjected to
MALDI-TOF-MS with the spectrometer in negative mode.
Sialylated N-glycans of CHO and CHO 4GalT mutants
4GalT-1,
4GalT-6, or Both Have a Normal
Complement of Glycolipids--
In in vitro assays, GlcCer
is a good acceptor for
4GalT-1 (11), and
4GalT-6 is a
lactosylceramide synthase (15). To identify in vivo
acceptors for these
4GalTs, we isolated glycolipids from
Gat
2 CHO, Gat
2Lec20, Pro
5
CHO, and Pro
5Lec20 mutants and performed high-performance
TLC with the standards GlcCer, LacCer, and GM3. Gat
2Lec8
CHO cells were used as a control because they have an inactive UDP-Gal
Golgi translocase (35) and do not add Gal to glycolipids (29). CHO
cells synthesize GM3 and a small amount of GlcCer and LacCer, but no
complex gangliosides (29), as shown in Fig. 8. As expected, the
Gat
2Lec8 mutant possessed a very small amount of GM3 and
LacCer and an increased amount of GlcCer compared with
Gat
2 CHO parental cells. Interestingly, the glycolipid
expression pattern of CHO cells that lack functional
4GalT-1
(Gat
2Lec20) or
4GalT-6 (Pro
5 CHO) or
both (Pro
5Lec20) was very similar to that of parental
Gat
2 CHO cells, which express all six
4GalTs. The
major glycolipid was GM3 in all CHO cell lines, and there was no
significant increase in GlcCer levels in cells lacking
4GalT-1,
4GalT-6, or
4GalT-1 and
4GalT-6. These results show that
although
4GalT-1 and
4GalT-6 have activity for GlcCer in
vitro, in CHO cells, neither is required for glycolipid synthesis.
4GalT-5 and/or
4GalT-4 seem likely to be responsible for
glycolipid synthesis in CHO cells.
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Fig. 8.
Thin-layer chromatography of
glycolipids. Purified glycolipids extracted from the four cell
lines and Gat 2Lec8 cells were spotted on a Silica Gel 60 high-performance TLC plate with standards (Std) of GlcCer
(20 µg), LacCer (27 µg), and GM3 (4 µg). The plate was developed
by ascending chromatography in chloroform, methanol, and 0.02%
CaCl2 (60:40:9) and stained with
resorcinol/H2SO4 reagent. Glycolipids are
marked with arrowheads. The band marked with an
asterisk in the Gat
2Lec8
lane is not GM3.
4GalT Acceptor Specificities of CHO Cells Lacking
4GalT-1,
4GalT-6, or Both--
To correlate the activities of
4GalT-2, -3, -4, and -5 in cell extracts with the glycans they
produce in vivo,
4GalT assays were performed under a
range of conditions. The mixture of
4GalTs present in Lec20 mutants
had little activity for the transfer of Gal to GlcNAc when Lec20 cell
extracts were assayed under various conditions of substrate
concentration and pH or in the presence of different nonionic
detergents (Fig. 9). When microsomal
membranes from Gat
2 CHO cells, which express the six
4GalT genes, were assayed under the optimized conditions determined
in Fig. 9, a specific activity of ~24 nmol/h/mg of protein was
obtained (Table IV). In
Pro
5 CHO cells, which are missing
4GalT-6, the
specific activity was slightly but significantly reduced,
suggesting that recombinant
4GalT-6 can use GlcNAc as a
substrate, although not efficiently. In Gat
2Lec20 cells
lacking functional
4GalT-1, the activity with GlcNAc was reduced to
~34%. In Pro
5Lec20 cells, with defective
4GalT-1
and
4GalT-6, activity with GlcNAc was only 28% of that in
Gat
2 CHO cells.
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Fig. 9.
4GalT activities of
Pro
5 CHO and
Pro
5Lec20 under different assay
conditions.
4GalT activities of Pro
5 CHO and
Pro
5Lec20 cell extracts were assayed using GlcNAc as
acceptor as described under "Experimental Procedures." The reaction
contained 5 µmol of MES, pH 6.5 (C) or pH 5.7 (B and D), 3 µmol of MnCl2, 1.2%
Triton X-100, 25 nmol of UDP-[6-3H]Gal (~10,000
cpm/nmol) (A-C), 0.5 µmol of GlcNAc (A,
C, and D), and ~100 µg of protein. In
A, MES was used for pH 5~6.5, and MOPS was used for pH
7-8. In C, all detergents were used at a final
concentration of 1%. TX-100, Triton X-100;
NP-40, Nonidet P-40; DOC, sodium
deoxycholate.
4GalT activities of CHO cells lacking
4GalT-1,
4GalT-6, or
both
4GalT activities were measured under optimized conditions using
microsomal membranes as described under "Experimental Procedures."
O-pNP, O-paranitrophenol.
4GalT-1 interacts with
-lactalbumin,
resulting in a change of acceptor specificity from GlcNAc to Glc and
the production of lactose.
4GalT-2 is also able to produce lactose
efficiently (9). However,
4GalT-2 in CHO cells does not appear to
associate with
-lactalbumin in vitro since
Gat
2Lec20 cells, which lack
4GalT-1 activity but have
a normal complement of
4GalT-2 transcripts (Fig. 5A), had
only 3% of the parental Gat
2 CHO activity for transfer
of Gal to Glc in the presence of
-lactalbumin (Table IV).
2 CHO extracts always had a higher specific activity
than Pro
5 CHO extracts, suggesting that
4GalT-6 is
acting on complex acceptors in vitro (Table IV). For
Gat
2Lec20 cells lacking functional
4GalT-1, the most
severe reduction in activity (~97%) was observed for the biantennary
N-glycan acceptor GnGn. Therefore, under the assay
conditions used, none of the five other
4GalTs efficiently
galactosylated a biantennary complex glycopeptide in vitro.
The tetraantennary N-glycan acceptor GnGnGnGn was more
effectively galactosylated in the Gat
2Lec20
4GalT-1
mutant (~27% compared with
Gat
2 CHO cells). However, the triantennary acceptor was a
significantly better acceptor for the mixture of
4GalT-2, -3, -4, -5, and -6 in Gat
2Lec20 cell extract (~54% compared
with wild-type Gat
2 CHO cells). Therefore, whereas
4GalT-1 appears to be the major activity transferring Gal to complex
N-glycans in CHO cell microsomes, other
4GalTs
efficiently transfer Gal to the triantennary complex acceptor
GnGn
4Gn.
4GalT-6 is also able to use GnGn
4Gn efficiently as an
acceptor since the absence of
4GalT-6 in Pro
5 CHO
resulted in a reduction of 43% activity (Table IV).
2 CHO cells and each mutant line (Table IV), as would
be predicted from the glycolipid analysis in Fig. 8. Since
4GalT-6
is known to be a lactosylceramide synthase (15), it was surprising that Pro
5 CHO cells, which lack
4GalT-6, had equivalent
in vitro activity for GlcCer. Similarly,
Gat
2Lec20 and Pro
5Lec20, which lack
functional
4GalT-1 or
4GalT-1 and
4GalT-6, respectively,
showed no decrease in transfer to GlcCer. Thus, neither
4GalT-1 nor
4GalT-6 appears to contribute to glycolipid synthesis in CHO cells.
4GalT-1 and
4GalT-6 contributed to the transfer of
Gal to the mucin core 2 acceptor (Table IV). Compared with Gat
2 CHO cells, Pro
5 CHO cells were reduced
~25%, suggesting that
4GalT-6 adds Gal to core 2;
Gat
2Lec20 cells were reduced ~40%, suggesting that
4GalT-1 also transfers Gal to core 2. Clearly, other
4GalTs such
as
4GalT-4, which has been identified as having a high degree of
specificity for a core 2 acceptor (12), contribute in CHO extracts to
the transfer of Gal to the core 2 oligosaccharide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4GalTs with overlapping
in vitro acceptor specificities (8, 36, 37) presents the challenge of sorting out their unique biological functions. An important question is the degree of redundancy between different members of the
4GalT family in transferring Gal to complex
glycoprotein and glycolipid acceptors. To begin to address this
question, we have identified N-glycan and glycolipid
structures synthesized in CHO cells that express the six
4GalTs
compared with mutant cells that lack
4GalT-1,
4GalT-6, or both.
We have shown that mutants of the Lec20 complementation group (21) lack
4GalT-1 activity. In Gat
2Lec20 CHO cells, it is due to
a deletion mutation that removes exons III and IV of the
4GalT-1
gene so that only the N-terminal 214 amino acids of
4GalT-1 can be
synthesized. Pro
5Lec20 CHO cells have a mutation that
results in extremely low steady-state levels of
4GalT-1 transcripts.
The overall phenotype of both Lec20 mutants is essentially identical
(21); and thus, it was of interest to discover that
Pro
5Lec20 cells have no detectable
4GalT-6
transcripts. The
4GalT-6 deficiency in Pro
5Lec20 cells
originated from the parental Pro
5 CHO cells, which are
also completely devoid of
4GalT-6 gene transcripts. Therefore, we
used the four cell lines to investigate the relative contributions of
4GalT-1,
4GalT-6, and the four remaining
4GalTs to in
vivo Gal transfer to glycoproteins and glycolipids and to in
vitro galactosyltransferase acceptor specificity for various acceptors.
4GalT-1 had the most
profound effect on in vitro lactose synthase activity and on
the transfer of Gal to the biantennary GnGn glycopeptide (Table IV).
One or more of the remaining five
4GalTs transferred Gal quite
efficiently to GlcNAc, tri- and tetraantennary glycopeptides, and the
core 2 oligosaccharide, although
4GalT-1 provided
50% of the
activity with these acceptors. Interestingly,
4GalT-1 did not
contribute to the transfer of Gal to GlcCer in CHO cell extracts, even
though recombinant
4GalT-1 uses GlcCer as an acceptor (9).
Thin-layer chromatography of glycolipids from both the Lec20 mutant
lines confirmed that
4GalT-1 does not contribute to the synthesis of
LacCer or GM3 in CHO cells (Fig. 8).
Summary of CHO 4GalT mutants
The in vitro results with microsomal membranes suggest that
4GalT-1 is the most important
4GalT in galactosylating
biantennary N-glycans (Table IV). This conclusion is
supported by MALDI-TOF-MS analysis of neutral and sialylated
N-glycans (Tables II and III). Gat
2 CHO cells,
which express all six
4GalTs, synthesize fully galactosylated tetra-
or triantennary neutral N-glycans, but have appreciable amounts of undergalactosylated biantennary N-glycans. In
Lec20 mutants that lack functional
4GalT-1, almost every possible
partially galactosylated N-glycan is synthesized, but the
predominant species is undergalactosylated biantennary
N-glycans. Thus,
4GalT-1 is required for efficiently
generating fully galactosylated bi-, tri-, and tetraantennary neutral
N-glycans. Most interestingly, only a very small proportion
of the partially galactosylated structures that predominate in Lec20
mutants acquire sialic acid (Table III). In fact, no biantennary
N-glycans containing 1 Gal residue capped with sialic acid
were detected. This strongly suggests that the first sialic acid is not
transferred by the
2,3 sialyltransferase in CHO cells
until both Gal residues are present on a biantennary structure. In
addition, it is apparent that only one partially galactosylated
triantennary structure
(SG2Gn3M3Gn2) and one
tetraantennary structure
(SG1Gn4M3Gn2) were
present in Lec20 mutants. None of the other neutral
N-glycans with 1, 2, or 3 Gal residues (see Table II) were
sialylated (see Table III). Also of interest are the several
N-glycans that appear to have polylactosamine sequences among the sialylated species.
The results of in vitro galactosyltransferase assays and
glycan analyses reveal subtle but significant effects of the absence of
4GalT-6 in CHO cells. The Pro
5 CHO cell extract, which
lacks
4GalT-6, had significantly, although slightly, reduced
activity with all acceptors except GlcCer (Table IV). By far the
biggest effect of the loss of
4GalT-6 in Pro
5 CHO was
in the ~44% reduced transfer of Gal to the triantennary acceptor
from fetuin (GnGn
4Gn). This was not reflected in an abundance of
undergalactosylated triantennary N-glycans in
Pro
5 CHO glycoproteins, however. In fact, there were only
minor peaks of bi- and triantennary neutral N-glycans
lacking Gal residues in Pro
5 CHO glycoproteins. By
contrast, for the sialylated N-glycans, the absence of
4GalT-6 gave rise to the same species of undergalactosylated tri-
and tetraantennary structures as did the absence of
4GalT-1. Thus,
Pro
5 CHO cells lacking only
4GalT-6 contained only a
subset of the fully galactosylated, sialylated N-glycans
synthesized by the full complement of six
4GalTs in
Gat
2 CHO cells. Finally, it can be seen from the spectrum
of complex N-glycans synthesized in the double mutant
Pro
5Lec20 that the effects of missing
4GalT-1 and
4GalT-6 are essentially additive.
Perhaps the most unexpected result with cells lacking 4GalT-6 was
the fact that this was not reflected in an increased amount of GlcCer
due to reduced synthesis of LacCer and GM3 (Fig. 8).
4GalT-6 was
called LacCer synthase when first cloned (15) and has been proposed to
be a major
4GalT responsible for LacCer synthesis. Although it is
true that all the
4GalTs can synthesize LacCer in vitro,
4GalT-6 and
4GalT-5 are more closely related to each other than
to the other
4GalTs at the amino acid level. Thus, it may be that
4GalT-5 is the
4GalT that synthesizes LacCer in CHO cells because
it is clear that neither
4GalT-1 nor
4GalT-6 is responsible.
In summary, the Gal transfer properties of Gat
2
CHO cells, which express all six
4GalTs, compared with those of
glycosylation mutants lacking functional
4GalT-1
(Gat
2Lec20),
4GalT-6 (Pro
5 CHO), or both
(Pro
5Lec20) show that
4GalT-1 is a key enzyme for the
galactosylation of complex N-glycans and that neither
4GalT-1 nor
4GalT-6 is involved in glycolipid synthesis in CHO
cells (Table V).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Joel H. Shaper for providing
bovine 4GalT-1 and Drs. Sung-Hae Park and Daniel Moloney for helpful comments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 CA36434 (to P. S.) and RO1 CA45799 (to N. L. S. and Joel H. Shaper) and in part by Albert Einstein Cancer Center Grant PO1 13330.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF318896.
To whom correspondence should be addressed: Dept. of
Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-430-8574;
E-mail: stanley@aecom.yu.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M010046200
2 J. H. Shaper, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
4GalT,
4-galactosyltransferase;
GlcCer, glucosylceramide;
LacCer, lactosylceramide;
Lc3, GlcNAc
1,3Gal
1,4GlcCer;
nLc5, GlcNAc
1,3Gal
1,4GlcNAc
1, 3Gal
1,4GlcCer;
GM3, NeuAc
2,3Gal
1,4GlcCer;
CHO, Chinese hamster ovary;
ConA, concanavalin A;
PHA-E, Phaseolus vulgaris erythroagglutinin;
PHA-L, P. vulgaris leukoagglutinin;
RCA, Ricinus
communis agglutinin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MES, 2-(N-morpholino)ethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid;
VSV, vesicular stomatitis virus;
GnGn, GlcNAc-terminating biantennary N-glycan,
GnGn
4Gn, GlcNAc-terminating triantennary N-glycan with
a
1,4-linked branch;
GnGnGnGn, GlcNAc-terminating tetraantennary
N-glycan;
PNGase F, peptide N-glycosidase F;
MALDI-TOF-MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-polymerase chain reaction;
kb, kilobase(s);
bp, base pair(s).
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