From the Department of Cell Biology, Albert Einstein
College of Medicine, New York, New York 10461 and
§ Genentech Inc.,
South San Francisco, California 94080
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ABSTRACT |
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LEC18 and LEC14 cells are gain-of-function
glycosylation mutants isolated from Chinese hamster ovary cells for
resistance to pea lectin. Structural studies have shown that LEC18
cells synthesize complex N-glycans with a GlcNAc residue
linked at the O-6 position of the core GlcNAc (Raju, T. S., Ray,
M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex
N-glycans with a GlcNAc residue linked at the O-2 position
of the core -linked Man residue (Raju, T. S., and Stanley, P. (1996) J. Biol. Chem. 271, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct
N-acetylglucosaminyltransferase (GlcNAc-T) activity. The
LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to
GlcNAc
1-O-pNP and to a GlcNAc-terminating, biantennary,
complex N-glycan, with or without a core fucose. By
contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have
significant activity with simple acceptors, and transfers GlcNAc
preferentially to a GlcNAc-terminating biantennary glycopeptide that
contains a core fucose residue. The acceptor specificities and other
biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from
previously characterized GlcNAc-transferases including GlcNAc-TIII,
indicating that they represent new members of the mammalian GlcNAc-T
group of transferases.
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INTRODUCTION |
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Glycosylation mutants of mammalian cells allow the identification of new molecules involved in complex glycan synthesis by revealing the nature of acceptor substrates, glycosyltransferases, co-factors, and regulatory molecules through mutations that alter, in each case, only one glycosyl transfer reaction. Both loss-of-function and gain-of-function mutants have revealed new aspects of glycosylation in mammals (1, 2).
Gain-of-function glycosylation mutants express an activity that is not
detectable in the parental cell. Each of several Chinese hamster ovary
(CHO)1 cell gain-of-function
mutants express a glycosyltransferase activity that is lacking in
parental CHO, and that synthesizes N-glycans with a sugar
modification absent from the N-glycans of parent cell
glycoproteins. Thus, the LEC10 CHO mutant (3, 4) expresses GlcNAc-TIII
(5), and the N-glycans of glycoproteins made in LEC10 cells
include a proportion that contain the bisecting GlcNAc (3). LEC11,
LEC12, LEC29, and LEC30 CHO mutants each express an
(1,3)fucosyltransferase activity that is not detectable in parent
CHO cells, and they synthesize N-glycans with fucose in O-3
linkage to the GlcNAc of lactosamine units (6-8).
LEC14 and LEC18 CHO mutants are gain-of-function mutants that were
obtained, after mutagenesis, by selection for resistance to pea lectin
(9). They have unique and distinct lectin resistance properties and
behave dominantly in somatic cell hybrids formed with parent CHO cells
(9). Structural analyses of complex N-glycans from LEC14 and
LEC18 revealed that both add a specific sugar residue to
N-glycans that is not present on N-glycans from
CHO cell glycoproteins (10, 11). In LEC18 cells, a proportion of the
complex, polylactosamine-containing N-glycans contain an
additional GlcNAc in the core region at the O-6 position of the
(1,4)-GlcNAc adjacent to the
-linked core Man residue (Ref. 10;
Fig. 1). This O-6-linked GlcNAc residue is absent from similar
N-glycans of parental CHO cells and has not been observed on
glycoproteins from other sources. In LEC14 cells, a proportion of the
complex, polylactosamine-containing N-glycans contain an
additional GlcNAc linked
(1,2) to the
(1,4)-Man residue of the
core (Ref. 11; Fig. 1). CHO cells lack this modification, which has to
date been observed only in N-glycans from LEC14 cells.
The novel N-glycan cores, which are synthesized by LEC14 and LEC18 mutants, suggested that each mutant expresses a GlcNAc-T activity that is silent in parental CHO cells, in a manner analogous to the previously described gain-of-function CHO mutants (2). In this paper we show that this is indeed the case. These novel activities, termed GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18), generate in vitro, the N-glycan core characteristic of LEC14 and LEC18 glycoproteins, respectively.
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EXPERIMENTAL PROCEDURES |
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Materials
UDP-6-[3H]GlcNAc (6.1 Ci/mmol), concanavalin A
(ConA)-Sepharose, and Sephadex G-25 were from Amersham Pharmacia
Biotech. Pea lectin (PSA)-agarose was from Vector Laboratories. Bio-Gel
P-2 (45-90 mesh), the Bradford protein reagent, and AG1-X4 resin
(200-400 mesh, Cl form) were from Bio-Rad.
N-Acetyl-
-D-glucosaminidases
(Diplococcus pneumoniae and bovine kidney), Pronase
(Streptomyces griseus), and protease inhibitor mixture
tablets were from Boehringer Mannheim or Prozyme, and jack bean
N-acetyl-
-D-glucosaminidase was from Oxford
GlycoSciences. D-(+)-Glc, D-(+)-Gal,
D-(+)-Man, D-(+)-Fuc, D-(+)-GlcN,
D-(+)-GalN, methyl
-D-mannoside (MM), methyl
-D-glucoside, GlcNAc
1-O-pNP,
Gal
1-O-pNP, Glc
1-O-pNP,
Man
1-O-pNP, GalNAc
1-O-pNP, GlcNAc
1,4GlcNAc
1-O-pNP, GlcNAc
1-S-pNP,
GlcNAc
1-O-benzyl, UDP-GlcNAc, UDP-Gal, UDP-Glc,
UDP-GalNAc, ATP, Triton X-100, Nonidet P-40, human IgG, human
fibrinogen, chicken ovalbumin, chitin, chloramine T, potassium
metabisulfite, and bovine serum albumin were from Sigma. Sodium
hydroxide (50%, w/v) and other reagent grade chemicals were from
Fisher.
Purification of Glycopeptides from Standard Glycoproteins
Oligomannosyl glycopeptides were prepared from chicken egg ovalbumin as described (12); biantennary N-linked glycopeptides (GnGn) with no fucose and terminating with GlcNAc were isolated from human fibrinogen as described (13) and the corresponding glycopeptides with fucose (GnGn(Fuc)) were prepared from human IgG. N,N'-Diacetylchitobiose and N,N',N"-triacetylchitotriose were isolated from chitin essentially as described by Rupley et al. (14). Glycopeptides with the structures shown in Fig. 1 were obtained from glycoproteins of LEC14 and LEC18 cells as described (10, 11).
Cell Lines and Cell Culture
CHO cells were grown in suspension at 37 °C in complete medium (Life Technologies, Inc.) containing 10% bovine calf serum. Parent CHO (Pro
5) and Pro
LEC18.21B,
Pro
LEC14.4A, and Pro
LEC10.3C mutant CHO
cells were described previously (3, 9).
Preparation of Cell Extracts
Postnuclear supernatant from LEC18, LEC14, and parent CHO cells
was prepared essentially as described (15). Briefly, cells (~6 × 106) were washed two times with saline, followed by one
wash with homogenizing buffer (10 mM Tris-HCl, pH 7.4, with
250 mM sucrose), and incubated in 1 ml of homogenizing
buffer on ice. After 20 min, the swollen cells were homogenized using a
Balch Homogenizer (Industrial Tectonics Inc., Dexter, Michigan; basic
diameter = 0.2489 inches, tungsten carbide, part p592492) 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 70 °C. For preparation of microsomal membranes,
postnuclear supernatant was centrifuged at 100,000 × g
for 1 h at 4 °C. For LEC10 cells, extraction after cell washing
was in 1.5% Triton X-100, to which glycerol was added to 20% final
volume before storage at
70 °C.
N-Acetylglucosaminyltransferase Assays
Enzyme assays with cell extracts were carried out in 1-ml
Eppendorf tubes in a 50-µl reaction volume containing 12.5 nmol of
UDP-[3H]GlcNAc (~5500 cpm/nmol), 1.5 µmol of
MnCl2, 0.5 µmol of ATP, 0.5% Triton X-100, 0.5 µmol of
sodium cacodylate buffer, pH 7.0, 5-10 µl of extract (~50-100
µg of protein), and one of the following as acceptor molecule: 50 nmol of GlcNAc, GlcNAc1-O-pNP, Gal
1-O-pNP, Glc
1-O-pNP, Man
1-O-pNP,
GlcNAc
1-S-pNP, GlcNAc
1-O-benzyl, or ~74
nmol of GnGn or GnGn(Fuc) for 90 min at 37 °C. To assay GlcNAc-TIII activity, ~50-100 µg of LEC10 extract was incubated in a final volume of 40 µl with ~40-180 nmol of GnGn or GnGn(Fuc), in the presence of 50 mM PIPES buffer, pH 7.0, protease inhibitors
(Boehringer Mannheim tablet) according to manufacturer's instructions,
0.5% Triton X-100, 10 mM MnCl2, 0.2 M GlcNAc, and 24 nmol of UDP-GlcNAc (~25,000 cpm/nmol)
for 2 h at 37 °C. Assays lacking acceptor were used to
determine incorporation into endogenous acceptors and degradation of
donor sugar. After incubation at 37 °C for 15-120 min, the reaction
was stopped by adding 950 µl of cold water. Reactions containing
simple sugars or the glycopeptides, GnGn or GnGn(Fuc) were passed
through a 1-ml column of AG1-X4 (Cl
form) that was
subsequently washed with 3 ml of water. Eluate and washings were
combined, mixed with 17 ml of Ecolume, and counted by liquid
scintillation spectroscopy. Reactions with GlcNAc
1-O-pNP, GlcNAc
1-S-pNP, and GlcNAc
1-O-benzyl
acceptors were passed through a Sep-Pak C18 cartridge
(Waters), and radiolabeled product was eluted with 50% aqueous
methanol as described (16). Radioactivity was measured by liquid
scintillation spectroscopy.
Product Characterization
Size-exclusion Chromatography-- To characterize GlcNAc-T reaction products, terminated reactions were pooled, concentrated to 2 ml, and desalted on a Bio-Gel P-2 column (1.5 cm × 70 cm). The column was eluted under pressure with glass distilled water at a flow rate of 15-20 ml/h, and fractions of 2 ml were collected. Under these conditions, charged molecules may interact with the acrylamide and elute anomalously rather than according to molecular weight. A portion of each fraction (0.5 ml) was mixed with 5 ml of Ecolume, and radioactivity was counted by liquid scintillation spectroscopy.
Lectin Affinity Chromatography--
Assay tubes, in which GnGn
or GnGn(Fuc) were acceptors, were adjusted to 1 ml with cold water and
passed through a 1-ml column of AG1-X4 resin (Cl form)
that was subsequently washed with 3 ml of water. Combined eluate and
washings were concentrated to ~250 µl, mixed with 2× ConA buffer
(0.2 M sodium acetate, 0.02 M
MgCl2, 0.02 M CaCl2, 0.02 M MnCl2, 0.04% sodium azide, pH 7.3), and
applied to a ConA-Sepharose column (0.5 cm × 20 cm). The column
was washed with at least 10 column volumes of ConA buffer before bound
glycopeptides were eluted with at least 4 column volumes of ConA buffer
containing 10 mM methyl-
-D-glucoside,
followed by ConA buffer containing 10 mM MM, and finally
200 mM MM in ConA buffer. Reaction products were also
fractionated on PSA-agarose (0.5 cm × 20 cm) and eluted with ConA
buffer followed by ConA buffer containing 200 mM MM. Fractions of 1 ml were collected, and a portion was counted by scintillation spectroscopy. Pooled products from lectin columns were
desalted on Bio-Gel P-2 (1.5 cm × 70 cm) and characterized by
glycopeptide mapping using a Dionex HPAEC-PAD.
Glycopeptide Mapping by Dionex HPAEC-PAD--
Product analysis
by HPAEC-PAD was performed using a Dionex Bio-LC gradient pump and a
pulsed amperometric model PAD-2 detector (Dionex Corp., Sunnyvale, CA)
as described (17). Briefly, GlcNAc-T reactions containing
GlcNAc1-O-pNP acceptor were passed through a Sep-Pak
C18 cartridge, and the 50% aqueous methanol eluant was dried using a Savant Speed Vac, resuspended in 50-100 µl of glass distilled water, passed through a Centrex filter (Schleicher & Schuell), and analyzed by HPAEC-PAD using a CarboPac PA-10 (4 mm × 250 mm) pellicular anion-exchange column equipped with a CarboPac
guard column. The column was eluted with 15 mM NaOH
generated from 15% eluant 1 (100 mM NaOH) and 85% eluant
2 (water) for 25 min, followed by eluant 3 (500 mM NaOH)
for 10 min, at a flow rate of 0.9 ml/min. The following pulse
potentials and durations were used: E1 = 0.05 V
(t1 = 300 ms); E2 = 0.65 V (t2 = 180 ms); E3 =
0.65 V (t3 = 60 ms). Detection was with 1000 nm full scale. A Dionex Advanced Computer Interface connected to a
Gateway 2000, 4SX-33V computer with Dionex AI-450 software (release
3.32.00) was used to collect the data. For radioactivity measurement,
fractions of 0.5 min (0.45 ml), were collected, mixed with 5 ml of
Ecolume, and counted by liquid scintillation spectrometry.
Temperature and Chemical Inactivation of GlcNAc-T Activity
The novel GlcNAc-T activity in LEC18 cells transferred GlcNAc to
both GlcNAc1-O-pNP and to a glycopeptide substrate (see "Results"). To investigate whether a single GlcNAc-T performed both
reactions, the activities were compared following treatment with
increasing temperatures or with the oxidizing agent chloramine T (18).
For heat inactivation, LEC18 cell extracts (~500 µg of protein in
50 µl of extraction buffer/tube in duplicate) were placed in a water
bath set at the appropriate temperature for 10 min before being
returned to 4 °C and then assayed at 37 °C under standard
conditions with both GlcNAc
1-O-pNP and glycopeptide acceptor GnGn, and in the absence of acceptor. For chemical
inactivation, LEC18 cell extract (~300 µg of protein in 30 µl of
extraction buffer/tube in duplicate) were incubated on ice with 15 µl
of buffer alone or 15 µl of buffer containing increasing
concentrations of chloramine T. After 5 min at 4 °C, 15 µl of
potassium metabisulfite at the same concentration as the chloramine T
was added to stop the oxidation reaction. Subsequently, treated
extracts were assayed under standard conditions with both
GlcNAc
1-O-pNP and GnGn acceptors, and in the absence of
acceptor.
Product Analysis following
N-Acetyl--D-Glucosaminidase Treatment
Transferase reactions containing GnGn or GnGn(Fuc) acceptors
were passed through a 1-ml column of AG1-X4 resin (Cl
form), washed with 3 ml of water, and the combined eluate was desalted
on Bio-Gel P-2, dried, and redissolved in buffer appropriate for
digestion with different
N-acetyl-
-D-glucosaminidases. Jack bean
N-acetyl-
-D-glucosaminidase digestion was
performed in 50-100 µl of sodium citrate phosphate buffer (pH 4.5)
at 37 °C under toluene for 48 h with a total of 50 milliunits
of enzyme. D. pneumoniae or bovine kidney
N-acetyl-
-D-hexosaminidase digestion was
performed in 50-100 µl of sodium citrate phosphate buffer (pH 5.0)
at 37 °C under toluene for 25-48 h with a total of 50-100
milliunits of enzyme. Digestion was stopped by heating in a boiling
water bath for 5 min, and products were desalted on a Bio-Gel P-2
column (1.5 cm × 70.0 cm) before analysis by HPAEC-PAD as
described above for GnGn and GnGn(Fuc) product analysis.
To determine if the LEC18 product from GlcNAc1-O-pNP was
susceptible to digestion with
N-acetyl-
-D-glucosaminidase, ~3,500 cpm
product was digested for 24 h under the conditions described above
with N-acetyl-
-D-glucosaminidase from
D. pneumoniae. As controls,
N,N'-diacetylchitobiose,
N,N',N"-triacetylchitotriose, and
N,N'-diacetylchitobiose
1-O-pNP (100 µg each)
were digested under the same conditions with either jack bean or bovine
testis or D. pneumoniae
N-acetyl-
-D-glucosaminidase. Chitobiose and chitotriose digests were dried, resuspended in 15 µl of 15% aqueous acetic acid containing 9-aminopyrene 1,3,5-trisulfonate to which ~5
µl of sodium cyanoborohydride in THF was added, and incubated at
65 °C for 2 h. The derivatization reaction was stopped by
adding 0.5 ml of cold water. The mixture was analyzed using a capillary electropherograph (P/ACE System 5000, Beckman Instruments, Palo Alto,
CA) equipped with an argon ion laser induced fluorescence detector and
a coated capillary at 20 kV. The digests of LEC18 product from
GlcNAc
1-O-pNP and the chitobiose
1-O-pNP
standard were analyzed by Dionex HPAEC-PAD as described above.
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RESULTS |
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LEC18 Cells Have a GlcNAc-T Activity Undetectable in CHO
Cells--
A subset of complex N-glycans in LEC18
glycoproteins carry a GlcNAc linked to the O-6 position of the core
GlcNAc residue (Fig. 1). A GlcNAc-T
capable of performing this transfer might act on simple monosaccharide
acceptors, similar to chitin synthases (19, 20), or to the GlcNAc-T
from the snail, Lymnaea stagnalis, that transfers GlcNAc in
1,4-linkage to simple GlcNAc derivatives and to terminal GlcNAc
residues of N- and O-glycans (21-23). Therefore, extracts from LEC18 cells were tested for their ability to transfer GlcNAc to simple monosaccharide acceptors.
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LEC18 and LEC14 Cells Have Distinct GlcNAc-Ts That Act on the
Biantennary N-Glycan GnGn(Fuc)--
Attempts to identify a GlcNAc-T
that transferred GlcNAc to simple sugar acceptors in LEC14 extracts
failed (data not shown). Therefore, acceptors that could potentially be
biosynthetic intermediates were tested. The novel GlcNAc residues found
in the core of LEC14 and LEC18 N-glycans (Fig. 1) are
somewhat analogous to the bisecting GlcNAc (Fig. 1), in the case of
LEC14, and to a fucose that modifies the core GlcNAc (24), in the case
of LEC18. GlcNAc-TIII transfers the bisecting GlcNAc to the O-4
position of Man in GnGn (3, 5), and GnGn is also the preferred
substrate of core (1,6)Fuc-T (25) and core
(1,2)xylosyl-T (26).
It has previously been established that parent CHO extract does not
transfer GlcNAc to GnGn under assay conditions in which GlcNAc-TIII in
LEC10 extract transfers the bisecting GlcNAc at a specific activity of
6-10 nmol/mg/h (3, 4). Therefore, the branching transferases, GlcNAc-TIV and GlcNAc-TV, which are certainly active in CHO cells based
on structural studies of CHO-derived glycoproteins (for example Ref.
27), and from assays with GnGn and CHO extracts performed under
different conditions (15), were not detected under the conditions
optimal for the LEC18 GlcNAc-T.
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GlcNAc-TVIII Is Responsible For Transferring GlcNAc to
GlcNAc1-O-pNP and to the Core of a N-Glycan--
Because LEC18
extract transfers GlcNAc to GlcNAc
1-O-pNP, the question
arose as to whether LEC18 extracts possess two novel GlcNAc-Ts:
GlcNAc-TVIII that transfers GlcNAc to the core of N-glycans and another GlcNAc-T that transfers GlcNAc to
GlcNAcb1-O-pNP. This question was addressed by comparing the
heat and chemical inactivation profiles of LEC18 extracts with both
acceptors. The data in Fig. 8
(upper panel) show that increasing temperature reduced
GlcNAc transfer by LEC18 cell extract to GlcNAc
1-O-pNP and to the N-glycan GnGn to a similar degree. In like
manner, when the oxidizing agent chloramine T was used to inactivate
enzyme activity (18), the reduction in GlcNAc transfer to
GlcNAc
1-O-pNP and to GnGn was essentially identical (Fig.
8, lower panel). Therefore, LEC18 cells contain one
activity, GlcNAc-TVIII, that can transfer GlcNAc to both a simple
-linked GlcNAc and to the predicted physiological acceptor,
GnGn.
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GlcNAc-TVII from LEC14 Cells, GlcNAc-TVIII from LEC18 Cells, and
GlcNAc-TIII from LEC10 Cells Exhibit Distinct Acceptor
Preferences--
The data in Table II show that GlcNAc-TVII (LEC14)
and GlcNAc-TVIII (LEC18) transfer GlcNAc to GnGn(Fuc) to different
extents, indicating that the GlcNAc-Ts may not be identical in their
ability to act on this acceptor. Therefore, specific activities were
determined over a range of GnGn(Fuc) concentrations (Fig.
9, upper panel). Lineweaver-Burk plots revealed apparent Km values of 5 mM and 6.3 mM with a
Vmax in each case of ~25 nmol/mg/h for GlcNAc-TVII and GlcNAc-TVIII, respectively. Most interestingly, when
the same experiment was performed with GnGn lacking the core Fuc, LEC18
GlcNAc-TVIII had approximately similar activities
(Vmax = 29 nmol/mg/h;
Km(app) = 11 mM), whereas LEC14
GlcNAc-TVII utilized this acceptor very poorly (Fig. 9, lower
panel). It seems that GlcNAc-TVII strongly prefers a core
glycopeptide acceptor that contains an (1,6)-linked core fucose, and
suggests that this transferase may not act until after the addition of
this fucose to N-glycan cores. This is entirely consistent
with the fact that LEC14 glycopeptides with the
1,2-linked core
GlcNAc were core fucosylated (11). Interestingly, glycopeptides
containing the core GlcNAc unique to LEC18 glycoproteins were also core
fucosylated (10). However, in contrast to GlcNAc-TVII, GlcNAc-TVIII
utilized GnGn and GnGn(Fuc) rather equivalently in vitro
(Fig. 9).
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DISCUSSION |
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Molecules that participate in glycosylation reactions responsible for the synthesis of complex glycans are identified from knowledge of glycan structures that act as acceptors in assays for glycosyltransferases, by characterizing the products of new glycosyltransferase genes, and by determining the biochemical basis of mutations that affect individual glycosylation reactions. The advantage of the mutant strategy is that it often leads to the identification of an activity for which existence would not be predicted from structural studies because the activity generates an intermediate never found on a mature glycoconjugate, or because glycoconjugates bearing the modification generated by that activity have never been isolated. This latter situation is the case with LEC14 and LEC18 CHO cells, which we have shown in this paper to express two new GlcNAc-T activities termed GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18).
Previous studies of the N-glycans present on LEC14 and LEC18 glycoproteins showed that each of these mutants adds a GlcNAc to the core of N-glycans in a linkage not observed in previously characterized N-glycans (Fig. 1; Refs. 10 and 11). Based on the properties of similar gain-of-function CHO glycosylation mutants (2), the biochemical basis for the LEC14 and LEC18 phenotypes was predicted to be the de novo expression of a glycosyltransferase activity (10, 11). In vitro assays with detergent extracts or microsomal membranes have now shown that GlcNAc-TVII and GlcNAc-TVIII are unique to LEC14 and LEC18 cells, respectively. They appear to be Golgi transferases because of their membrane association and because biantennary glycopeptides that are generated in the medial or trans Golgi are acceptors.
The major product obtained from GnGn(Fuc) by GlcNAc-TVIII behaved
exactly as authentic LEC18-derived N-glycans described
previously (10); it did not bind to either ConA-Sepharose or
PSA-agarose, eluted at the expected position from CarboPac PA-1, and
digestion with N-acetyl--glucosaminidases released only
the arm GlcNAcs (Fig. 7). GlcNAc-TVIII is a GlcNAc-to-GlcNAc
transferase as is the snail
(1,4)GlcNAc-T (21-23), and, like the
snail enzyme, GlcNAc-TVIII utilizes simple GlcNAc
1-O-pNP
and glycopeptides as acceptors (Table I). The snail
(1,4)GlcNAc-T is
cloned, and it is therefore quite clear that the same enzyme transfers
to both simple and complex acceptors. The heat and chemical
inactivation profiles of LEC18 cell extract (Fig. 8) provide strong
evidence that GlcNAc-TVIII transfers to GlcNAc
1-O-pNP and
to N-glycan acceptors.
GlcNAc-TVII in LEC14 cells does not act on simple acceptors and is
similar in this regard to most GlcNAc-Ts (31). Neither parent CHO nor
LEC18 cells possess GlcNAc-TVII activity, which is unique to LEC14. The
major product obtained from the action of GlcNAc-TVII on GnGn(Fuc)
behaved as expected (11); it did not bind to ConA-Sepharose or
PSA-agarose, it eluted from CarboPac PA-1 at the same position as the
authentic LEC14 glycopeptide shown in Fig. 1, and it was completely
resistant to digestion with N-acetyl--glucosaminidases
(Fig. 7). GlcNAc-TVII is a GlcNAc-to-Man transferase and catalyzes a
reaction most similar to a
(1,2)xylosyltransferase recently purified
from plants (26). However, in contrast to the
(1,2)xylosyltransferase, which utilizes GnGn and GnGn(Fuc) equivalently as acceptors, GlcNAc-TVII strongly prefers GnGn(Fuc).
Two outcomes of this study are of some interest with respect to the
properties of mammalian GlcNAc-Ts. The first is that GlcNAc-TI and
GlcNAc-TIII are not active under the in vitro conditions
optimal for GlcNAc-TVII and GlcNAc-TVIII (Table
I),2 and GlcNAc-TIV and
GlcNAc-TV activities are minimal under these conditions (Fig. 6). The
second is the clear difference in acceptor preference of the three
GlcNAc-Ts that modify the core of N-glycans. When
presented with GnGn versus GnGn(Fuc), GlcNAc-TIII clearly prefers GnGn and GlcNAc-TVII clearly prefers GnGn(Fuc), whereas GlcNAc-TVIII uses both acceptors equivalently (Figs. 9 and 10). These
results provide additional evidence of the distinct nature of
GlcNAc-TIII, GlcNAc-TVII, and GlcNAc-TVIII. In addition, they indicate
that the in vivo action of these GlcNAc-Ts may depend on the
prior action of other glycosyltransferases, such as
(1,6)fucosyltransferase or branching GlcNAc-Ts. In this context, it
is of note that monoclonal antibodies that recognize the
(1,6)fucose
residue in an N-glycan core do not bind well to LEC14 or
LEC18 glycoproteins (32).
It will now be important to clone the genes that encode GlcNAc-TVII and
GlcNAc-TVIII. This should be possible by expression cloning using a
cDNA library from LEC14 or LEC18 to complement CHO cells. We have
recently shown that expression of GlcNAc-TIII in LEC10 CHO cells
corresponds to transcriptional activation of the Mgat3 gene.
Northern blot analyses, with probes from the mouse Mgat3
gene that encodes GlcNAc-TIII (33), revealed the predicted ~4.7-kilobase Mgat3 gene transcript in LEC10 cells and no
signal from the silent Mgat3 gene of CHO
cells.3 Expression of the
(1,3)Fuc-T in LEC11 cells also corresponds to transcriptional
activation of a Chinese hamster FUT gene. Three independent
LEC11 mutants have been shown to possess an ~1.8-kilobase transcript
that hybridizes to a CHO FUT gene probe, whereas this gene
is transcriptionally inactive in parent CHO cells (34). It therefore
seems likely that GlcNAc-TVII and GlcNAc-TVIII reflect transcriptional
activation of new glycosyltransferase genes. Although it is possible
that, like the blood group A, B, and O transferases that represent
alleles of a single gene (35), GlcNAc-TVII and/or GlcNAc-TVIII might
arise from the alteration of a known GlcNAc-T gene, the properties of
genes encoding GlcNAc-Ts make this unlikely. To date, any GlcNAc-T that
transfers GlcNAc to a unique substrate (e.g. GlcNAc TI
versus GlcNAc-TII; see Ref. 28), or in a specific linkage,
is encoded by a unique gene. There is no significant sequence homology
between the genes encoding GlcNAc-TI, GlcNAc-TII, GlcNAc-TIII,
GlcNAc-TV, or core 2 GlcNAc-T (reviewed in Ref. 36). Although there is
homology between core 2 GlcNAc-T and the GlcNAc-T that creates the I
antigen (37), these genes encode transferases that perform essentially
identical reactions. By contrast, GlcNAc-TVII (LEC14) and GlcNAc-TVIII
(LEC18) perform unique reactions, distinct from all the other GlcNAc-Ts
described to date, and therefore are predicted to be encoded by
distinct genes.
Cloning of both genes is an essential step to determining biological functions for the GlcNAc residues they add to N-glycans. The reason N-glycans with these GlcNAc residues have not previously been observed is presumably because they are present on a limited number of tissue specific glycoproteins or serum glycoproteins, or present in small amounts, or present only at particular times of development. Cloning the genes that encode GlcNAc-TVII and GlcNAc-TVIII will allow their spatio-temporal expression patterns to be determined and their expression to be altered or ablated in a complex organism such as the mouse.
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ACKNOWLEDGEMENTS |
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We thank Subha Sundaram for superb technical assistance and E. Richard Stanley for suggesting the chloramine T experiment.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 36434 (to P. S.).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.
¶ To whom correspondence and reprint requests should be addressed: Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-829-7619; E-mail: stanley{at}aecom.yu.edu.
1
The abbreviations used are: CHO, Chinese hamster
ovary; ConA, concanavalin A agglutinin; PSA, Pisum sativum
(pea) agglutinin; GlcNAc-T, N-acetylglucosaminyltransferase;
GlcNAc1-O-pNP,
p-nitrophenyl-N-acetyl-D-glucosamine; MM, methyl-
-D-mannoside; GnGn, biantennary
N-linked glycopeptide terminating with GlcNAc; GnGn(Fuc),
biantennary N-linked glycopeptide (GnGn) containing a core
fucose residue linked
1,6 to the Asn-linked GlcNAc; HPAEC-PAD, high
performance anion-exchange chromatography with pulsed amperometric
detection; PIPES, 1,4-piperazinediethanesulfonic acid.
2 T. S. Raju, unpublished observations.
3 M. Bhaumik, J. Chang, X. Zhang and P. Stanley, unpublished observations.
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REFERENCES |
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